Back to overview

SURFE²R N1

Search on our data base

Filter your search

  • Products

  • Resource type

  • Application Areas

  • Therapeutic Areas

  • Reset filters
Search results153
Case study PDF
Replacing radiolabeling techniques with the SURFE2R N1
Flyer PDF
Mitochondrial Membrane Research
Publication link
2024 – Transport mechanism of DgoT, a bacterial homolog of SLC17 organic anion transporters
SURFE2R N1 Preprint Publication in bioRxiv (2024) Authors: Dmitrieva N., Gholami S., Alleva C., Carloni P., Alfonso-Prieto M., Fahlke C.

The solute carrier 17 (SLC17) family contains anion transporters that accumulate neurotransmitters in secretory vesicles, remove carboxylated monosaccharides from lysosomes, or extrude organic anions from the kidneys and the liver. We combined experimental and computational approaches to describe the transport mechanisms of a model bacterial protein, the D-galactonate transporter DgoT, at atomic resolution. We found that protonation of D46 and E133 precedes galactonate binding and that substrate binding induces closure of the extracellular gate, with the conserved R47 coupling substrate binding to transmembrane helix movement. After isomerization to an inward-facing conformation, deprotonation of E133 and subsequent proton trans-fer from D46 to E133 opens the intracellular gate and permits galactonate dissociation. After release of the second proton, apo DgoT returns to the outward-facing conformation. Our results provide a framework to understand how various SLC17 transport functions with distinct transport stoichiometries can be attained through subtle variations in proton and substrate binding/unbinding.

Publication link
2024 – Unraveling the kinetics and pharmacology of human PepT1 using solid supported membrane-based electrophysiology
SURFE2R N1 and SURFE2R 96SE Publication in Bioelectrochemistry (2024) Authors: Körner A., Bazzone A., Wichert M., Barthmes M., Dondapati S.K., Fertig N., Kubick S.

The human Peptide Transporter 1 (hPepT1) is known for its broad substrate specificity and its ability to transport (pro-)drugs. Here, we present an in-depth comprehensive study of hPepT1 and its interactions with various substrates via solid supported membrane-based electrophysiology (SSME). Using hPepT1-containing vesicles, we could not identify any peptide induced pre-steady-state currents, indicating that the recorded peak currents reflect steady-state transport. Electrogenic co-transport of H+/glycylglycine (GlyGly) was observed across a pH range of 5.0 to 9.0. The pH dependence is described by a bell-shaped activity curve and two pK values. KM and relative Vmax values of various canonical and non-canonical peptide substrates were contextualized with current mechanistic understandings of hPepT1. Finally, specific inhibition was observed for various inhibitors in a high throughput format, and IC50 values are reported. Taken together, these findings contribute to promoting the design and analysis of pharmacologically relevant substances.

Publication link
2024 – Functional and structural asymmetry suggest a unifying principle for catalysis in membrane-bound pyrophosphatases
SURFE2R N1 Publication in EMBO Rep. (2024) Authors: Strauss J., Wilkinson C., Vidilaseris K., de Castro Ribeiro O.M., Liu J., Hillier J., Wichert M., Malinen A.M., Gehl B., Jeuken L.J.C., Pearson A.R., Goldman A.

Membrane-bound pyrophosphatases (M-PPases) are homodimeric primary ion pumps that couple the transport of Na+- and/or H+ across membranes to the hydrolysis of pyrophosphate. Their role in the virulence of protist pathogens like Plasmodium falciparum makes them an intriguing target for structural and functional studies. Here, we show the first structure of a K+-independent M-PPase, asymmetric and time-dependent substrate binding in time-resolved structures of a K+-dependent M-PPase and demonstrate pumping-before-hydrolysis by electrometric studies. We suggest how key residues in helix 12, 13, and the exit channel loops affect ion selectivity and K+-activation due to a complex interplay of residues that are involved in subunit-subunit communication. Our findings not only explain ion selectivity in M-PPases but also why they display half-of-the-sites reactivity. Based on this, we propose, for the first time, a unified model for ion-pumping, hydrolysis, and energy coupling in all M-PPases, including those that pump both Na+ and H+.

Publication link
2023 – Structure and electromechanical coupling of a voltage-gated Na+/H+ exchanger
SURFE2R N1 Publication in Nature (2023) Authors: Yeo H., Mehta V., Gulati A., Drew D.

Voltage-sensing domains control the activation of voltage-gated ion channels, with a few exceptions. One such exception is the sperm-specific Na+/H+ exchanger SLC9C1, which is the only known transporter to be regulated by voltage-sensing domains. After hyperpolarization of sperm flagella, SLC9C1 becomes active, causing pH alkalinization and CatSper Ca2+ channel activation, which drives chemotaxis. SLC9C1 activation is further regulated by cAMP, which is produced by soluble adenyl cyclase (sAC). SLC9C1 is therefore an essential component of the pH–sAC–cAMP signalling pathway in metazoa, required for sperm motility and fertilization. Despite its importance, the molecular basis of SLC9C1 voltage activation is unclear. Here we report cryo-electron microscopy (cryo-EM) structures of sea urchin SLC9C1 in detergent and nanodiscs. We show that the voltage-sensing domains are positioned in an unusual configuration, sandwiching each side of the SLC9C1 homodimer. The S4 segment is very long, 90 Å in length, and connects the voltage-sensing domains to the cytoplasmic cyclic-nucleotide-binding domains. The S4 segment is in the up configuration—the inactive state of SLC9C1. Consistently, although a negatively charged cavity is accessible for Na+ to bind to the ion-transporting domains of SLC9C1, an intracellular helix connected to S4 restricts their movement. On the basis of the differences in the cryo-EM structure of SLC9C1 in the presence of cAMP, we propose that, upon hyperpolarization, the S4 segment moves down, removing this constriction and enabling Na+/H+ exchange.

Publication link
2023 – A Comparative Study on the Lysosomal Cation Channel TMEM175 Using Automated Whole-Cell Patch-Clamp, Lysosomal Patch-Clamp, and Solid Supported Membrane-Based Electrophysiology: Functional Characterization and High-Throughput Screening Assay Development
SURFE2R 96SE, SURFE2R N1, SyncroPatch 384 Publication in International Journal of Molecular Sciences (2023) Authors: Bazzone A., Barthmes M., George C., Brinkwirth N., Zerlotti R., Prinz V., Cole K., Friis S., Dickson A., Rice S., Lim J., Toh M., Mohammadi M. Pau D., Stone D., Renger J., Fertig N.
The lysosomal cation channel TMEM175 is a Parkinson’s disease-related protein and a promising drug target. Unlike whole-cell automated patch-clamp (APC), lysosomal patch-clamp (LPC) facilitates physiological conditions, but is not yet suitable for high-throughput screening (HTS) applications. Here, we apply solid supported membrane-based electrophysiology (SSME), which enables both direct access to lysosomes and high-throughput electrophysiological recordings. In SSME, ion translocation mediated by TMEM175 is stimulated using a concentration gradient at a resting potential of 0 mV. The concentration-dependent K+ response exhibited an I/c curve with two distinct slopes, indicating the existence of two conducting states. We measured H+ fluxes with a permeability ratio of PH/PK = 48,500, which matches literature findings from patch-clamp studies, validating the SSME approach. Additionally, TMEM175 displayed a high pH dependence. Decreasing cytosolic pH inhibited both K+ and H+ conductivity of TMEM175. Conversely, lysosomal pH and pH gradients did not have major effects on TMEM175. Finally, we developed HTS assays for drug screening and evaluated tool compounds (4-AP, Zn as inhibitors; DCPIB, arachidonic acid, SC-79 as enhancers) using SSME and APC. Additionally, we recorded EC50 data for eight blinded TMEM175 enhancers and compared the results across all three assay technologies, including LPC, discussing their advantages and disadvantages.
Publication link
2023 – Structure and function of a YeeE-YeeD complex for sophisticated thiosulfate uptake
SURFE2R N1 Publication in bioRxiv (2023) Authors: Ikei M., Miyazaki R., Monden K., Naito Y., Takeuchi A., Takahashi Y., Tanaka Y., Mori T., Ichikawa M., Tsukazaki T.

Uptake of thiosulfate ions as an inorganic sulfur source from the environment is important for bacterial sulfur assimilation. Recently, a selective thiosulfate uptake pathway involving membrane protein YeeE (TsuA) was characterized. However, the precise function of YeeE and a putative cofactor in the
thiosulfate ion uptake pathway remained unclear. Here, we assessed selective thiosulfate transport via YeeE in vitro and characterized YeeD (TsuB) as an adjacent and essential cofactor for YeeE-mediated thiosulfate uptake in vivo. We further showed that YeeD possesses thiosulfate decomposition activity
and that a conserved cysteine in YeeD was modified in several forms in the presence of thiosulfate. Finally, the crystal structure of a YeeE-YeeD fusion protein at 2.6-Å resolution revealed their interactions. The association was evaluated by a binding assay using purified proteins. Based on these results, a model of the sophisticated uptake of thiosulfate ions by YeeE and YeeD is proposed.

Publication link
2023 – Functional Characterization of the Lysosomal Peptide/Histidine Transporter PHT1 (SLC15A4) by Solid Supported Membrane Electrophysiology (SSME)
SURFE2R N1 Publication in Preprints.org (2023) Authors: Pujol-Giménez J., Hediger M.A.

Peptide/Histidine Transporter PHT1 (SLC15A4) is expressed in lysosomal membranes of immune cells where it plays an important role in metabolic and inflammatory signaling. PHT1 is an H+-coupled/histidine symporter that can transport a broad range of oligopeptides, including a variety of bacterial-derived peptides. Moreover, it enables the scaffolding of various metabolic signaling molecules and interacts with key regulatory elements of the immune response. Therefore, it is not surprising that PHT1 is associated with the development of autoimmune diseases, such as systemic lupus erythematosus (SLE). Unfortunately, the pharmacological development of PHT1 has been hampered by the lack of appropriate transport assays. With the aim to address this shortcoming, a novel transport assay based on solid supported membrane-based electrophysiology (SSME) is presented. Key findings of the present SSME studies include the first recordings of electrophysiological properties, a pH dependence analysis, an assessment of PHT1 substrate selectivity, as well as the transport kinetics of the identified substrates. In contrast to previous works, PHT1 is studied its native lysosomal environment. Moreover, observed substrate selectivity is validated by molecular docking. Overall, this new SSME-based assay is expected to contribute to unlock the pharmacological potential of PHT1 and to deepen the understanding of its functional properties.

Poster PDF
2023 – Functional characterization of human GAT-1 using solid supported membrane
SURFE2R N1 and Patchliner Poster
Publication link
2023 – A Small Multidrug Resistance Transporter in Pseudomonas aeruginosa Confers Substrate-Specific Resistance or Susceptibility
SURFE2R N1 Pre-Print Publication in BioRxiv (2023) Authors: Wegrzynowicz A.K., Heelan W.J., Demas S.P., McLean M.S., Peters J.M., Henzler-Wildman K.A.

Voltage-sensing domains control the activation of voltage-gated ion channels, with a few exceptions. One such exception is the sperm-specific Na+/H+ exchanger SLC9C1, which is the only known transporter to be regulated by voltage-sensing domains. After hyperpolarization of sperm flagella, SLC9C1 becomes active, causing pH alkalinization and CatSper Ca2+ channel activation, which drives chemotaxis. SLC9C1 activation is further regulated by cAMP, which is produced by soluble adenyl cyclase (sAC). SLC9C1 is therefore an essential component of the pH–sAC–cAMP signalling pathway in metazoa, required for sperm motility and fertilization. Despite its importance, the molecular basis of SLC9C1 voltage activation is unclear. Here we report cryo-electron microscopy (cryo-EM) structures of sea urchin SLC9C1 in detergent and nanodiscs. We show that the voltage-sensing domains are positioned in an unusual configuration, sandwiching each side of the SLC9C1 homodimer. The S4 segment is very long, 90 Å in length, and connects the voltage-sensing domains to the cytoplasmic cyclic-nucleotide-binding domains. The S4 segment is in the up configuration—the inactive state of SLC9C1. Consistently, although a negatively charged cavity is accessible for Na+ to bind to the ion-transporting domains of SLC9C1, an intracellular helix connected to S4 restricts their movement. On the basis of the differences in the cryo-EM structure of SLC9C1 in the presence of cAMP, we propose that, upon hyperpolarization, the S4 segment moves down, removing this constriction and enabling Na+/H+ exchange.

Publication link
2023 – PI-(3,5)P2-mediated oligomerization of the endosomal sodium/proton exchanger NHE9
SURFE2R N1 Pre-Print Publication in BioRxiv (2023) Authors: Kokane S., Meier P.F., Gulati A., Matsuoka R., Pipatpolkai T., Delemotte L., Drew D.

Na+/H+ exchangers (NHE) are found in all cells to regulate intracellular pH, sodium levels and cell volume. The NHE isoform 9 (SLC9A9) fine-tunes endosomal pH, and its activity is linked to glioblastoma, epilepsy, autism spectrum and attention-deficit-hyperactivity disorders. Here, we report cryo-EM structures of horse NHE9 and a cysteine-variant at 3.6 and 3.1 Å resolution, respectively. We show how lysine residues, from a previously unresolved TM2-TM3 β-hairpin loop domain, are positioned above the dimerization interface and interact with the endosomal-specific PI-(3,5)P2 lipid, together with residues located on dimer domain helices. Thermal-shift assays, solid-state membrane (SSM) electrophysiology and MD simulations, corroborates that NHE9 can specifically bind PI-(3,5)P2, and that its addition stabilizes the homodimer and enhances NHE9 activity. We have further determined the cryo-EM structure of E. coli NhaA, confirming the expected coordination of cardiolipin at the dimerization interface, solidifying the concept that Na+/H+ exchanger dimerization and transporter activity can be regulated by specific lipids. Taken together, we propose that the activity of NHE9 is regulated by the PI-(3,5)P2 lipid upon reaching endosomes, which we refer to as an lipid-activation-upon-arrival model.

Publication link
2023 – Plasticity of the binding pocket in peptide transporters underpins promiscuous substrate recognition
SURFE2R N1 Publication in Cell Reports (2023) Authors: Kotov V. Killer M. Jungnickel K.E.J., Lei J., Finocchio G., Steinke J., Bartels K., Strauss J., Dupeux F., Humm A-S., Cornaciu I., Márquez J.A., Pardon E., Steyaert J., Löw C.

Proton-dependent oligopeptide transporters (POTs) are promiscuous transporters of the major facilitator superfamily that constitute the main route of entry for a wide range of dietary peptides and orally administrated peptidomimetic drugs. Given their clinical and pathophysiological relevance, several POT homologs have been studied extensively at the structural and molecular level. However, the molecular basis of recognition and transport of diverse peptide substrates has remained elusive. We present 14 X-ray structures of the bacterial POT DtpB in complex with chemically diverse di- and tripeptides, providing novel insights into the plasticity of the conserved central binding cavity. We analyzed binding affinities for more than 80 peptides and monitored uptake by a fluorescence-based transport assay. To probe whether all 8400 natural di- and tripeptides can bind to DtpB, we employed state-of-the-art molecular docking and machine learning and conclude that peptides with compact hydrophobic residues are the best DtpB binders.

Publication link
2023 – Transport of metformin metabolites by guanidinium exporters of the Small Multidrug Resistance family
SURFE2R N1 Pre-Print Publication in BioRxiv (2023) Authors: Lucero R.M., Demirer K., Yeh T.J., Stockbridge R.B.

Proteins from the Small Multidrug Resistance (SMR) family are frequently associated with horizontally transferred multidrug resistance gene arrays found in bacteria from wastewater and the human-adjacent biosphere. Recent studies suggest that a subset of SMR transporters might participate in metabolism of the common pharmaceutical metformin by bacterial consortia. Here, we show that both genomic and plasmid-associated transporters of the SMRGdx functional subtype export byproducts of microbial metformin metabolism, with particularly high export efficiency for guanylurea. We use solid supported membrane electrophysiology to evaluate the transport kinetics for guanylurea and native substrate guanidinium by four representative SMRGdx homologues. Using an internal reference to normalize independent electrophysiology experiments, we show that transport rates are comparable for genomic and plasmid-associated SMRGdx homologues, and using a proteoliposome-based transport assay, we show that 2 proton:1 substrate transport stoichiometry is maintained. Additional characterization of guanidinium and guanylurea export properties focuses on the structurally characterized homologue, Gdx-Clo, for which we examined the pH dependence and thermodynamics of substrate binding and solved an x-ray crystal structure with guanylurea bound. Together, these experiments contribute in two main ways. By providing the first detailed kinetic examination of the structurally characterized SMRGdx homologue Gdx-Clo, they provide a functional framework that will inform future mechanistic studies of this model transport protein. Second, this study casts light on a potential role for SMRGdx transporters in microbial handling of metformin and its microbial metabolic byproducts, providing insight into how native transport physiologies are co-opted to contend with new selective pressures.

Publication link
2023 – Insight into the mechanism of H+-coupled nucleobase transport
SURFE2R N1 Publication in PNAS (2023) Authors: Weng J., Zhou X., Wiriyasermkul P., Ren Z., Chen K., Gil-Iturbe E., Zhou M., Quick M.

Members of the nucleobase/ascorbic acid transporter (NAT) gene family are found in all kingdoms of life. In mammals, the concentrative uptake of ascorbic acid (vitamin C) by members of the NAT family is driven by the Na+ gradient, while the uptake of nucleobases in bacteria is powered by the H+ gradient. Here, we report the structure and function of PurTCp, a NAT family member from Colwellia psychrerythraea. The structure of PurTCp was determined to 2.80 Å resolution by X-ray crystallography. PurTCp forms a homodimer, and each protomer has 14 transmembrane segments folded into a transport domain (core domain) and a scaffold domain (gate domain). A purine base is present in the structure and defines the location of the substrate binding site. Functional studies reveal that PurTCp transports purines but not pyrimidines and that purine binding and transport is dependent on the pH. Mutation of a conserved aspartate residue close to the substrate binding site reveals the critical role of this residue in H+-dependent transport of purines. Comparison of the PurTCp structure with transporters of the same structural fold suggests that rigid-body motions of the substrate-binding domain are central for substrate translocation across the membrane.

Publication link
2022 – Full opening of helix bundle crossing does not lead to NaK channel activation
SURFE2R N1 Publication in J. Gen. Physiol. (2022) Authors: Kurauskas V., Tonelli M., Henzler-Wildman K.

A critical part of ion channel function is the ability to open and close in response to stimuli and thus conduct ions in a regulated fashion. While x-ray diffraction studies of ion channels suggested a general steric gating mechanism located at the helix bundle crossing (HBC), recent functional studies on several channels indicate that the helix bundle crossing is wide-open even in functionally nonconductive channels. Two NaK channel variants were crystallized in very different open and closed conformations, which served as important models of the HBC gating hypothesis. However, neither of these NaK variants is conductive in liposomes unless phenylalanine 92 is mutated to alanine (F92A). Here, we use NMR to probe distances at near-atomic resolution of the two NaK variants in lipid bicelles. We demonstrate that in contrast to the crystal structures, both NaK variants are in a fully open conformation, akin to Ca2+-bound MthK channel structure where the HBC is widely open. While we were not able to determine what a conductive NaK structure is like, our further inquiry into the gating mechanism suggests that the selectivity filter and pore helix are coupled to the M2 helix below and undergo changes in the structure when F92 is mutated. Overall, our data show that NaK exhibits coupling between the selectivity filter and HBC, similar to K+ channels, and has a more complex gating mechanism than previously thought, where the full opening of HBC does not lead to channel activation.

Read more in the publication here.

Conference poster
2023 – Investigating lysosomal membrane proteins using SSM based electrophysiology: Improving amplification and accessibility
Poster presented at Physiology 2023 in Harrogate, UK
Publication link
2023 – Structure and sucrose binding mechanism of the plant SUC1 sucrose transporter
SURFE2R N1 Publication in Nature Plants (2023) Authors: Bavnhøj L., Driller J., Zuzic L., Dyrholm Stange A., Schiøtt B., Panyella Pedersen B.

Sucrose import from photosynthetic tissues into the phloem is mediated by transporters from the low-affinity sucrose transporter family (SUC/SUT family). Furthermore, sucrose redistribution to other tissues is driven by phloem sap movement, the product of high turgor pressure created by this import activity. Additionally, sink organs such as fruits, cereals and seeds that accumulate high concentrations of sugar also depend on this active transport of sucrose. Here we present the structure of the sucrose–proton symporter, Arabidopsis thaliana SUC1, in an outward open conformation at 2.7 Å resolution, together with molecular dynamics simulations and biochemical characterization. We identify the key acidic residue required for proton-driven sucrose uptake and describe how protonation and sucrose binding are strongly coupled. Sucrose binding is a two-step process, with initial recognition mediated by the glucosyl moiety binding directly to the key acidic residue in a stringent pH-dependent manner. Our results explain how low-affinity sucrose transport is achieved in plants, and pinpoint a range of SUC binders that help define selectivity. Our data demonstrate a new mode for proton-driven symport with links to cation-driven symport and provide a broad model for general low-affinity transport in highly enriched substrate environments.

Read more in the publication here.

 

Application note
TMEM175 – “TMEM 175 activity measurement in lysosomes on Nanion’s SURFE2R N1″
SURFE2R N1 application note:

TMEM175 is a lysosomal cation leak channel, which impairment has been linked to Parkinson’s disease and other neurodegenerative disorders; thereby making it an interesting drug target. The presented recordings illustrate SURFE2R’s potential as means for target validation and compound screening against TMEM175 channels residing in lysosomal membranes.

Publication Link
2023 – Symport and antiport mechanisms of human glutamate transporters
SURFE2R N1 Publication in Nature Communications (2023) Qiu B., Boudker O.

Excitatory amino acid transporters (EAATs) uptake glutamate into glial cells and neurons. EAATs achieve million-fold transmitter gradients by symporting it with three sodium ions and a proton, and countertransporting a potassium ion via an elevator mechanism. Despite the availability of structures, the symport and antiport mechanisms still need to be clarified. We report high-resolution cryo-EM structures of human EAAT3 bound to the neurotransmitter glutamate with symported ions, potassium ions, sodium ions alone, or without ligands. We show that an evolutionarily conserved occluded translocation intermediate has a dramatically higher affinity for the neurotransmitter and the countertransported potassium ion than outward- or inward-facing transporters and plays a crucial role in ion coupling. We propose a comprehensive ion coupling mechanism involving a choreographed interplay between bound solutes, conformations of conserved amino acid motifs, and movements of the gating hairpin and the substrate-binding domain.

Read more in the paper here.

Publication Link
2023 – A bioelectrochemical approach based on a solid supported membrane to evaluate the effect of natural products on Ca2+-ATPase: The case of 6-gingerol
SURFE2R ONE (a predecessor of the SURFE2R N1) Publication in Biochimica Acta (2023) Authors: Sfragano P.S.., Palchetti I., Tadini-Buoninsegni F.

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) plays an essential role in maintaining the low cytosolic Ca2+ level that enables a variety of cellular processes. SERCA couples ATP hydrolysis to the transport of two Ca2+ ions against their electrochemical potential gradient from the cytoplasm into the lumen of the sarco/endoplasmic reticulum (SR/ER). Because of its central role in regulating cytoplasmic Ca2+ concentration, SERCA dysfunction has been associated with several pathological conditions. Stimulation of SERCA activity may represent a potential therapeutic strategy in various disease states connected with dysfunctional SERCA. The natural phenolic compound 6-gingerol, the most abundant and the major biologically active compound of ginger, was reported to activate the SERCA enzyme. The present study aimed at investigating the effect of 6-gingerol on SERCA transport activity using a bioelectrochemical approach based on a solid supported membrane (SSM). We first performed a voltammetric characterization of 6-gingerol to better understand its electrochemical behavior. We then studied the interaction of 6-gingerol with SR vesicles containing SERCA adsorbed on the SSM electrode. The measured current signals indicated that ATP-dependent Ca2+ translocation by SERCA was remarkably increased in the presence of 6-gingerol at low micromolar concentration. We also found that 6-gingerol has a rather high affinity for SERCA (EC50 of 1.8 ± 0.3 µM), and SERCA activation by 6-gingerol is reversible. The observed stimulatory effect of 6-gingerol on SERCA Ca2+-translocating activity may be beneficial in the prevention and/or treatment of pathological conditions related to SERCA dysfunction.

Publication Link
2023 – Mutation in glutamate transporter homologue GltTk provides insights into pathologic mechanism of episodic ataxia 6
SURFE2R N1 Publication in Nature Communications (2023) Colucci E., Anshari Z., Patiño-Ruiz M., Nemchinova M., Whittaker J., Slotboom D., Guskov A.

Episodic ataxias (EAs) are rare neurological conditions affecting the nervous system and typically leading to motor impairment. EA6 is linked to the mutation of a highly conserved proline into an arginine in the glutamate transporter EAAT1. In vitro studies showed that this mutation leads to a reduction in the substrates transport and an increase in the anion conductance. It was hypothesised that the structural basis of these opposed functional effects might be the straightening of transmembrane helix 5, which is kinked in the wild-type protein. In this study, we present the functional and structural implications of the mutation P208R in the archaeal homologue of glutamate transporters GltTk. We show that also in GltTk the P208R mutation leads to reduced aspartate transport activity and increased anion conductance, however a cryo-EM structure reveals that the kink is preserved. The arginine side chain of the mutant points towards the lipidic environment, where it may engage in interactions with the phospholipids, thereby potentially interfering with the transport cycle and contributing to stabilisation of an anion conducting state.

Publication Link
2022 – Wastewater bacteria remediating the pharmaceutical metformin: Genomes, plasmids and products
SURFE2R N1 Publication in Front. Physiol. (2022) Authors: Martinez-Vaz B.M., Dodge A.G., Lucero R.M., Stockbridge R.B., Robinson A.A., Tassoulas L.J., Wackett L.P.
1Department of Biolog

Metformin is used globally to treat type II diabetes, has demonstrated anti-ageing and COVID mitigation effects and is a major anthropogenic pollutant to be bioremediated by wastewater treatment plants (WWTPs). Metformin is not adsorbed well by activated carbon and toxic N-chloro derivatives can form in chlorinated water. Most earlier studies on metformin biodegradation have used wastewater consortia and details of the genomes, relevant genes, metabolic products, and potential for horizontal gene transfer are lacking. Here, two metformin-biodegrading bacteria from a WWTP were isolated and their biodegradation characterized. Aminobacter sp. MET metabolized metformin stoichiometrically to guanylurea, an intermediate known to accumulate in some environments including WWTPs. Pseudomonas mendocina MET completely metabolized metformin and utilized all the nitrogen atoms for growth. Pseudomonas mendocina MET also metabolized metformin breakdown products sometimes observed in WWTPs: 1-N-methylbiguanide, biguanide, guanylurea, and guanidine. The genome of each bacterium was obtained. Genes involved in the transport of guanylurea in Aminobacter sp. MET were expressed heterologously and shown to serve as an antiporter to expel the toxic guanidinium compound. A novel guanylurea hydrolase enzyme was identified in Pseudomonas mendocina MET, purified, and characterized. The Aminobacter and Pseudomonas each contained one plasmid of 160 kb and 90 kb, respectively. In total, these studies are significant for the bioremediation of a major pollutant in WWTPs today.

Publication link
2023- Functional characterization of SGLT1 using SSM-based electrophysiology: Kinetics of sugar binding and translocation
SURFE2R- N1 Publication in Front. Physiol. (2023) Authors: Bazzone A., Zerlotti R., Barthmes M., Fertig N.

Beside the ongoing efforts to determine structural information, detailed functional studies on transporters are essential to entirely understand the underlying transport mechanisms. We recently found that solid supported membrane-based electrophysiology (SSME) enables the measurement of both sugar binding and transport in the Na+/sugar cotransporter SGLT1 (Bazzone et al, 2022a). Here, we continued with a detailed kinetic characterization of SGLT1 using SSME, determining KM and KDapp for different sugars, kobs values for sugar-induced conformational transitions and the effects of Na+, Li+, H+ and Cl on sugar binding and transport. We found that the sugar-induced pre-steady-state (PSS) charge translocation varies with the bound ion (Na+, Li+, H+ or Cl), but not with the sugar species, indicating that the conformational state upon sugar binding depends on the ion. Rate constants for the sugar-induced conformational transitions upon binding to the Na+-bound carrier range from 208 s−1 for D-glucose to 95 s−1 for 3-OMG. In the absence of Na+, rate constants are decreased, but all sugars bind to the empty carrier. From the steady-state transport current, we found a sequence for sugar specificity (Vmax/KM): D-glucose > MDG > D-galactose > 3-OMG > D-xylose. While KM differs 160-fold across tested substrates and plays a major role in substrate specificity, Vmax only varies by a factor of 1.9. Interestingly, D-glucose has the lowest Vmax across all tested substrates, indicating a rate limiting step in the sugar translocation pathway following the fast sugar-induced electrogenic conformational transition. SGLT1 specificity for D-glucose is achieved by optimizing two ratios: the sugar affinity of the empty carrier for D-glucose is similarly low as for all tested sugars (KD,Kapp = 210 mM). Affinity for D-glucose increases 14-fold (KD,Naapp = 15 mM) in the presence of sodium as a result of cooperativity. Apparent affinity for D-glucose during transport increases 8-fold (KM = 1.9 mM) compared to KD,Naapp due to optimized kinetics. In contrast, KM and KDapp values for 3-OMG and D-xylose are of similar magnitude. Based on our findings we propose an 11-state kinetic model, introducing a random binding order and intermediate states corresponding to the electrogenic transitions detected via SSME upon substrate binding.

 

Read more in the paper and find out more about other Solute Carriers in the article collection here.

 

Poster PDF
2015 – Organellar Transporters and Ion Channels – How to access their electrophysiology by using the SURFE2R technology and Planar Patch Clamp
SURFE2R N1, SyncroPatch 96 (a predecessor model of the SyncroPatch 384) and Port-a-Patch poster, GRC - Organellar Channels and Transporters 2015
Webinar
2018 – Electrophysiological Characterization of Sugar Transporters using SSM-based Electrophysiology
Presenter:
Dr. Andre Bazzone, Application Scientist, Nanion Technologies GmbH, Germany
Andre is an expert in the field of SSM-based electrophysiology: He made his PhD at the Max-Planck-Institute of Biophysics in Frankfurt, Germany on the electrophysiological characterization of sugar transporters using the SSM-based electrophysiology in 2016. Right afterwards, he started as Application Scientist at Nanion Technologies and today he is an important member of the Nanion SURFE2R team.
Source:
Webinar: "The SURFE2R Technology: Assays for Pharmacological and Functional Characterization of Membrane Transporters"

SSM-based electrophysiology helps to understand the mechanisms of different transporters. The technique was used to characterize and compare different sugar transporters and their transport deficient mutants. Proton-coupled (LacY, XylE, FucP), sodium-coupled (MelB) and loosely coupled (GlcP) sugar transporters were analyzed. A general transport model was concluded from the electrophysiological data. Here we present the most intriguing results for these transporters as well as our conclusions regarding the transport mechanism. We will discuss (1) substrate specifity, (2) protonation and coupling mechanisms, (3) the impact of different driving forces, (4) sugar binding kinetics and (5) the significance of specific amino acids for the transport cycle. All together SSM-based electrophysiology helped to conclude a detailed kinetic model for sugar transporters.

Webinar
2018 – Transporters Investigated Using the SURFE2R Instruments
Presenter:
Dr. Maria Barthmes, Product Manager SURFE2R product family, Nanion Technologies GmbH, Germany

Source:
Webinar: "The SURFE2R Technology: Assays for Pharmacological and Functional Characterization of Membrane Transporters"
January 30, 2018

Maria explains the solid supported membrane (SSM)-based methodology and gives a brief introduction on our two devices, the SURFE2R 96SE and the SURFE2R N1.

Publication link
2016 – Electrophysiological characterization of the archaeal transporter NCX_Mj using solid supported membrane technology
SURFE2R N1 and Vesicle Prep Pro Publication in Journal of General Physiology (2016) Authors: Barthmes M., Liao J., Jiang Y., Brüggemann A., Wahl-Schott C.

Sodium–calcium exchangers (NCXs) are membrane transporters that play an important role in Ca2+ homeostasis and Ca2+ signaling. The recent crystal structure of NCX_Mj, a member of the NCX family from the archaebacterium Methanococcus jannaschii, provided insight into the atomistic details of sodium–calcium exchange. Here, we extend these findings by providing detailed functional data on purified NCX_Mj using solid supported membrane (SSM)–based electrophysiology, a powerful but unexploited tool for functional studies of electrogenic transporter proteins. We show that NCX_Mj is highly selective for Na+, whereas Ca2+ can be replaced by Mg2+ and Sr2+ and that NCX_Mj can be inhibited by divalent ions, particularly Cd2+. By directly comparing the apparent affinities of Na+ and Ca2+ for NCX_Mj with those for human NCX1, we show excellent agreement, indicating a strong functional similarity between NCX_Mj and its eukaryotic isoforms. We also provide detailed instructions to facilitate the adaption of this method to other electrogenic transporter proteins. Our findings demonstrate that NCX_Mj can serve as a model for the NCX family and highlight several possible applications for SSM-based electrophysiology.

Poster PDF
2020 – A novel approach to detect electrogenic transporter activity in intact cells applied to investigate iPSC derived cardiomyocytes and neurons
SURFE2R N1 and SURFE2R 96SE poster, 64th Annual Meeting of the Biophysical Society
Poster PDF
2018 – Label-free analysis of Na+/Ca2+– exchanger (NCX) isolated from iPSC-derived cardiomyocytes
SURFE2R N1 and SURFE2R 96SE poster, Europhysiology Meeting 2018
Application Note PDF
PepT1 – “Electrophysiological recordings of PepT1 (SLC15A1) activity on Nanion’s SURFE2R”
SURFE2R N1 application note:   

The human peptide transporter PepT1 is an uptake transporter responsible for initial absorption and renal reabsorption of dietary oligopeptides. It is primarily located in the plasma membranes of enterocytes of the small intestine as well as the renal proximal tubular cells. PepT1 functions as a co-transporter, coupling the uphill peptide transport into the cells to the electrochemical proton gradient. Due to the movement of protons, PepT1 is an electrogenic transporter. PepT1 shows a very high capacity but a low affinity and substrate specificity. Its ability to transport a large range of compounds has enabled the rational design of drugs and pro-drugs (e.g. penicillins, ACE inhibitors) which have good oral bio-availability using delivery via PepT1. Designing pro-drugs with higher affinity for PepT1 is a successful strategy to increase the bio-availability of poorly absorbed drugs. Here we present electric real-time PepT1 activity measurements on the SURFE2R instruments using purified plasma membranes of CHO cells overexpressing PepT1. Peptide transport was activated on the SURFE2R N1 using a sensor with attached PepT1- containing membrane fragments which was inserted into the device. This was perfused with a buffer containing the dipeptide glycyl-glycine as the substrate. The data presented here show activation of PepT1 by glyclyglycine and inhibition by Lys[Z(No2)]-Val on the SURFE2R N1 and scale-up of the assay on the SURFE2R N96.

Poster PDF
2017 – An emerging technique for the characterization of transport proteins: SSM-based electrophysiology
SURFE2R N1 and SURFE2R SE96 poster, 19th IUPAB / 11th EBSA congress 2017
Application Note PDF
EAAT3 – “Electrophysiological recordings of EAAT3 (SLC1A1) activity on Nanion’s SURFE2R N1″
SURFE2R N1 application note:

The excitatory amino acid transporter 3 (EAAT3; also known as EAAC1) is a sodium-dependent neuronal uptake transporter encoded by the slc1a1 gene. It plays amajor role in the reuptake of glutamate from the synaptic cleft, thereby maintaining a low extra- cellular concentration of glutamate and regulating the excitatory neurotransmission. EAAT3 is also involved in the uptake of aspartate and cysteine into the cells. The transporter is highly expressed in mature neurons, where it is distributed in somata and dendrites. EAAT3 functions as a cotransporter, coupling the uphill substrate transport into the cells to the electrochemical gradients of sodium and potassium. The stoichiometry of transport is 1 glutamate with 3 Na+ and 1 H+ moving into the cell to 1 K+ moving out of the cell. Therefore EAAT3 is an electrogenic transporter, generating a net charge flow.Dysfunction of glutamate transporters leads to increased extracellular glutamate levels, thereby causing neuro- toxicity and neurodegeneration. Regulatory mechanisms facilitating EAAT3 function are, therefore, interesting as targets for the treatment of neurodegenerative diseases.Here we present EAAT3 activity measurements on the SURFE2R N1 instrument using purified plasma membrane of CHO cells expressing EAAT3.

Application Note PDF
GAT1 – “Electrophysiological recordings of hGAT1 (SLC6A1) activity on Nanion’s SURFE2R N1″
SURFE2R N1 application note:

GABA is the major inhibitory neurotransmitter in the brain and is important in controlling excitability. After release, GABA is removed from the extracellular space by GABA transporters (GATs), thus terminating inhibitory synaptic transmission. The GABA transporters belong to the family of neurotransmitter:sodium symporters referred to as the solute carrier 6 (SLC6) family in humans. GATs co-transport GABA, Na+ and Cl- with the proposed stiochiometry 1 GABA: 2 Na+: 1 Cl-, resulting in a net influx of 1 positive charge per cycle. So far, 4 GATs have been identified, GAT1, GAT2, GAT3 and BGT1.GAT1 is expressed throughout the brain in both GABAergic and non-GABAergic neurons, and is expressed in particularly high levels in the olfactory bulb, basal ganglia, cerebellum and retina. The physiological function of GAT1 is primarily to terminate synaptic transmission but also to ensure the fidelity of synaptic transmission by preventing the spread of neurotransmitter to neighbouring synapses. GABA transporters also play an important role in neurotransmitter reutilization. In certain circumstances, e.g. when the sodium gradient increases during ischemia or following seizures, GATs can act in reverse which may have a protective effect during seizures, by inhibiting electrical excitability. There is some evidence that GATs may play a role in neurodegenerative diseases such as Parkinson’s and Alzheimer’s and may provide a novel target for treating these conditions.Here we present human GAT1 activity measurements on the SURFE2R N1 instrument using purified plasma membranes from HEK cells. GABA affinity and effect of inhibitors were investigated.

Application Note PDF
CIC – “Electrophysiological recordings of the H+/ Cl- exchanger Ec-ClC on the SURFE2R N1”
SURFE2R N1 application note:   Samples kindly provided by Dr. Merritt Maduke, Stanford University, USA

ClCs are a family of chloride ion channels and transporters with important physiological roles including regulation of the membrane potential, transepithelial salt transport and ion homeostasis1,2. To date, 9 members of the ClC family have been identified in mammals1,2, the first 4 (ClC-1, ClC-2, ClC-Ka, and ClC-Kb) are located on the plasma membrane where they act as chloride ion channels whereas the remaining 5 are located in intracellular organelles (ClC-3-7) and are chloride-proton exchangers1-3. These transporters are important for endosome, lysosome and synaptic vesicle acidification1,2, and mutations in, e.g. ClC-5 underlie the rare chronic kidney disorder, Dent’s Disease, and mutations in ClC-7 underlie osteopetrosis 2,4, a rare inherited bone hardening disorder. Given their ubiquitous expression and importance in physiological processes, they are important potential drug targets.ClC from Escherichia coli (Ec-ClC or ClC-ec1) is closely ClC from Escherichia coli (Ec-ClC or ClC-ec1) is closely related to its mammalian counterparts and is a Cl-/H+ exchanger3,5. It transports 2 Cl- into the cell, coupled to the efflux of 1 H+3,5. Therefore Ec-ClC is an electrogenic transporter, generating a net charge flow. In E. coli, the Ec-ClC mediates acid resistance of enteric bacteria by promoting H+ extrusion1,5,6. Here we present Ec-ClC activity measurements on the SURFE2R N1 instrument using proteoliposomes reconstituted with purified Ec-ClC at different lipid-to-protein ratios.

Application Note PDF
CNT1 – “Electrophysiological recordings of CNT1 (SLC28A1) activity on Nanion’s SURFE2R N1″
SURFE2R N1 application note:

The concentrative nucleoside transporter 1 (CNT1) is a sodium-dependent uptake transporter encoded by the SLC28A1 gene. CNT1 functions as a co-transporter, coupling the uphill nucleoside transport into the cells to the electrochemical gradient of sodium. The stoichiometry of transport is proposed to be 1:1, but a stoichiometry of 2 Na+: 1 nucleoside has also been suggested. CNT1 is an electrogenic transporter, generating a net charge flow. It plays a major role in the uptake of pyrimidines, including uridine and cytidine, from the extracellular milieu into the cytoplasm in nucleoside salvage pathways which is the first step of nucleoside biosynthesis. The transporter is expressed in epithelial tissues including liver, kidney and small intestine where it is localized to the apical membrane. CNTs are important targets for many antiviral and anticancer agents, and CNT1 has been proposed to play a role in tumor biology via a mechanism beyond nucleoside transport. In fact, tumors expressing high levels of CNT1 can indicate a higher risk of relapse for breast cancer  patients, suggesting that nucleoside salvage may interfere with chemosensitivity. On the other hand, high expression of the CNT1 protein could promote drug- induced cytotoxicity if patients were treated with suitable hCNT substrates. In any case, hCNT1 is an important mediator in the transport of anticancer and antiviral nucleoside drugs by mechanisms that require further study.Here we present CNT1 activity measurements on the SURFE2R N1 instrument using purified plasma membrane of CHO cells expressing CNT1. 

Publication link
2022 – Structure, mechanism and lipid-mediated remodeling of the mammalian Na+/H+ exchanger NHA2
SURFE2R N1 Publication in Nature Structural & Molecular Biology (2022) Authors: Matsuoka R., Fudim R., Jung S., Zhang C., Bazzone A., Chatzikyriakidou Y., Robinson C. V., Nomura N., Iwata S., Landreh M., Orellana L., Beckstein O., Drew D.

The Na+/H+ exchanger SLC9B2, also known as NHA2, correlates with the long-sought-after Na+/Li+ exchanger linked to the pathogenesis of diabetes mellitus and essential hypertension in humans. Despite the functional importance of NHA2, structural information and the molecular basis for its ion-exchange mechanism have been lacking. Here we report the cryo-EM structures of bison NHA2 in detergent and in nanodiscs, at 3.0 and 3.5 Å resolution, respectively. The bison NHA2 structure, together with solid-state membrane-based electrophysiology, establishes the molecular basis for electroneutral ion exchange. NHA2 consists of 14 transmembrane (TM) segments, rather than the 13 TMs previously observed in mammalian Na+/H+ exchangers (NHEs) and related bacterial antiporters. The additional N-terminal helix in NHA2 forms a unique homodimer interface with a large intracellular gap between the protomers, which closes in the presence of phosphoinositol lipids. We propose that the additional N-terminal helix has evolved as a lipid-mediated remodeling switch for the regulation of NHA2 activity.

Publication link
2022 – Structures and mechanism of the plant PIN-FORMED auxin transporter
SURFE2R N1 Publication in Nature (2022) Authors: Ung K.L., Winkler M., Schulz L., Kolb M., Janacek D.P., Dedic E., Stokes D.L., Hammes U.Z., Pedersen B.P.

Auxins are hormones that have central roles and control nearly all aspects of growth and development in plants. The proteins in the PIN-FORMED (PIN) family (also known as the auxin efflux carrier family) are key participants in this process and control auxin export from the cytosol to the extracellular space. Owing to a lack of structural and biochemical data, the molecular mechanism of PIN-mediated auxin transport is not understood. Here we present biophysical analysis together with three structures of Arabidopsis thaliana PIN8: two outward-facing conformations with and without auxin, and one inward-facing conformation bound to the herbicide naphthylphthalamic acid. The structure forms a homodimer, with each monomer divided into a transport and scaffold domain with a clearly defined auxin binding site. Next to the binding site, a proline–proline crossover is a pivot point for structural changes associated with transport, which we show to be independent of proton and ion gradients and probably driven by the negative charge of the auxin. The structures and biochemical data reveal an elevator-type transport mechanism reminiscent of bile acid/sodium symporters, bicarbonate/sodium symporters and sodium/proton antiporters. Our results provide a comprehensive molecular model for auxin recognition and transport by PINs, link and expand on a well-known conceptual framework for transport, and explain a central mechanism of polar auxin transport, a core feature of plant physiology, growth and development.

Publication link
2022 – SSM-based electrophysiology, a label-free real-time method reveals sugar binding & transport events in SGLT1
SURFE2R N1 Publication in Biosensors and Bioelectronics (2022) Authors: Bazzone A., Körner A., Meincke M., Bhatt M., Srujan S., Barthmes M., Kubick S., Fertig N.

Here, we present a solid-supported membrane (SSM)-based electrophysiological approach to study sugar binding and Na+/glucose cotransport by SGLT1 in membrane vesicles. SSM-based electrophysiology delivers a cumulative real-time current readout from numerous SGLT1 proteins simultaneously using a gold-coated sensor chip.In contrast to conventional techniques, which mainly operate with voltage steps, currents are triggered by sugar or sodium addition. Sugar concentration jumps in the presence of sodium lead to transport currents between 5 and 10 nA. Remarkably, in the absence of sodium (i.e. no transport), we observed fast pre-steady-state (PSS) currents with time constants between 3 and 10 ms. These PSS currents mainly originate from sugar binding. Sodium binding does not induce PSS currents. Due to high time resolution, PSS currents were distinguished from transport and eventually correlated with conformational transitions within the sugar translocation pathway.In addition, we analyzed the impact of driving forces on transport and binding currents, showing that membrane voltage and sodium concentration gradients lead to an increased transport rate without affecting sugar binding kinetics. We also compared Na+/sugar efflux with physiologically relevant influx and found similar transport rates, but lower affinity in efflux mode.SSM-based electrophysiology is a powerful technique, which overcomes bottlenecks for transport measurements observed in other techniques such as the requirement of labels or the lack of real-time data. Rapid solution exchange enables the observation of substrate-induced electrogenic events like conformational transitions, opening novel perspectives for in-depth functional studies of SGLT1 and other transporters.

Publication link
2022 – Structure and lipid-mediated remodelling mechanism of the Na+/H+ exchanger NHA2
SURFE2R N1 Publication in Nature Structural & Molecular Biology (2022) Authors: Matsuoka R., Fudim R., Jung S., Zhang C., Bazzone A., Chatzikyriakidou Y., Nomura N., Iwata S., Orellana L., Beckstein O., Drew D.

Na+/H+ exchangers catalyse an ion-exchange activity that is carried out in most, if not all cells. SLC9B2, also known as NHA2, correlates with the long-sought after sodium/lithium (Na+/Li+) exchanger linked to the pathogenesis of diabetes mellitus and essential hypertension in humans. Despite its functional importance, structural information and the molecular basis of its ion-exchange mechanism have been lacking. Here, we report the cryo EM structures of bison NHA2 in detergent and in nanodiscs at 3.0 and 3.5 Å resolution, respectively. NHA2 shares closest structural similarity to the bacterial electrogenic Na+/H+ antiporter NapA, rather than other mammalian SLC9A members. Nevertheless, SSM-based electrophysiology results with NHA2 show the catalysis of electroneutral rather than electrogenic ion exchange, and the ion-binding site is quite distinctive, with a tryptophan-arginine- glutamate triad separated from the well-established ion-binding aspartates. These triad residues fine-tune ion binding specificity, as demonstrated by a salt-bridge swap mutant that converts NHA2 into a Li+-specific transporter. Strikingly, an additional N-terminal helix in NHA2 establishes a unique homodimer with a large ∼ 25 Å intracellular gap between protomers. In the presence of phosphatidylinositol lipids, the N-terminal helix rearranges and closes this gap. We confirm that dimerization of NHA2 is required for activity in vivo, and propose that the N- terminal helix has evolved as a lipid-mediated remodelling switch for regulation of transport activity.

Publication link
2022 – Mechanistic basis of choline import involved in teichoic acids and lipopolysaccharide modification
SURFE2R N1 Publication in Science Advances (2022) Authors: Bärland N., Rueff A.S., Cebrero G., Hutter C.A.J., Seeger M.A., Veening J.W., Perez C.

Phosphocholine molecules decorating bacterial cell wall teichoic acids and outer-membrane lipopolysaccharide have fundamental roles in adhesion to host cells, immune evasion, and persistence. Bacteria carrying the operon that performs phosphocholine decoration synthesize phosphocholine after uptake of the choline precursor by LicB, a conserved transporter among divergent species. Streptococcus pneumoniae is a prominent pathogen where phosphocholine decoration plays a fundamental role in virulence. Here, we present cryo–electron microscopy and crystal structures of S. pneumoniae LicB, revealing distinct conformational states and describing architectural and mechanistic elements essential to choline import. Together with in vitro and in vivo functional characterization, we found that LicB displays proton-coupled import activity and promiscuous selectivity involved in adaptation to choline deprivation conditions, and describe LicB inhibition by synthetic nanobodies (sybodies). Our results provide previously unknown insights into the molecular mechanism of a key transporter involved in bacterial pathogenesis and establish a basis for inhibition of the phosphocholine modification pathway across bacterial phyla.

Publication link
2022 – Mechanistic basis of choline import involved in teichoic acids and lipopolysaccharide modification
SURFE2R N1 Publication in Sci Adv. (2022) Authors: Bärland N., Rueff A-S., Cebrero G., Hutter C. A. J., Seeger M. A., Veening J-W., Perez C.

Phosphocholine molecules decorating bacterial cell wall teichoic acids and outer-membrane lipopolysaccharide have significant roles in adhesion to host cells, immune evasion, and persistence. Bacteria carrying the operon that performs phosphocholine decoration, synthesize phosphocholine after uptake of the choline precursor by LicB, a conserved transporter among divergent species. Streptococcus pneumoniae is a prominent pathogen where phosphocholine decoration plays a fundamental role in virulence. Here we present cryo-electron microscopy and crystal structures of S. pneumoniae LicB, revealing distinct conformational states and describing architectural and mechanistic elements essential to choline import. Together with in vitro and in vivo functional characterization, we found that LicB displays proton-coupled import activity and promiscuous selectivity involved in adaptation to choline deprivation conditions, and describe LicB inhibition by synthetic nanobodies (sybodies) and hemicholinium-3. Our results provide novel insights into the molecular mechanism of a key transporter involved in bacterial pathogenesis and establish a basis for inhibition of the phosphocholine modification pathway across bacterial phyla.

Publication link
2022 – Electrogenic reaction step and phospholipid translocation pathway of the mammalian P4-ATPase ATP8A2
SURFE2R N1 Publication in FEBS Letters (2022) Authors: Tadini-Buoninsegni F., Mikkelsen S.A., Mogensen L.S., Holm R., Molday R.S., Andersen J.P.

ATP8A2 is a mammalian P4-ATPase (flippase) that translocates the negatively charged lipid substrate phosphatidylserine from the exoplasmic leaflet to the cytoplasmic leaflet of cellular membranes. Using an electrophysiological method based on solid supported membranes, we investigated the electrogenicity of specific reaction steps of ATP8A2 and explored a potential phospholipid translocation pathway involving residues with positively charged side chains. Changes to the current signals caused by mutations show that the main electrogenic event occurs in connection with release of the bound phosphatidylserine to the cytoplasmic leaflet and support the hypothesis that the phospholipid interacts with specific lysine and arginine residues near the cytoplasmic border of the lipid bilayer during the translocation and/or reorientation required for insertion into the cytoplasmic leaflet.

Publication link
2022 – Expression, purification and characterization of human proton-coupled oligopeptide transporter 1 hPEPT1
SURFE2R N1 Publication in Protein Expression and Purification (2022) Authors: Rafiq M., Ernst H.A., Aduri N.G., Prabhala B.K., Tufail S., Rahman M., Bloch M.B., Mirza N., Taylor N., Boesen T., Gajhede M., Mirzaa O.

The human peptide transporter hPEPT1 (SLC15A1) is responsible for uptake of dietary di- and tripeptides and a number of drugs from the small intestine by utilizing the proton electrochemical gradient, and hence an important target for peptide-like drug design and drug delivery. hPEPT1 belongs to the ubiquitous major facilitator superfamily that all contain a 12TM core structure, with global conformational changes occurring during the transport cycle. Several bacterial homologues of these transporters have been characterized, providing valuable insight into the transport mechanism of this family. Here we report the overexpression and purification of recombinant hPEPT1 in a detergent-solubilized state. Thermostability profiling of hPEPT1 at different pH values revealed that hPEPT1 is more stable at pH 6 as compared to pH 7 and 8. Micro-scale thermophoresis (MST) confirmed that the purified hPEPT1 was able to bind di- and tripeptides respectively. To assess the in-solution oligomeric state of hPEPT1, negative stain electron microscopy was performed, demonstrating a predominantly monomeric state.

Publication link
2022 – Coexistence of Ammonium Transporter and Channel Mechanisms in Amt-Mep-Rh Twin-His Variants Impairs the Filamentation Signaling Capacity of Fungal Mep2 Transceptors
SURFE2R N1 Publication in mBio (2022) Authors: Williamson G., Brito A. S., Bizior A., Tamburrino G., Mirandela G. D., Harris T., Hoskisson P. A., Zachariae U., Marini A. M., Boeckstaens M., Javelle A.

Ammonium translocation through biological membranes, by the ubiquitous Amt-Mep-Rh family of transporters, plays a key role in all domains of life. Two highly conserved histidine residues protrude into the lumen of the pore of these transporters, forming the family’s characteristic Twin-His motif. It has been hypothesized that the motif is essential to confer the selectivity of the transport mechanism. Here, using a combination of in vitro electrophysiology on Escherichia coli AmtB, in silico molecular dynamics simulations, and in vivo yeast functional complementation assays, we demonstrate that variations in the Twin-His motif trigger a mechanistic switch between a specific transporter, depending on ammonium deprotonation, to an unspecific ion channel activity. We therefore propose that there is no selective filter that governs specificity in Amt-Mep-Rh transporters, but the inherent mechanism of translocation, dependent on the fragmentation of the substrate, ensures the high specificity of the translocation. We show that coexistence of both mechanisms in single Twin-His variants of yeast Mep2 transceptors disrupts the signaling function and so impairs fungal filamentation. These data support a signaling process driven by the transport mechanism of the fungal Mep2 transceptors.

Publication link
2022 – Crystal structures of bacterial Small Multidrug Resistance transporter EmrE in complex with structurally diverse substrates
SURFE2R N1 Publication in BioRxiv (2022) Authors: Kermani A.A., Burata O.E., Koff B.B., Koide A., Koide S., Stockbridge R.B.

Proteins from the bacterial small multidrug resistance (SMR) family are proton-coupled exporters of diverse antiseptics and antimicrobials, including polyaromatic cations and quaternary ammonium compounds. The transport mechanism of the Escherichia coli transporter, EmrE, has been studied extensively, but a lack of high-resolution structural information has impeded a structural description of its molecular mechanism. Here we apply a novel approach, multipurpose crystallization chaperones, to solve several structures of EmrE, including a 2.9 Å structure at low pH without substrate. We report five additional structures in complex with structurally diverse transported substrates, including quaternary phosphonium, quaternary ammonium, and planar polyaromatic compounds. These structures show that binding site tryptophan and glutamate residues adopt different rotamers to conform to disparate structures without requiring major rearrangements of the backbone structure. Structural and functional comparison to Gdx-Clo, an SMR protein that transports a much narrower spectrum of substrates, suggests that in EmrE, a relatively sparse hydrogen bond network among binding site residues permits increased sidechain flexibility.

Publication link
2021 – Structure and dynamics of the drug-bound bacterial transporter EmrE in lipid bilayers
SURFE2R N1 Publication in Nature Communications (2021) Authors: Shcherbakov A.A., Hisao G., Mandala V.S., Thomas N.E., Soltani M., Salter E.A., Davis Jr J.H., Henzler-Wildman K.A., Hong M.

The dimeric transporter, EmrE, effluxes polyaromatic cationic drugs in a proton-coupled manner to confer multidrug resistance in bacteria. Although the protein is known to adopt an antiparallel asymmetric topology, its high-resolution drug-bound structure is so far unknown, limiting our understanding of the molecular basis of promiscuous transport. Here we report an experimental structure of drug-bound EmrE in phospholipid bilayers, determined using 19F and 1H solid-state NMR and a fluorinated substrate, tetra(4-fluorophenyl) phosphonium (F4-TPP+). The drug-binding site, constrained by 214 protein-substrate distances, is dominated by aromatic residues such as W63 and Y60, but is sufficiently spacious for the tetrahedral drug to reorient at physiological temperature. F4-TPP+ lies closer to the proton-binding residue E14 in subunit A than in subunit B, explaining the asymmetric protonation of the protein. The structure gives insight into the molecular mechanism of multidrug recognition by EmrE and establishes the basis for future design of substrate inhibitors to combat antibiotic resistance.

Publication link
2021 – The CopA2-Type P1B-Type ATPase CcoI Serves as Central Hub for cbb3-Type Cytochrome Oxidase Biogenesis
SURFE2R N1 Publication in Frontiers in Microbiology (2021) Authors: Andrei A., Di Renzo M.A., Öztürk Y., Meisner A., Daum N., Frank F., Rauch J., Daldal F., Andrade S.L.A., and Koch H-G.

Copper (Cu)-transporting P1B-type ATPases are ubiquitous metal transporters and crucial for maintaining Cu homeostasis in all domains of life. In bacteria, the P1B-type ATPase CopA is required for Cu-detoxification and exports excess Cu(I) in an ATP-dependent reaction from the cytosol into the periplasm. CopA is a member of the CopA1-type ATPase family and has been biochemically and structurally characterized in detail. In contrast, less is known about members of the CopA2-type ATPase family, which are predicted to transport Cu(I) into the periplasm for cuproprotein maturation. One example is CcoI, which is required for the maturation of cbb3-type cytochrome oxidase (cbb3-Cox) in different species. Here, we reconstituted purified CcoI of Rhodobacter capsulatus into liposomes and determined Cu transport using solid-supported membrane electrophysiology. The data demonstrate ATP-dependent Cu(I) translocation by CcoI, while no transport is observed in the presence of a non-hydrolysable ATP analog. CcoI contains two cytosolically exposed N-terminal metal binding sites (N-MBSs), which are both important, but not essential for Cu delivery to cbb3-Cox. CcoI and cbb3-Cox activity assays in the presence of different Cu concentrations suggest that the glutaredoxin-like N-MBS1 is primarily involved in regulating the ATPase activity of CcoI, while the CopZ-like N-MBS2 is involved in Cu(I) acquisition. The interaction of CcoI with periplasmic Cu chaperones was analyzed by genetically fusing CcoI to the chaperone SenC. The CcoI-SenC fusion protein was fully functional in vivo and sufficient to provide Cu for cbb3-Cox maturation. In summary, our data demonstrate that CcoI provides the link between the cytosolic and periplasmic Cu chaperone networks during cbb3-Cox assembly.

Publication link
2021 – Stimulation of Ca2+‐ATPase Transport Activity by a Small‐Molecule Drug
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in ChemMedChem(2021) Authors: Sordi G., Goti A., Young H.S., Palchetti I., Tadini‐Buoninsegni F.

The sarco(endo)plasmic reticulum Ca2+−ATPase (SERCA) hydrolyzes ATP to transport Ca2+ from the cytoplasm to the sarcoplasmic reticulum (SR) lumen, thereby inducing muscle relaxation. Dysfunctional SERCA has been related to various diseases. The identification of small‐molecule drugs that can activate SERCA may offer a therapeutic approach to treat pathologies connected with SERCA malfunction. Herein, we propose a method to study the mechanism of interaction between SERCA and novel SERCA activators, i. e. CDN1163, using a solid supported membrane (SSM) biosensing approach. Native SR vesicles or reconstituted proteoliposomes containing SERCA were adsorbed on the SSM and activated by ATP concentration jumps. We observed that CDN1163 reversibly interacts with SERCA and enhances ATP‐dependent Ca2+ translocation. The concentration dependence of the CDN1163 effect provided an EC50=6.0±0.3 μM. CDN1163 was shown to act directly on SERCA and to exert its stimulatory effect under physiological Ca2+ concentrations. These results suggest that CDN1163 interaction with SERCA can promote a protein conformational state that favors Ca2+ release into the SR lumen.

Publication link
2021 – Structural basis for Potassium transport by KdpFABC
SURFE2R N1 Publication in PNAS (2021) Authors: Sweet M.E., Larsen C., Zhang X., Schlame M., Pedersen B.P., Stokes D.L.

KdpFABC is an oligomeric K+ transport complex in prokaryotes that maintains ionic homeostasis under stress conditions. The complex comprises a channel-like subunit (KdpA) from the Superfamily of K+ Transporters and a pump-like subunit (KdpB) from the superfamily of P-type ATPases. Recent structural work has defined the architecture and generated contradictory hypotheses for the transport mechanism. Here, we use substrate analogs to stabilize four key intermediates in the reaction cycle and determine the corresponding structures by cryo-EM. We find that KdpB undergoes conformational changes consistent with other representatives from the P-type superfamily, whereas KdpA, KdpC and KdpF remain static. We observe a series of spherical densities that we assign as K+ or water and which define a pathway for K+ transport. This pathway runs through an intramembrane tunnel in KdpA and delivers ions to sites in the membrane domain of KdpB. Our structures suggest a mechanism where ATP hydrolysis is coupled to K+ transfer between alternative sites in the membrane domain of KdpB, ultimately reaching a low-affinity site where a water-filled pathway allows release of K+ to the cytoplasm.

Publication link
2021 – Functional Characterization of SLC Transporters Using Solid Supported Membranes
SURFE2R N1 Chapter in Biophysics of Membrane Proteins (2021) Authors: Bazzone A., Barthmes M.

Here, we present a protocol for the functional characterization of the H+-coupled human peptide transporter PepT1 and sufficient notes to transfer the protocol to the Na+-coupled sugar transporter SGLT1, the organic cation transporter OCT2, the Na+/Ca2+ exchanger NCX, and the neuronal glutamate transporter EAAT3.The assay was developed for the commercially available SURFE2R N1 instrument (Nanion Technologies GmbH) which applies solid supported membrane (SSM)-based electrophysiology. This technique is widely used for the functional characterization of membrane transporters with more than 100 different transporters characterized so far. The technique is cost-effective, easy to use, and capable of high-throughput measurements.SSM-based electrophysiology utilizes SSM-coated gold sensors to physically adsorb membrane vesicles containing the protein of interest. A fast solution exchange provides the substrate and activates transport. For the measurement of PepT1 activity, we applied a peptide concentration jump to activate H+/peptide symport. Proton influx charges the sensor. A capacitive current is measured reflecting the transport activity of PepT1. Multiple measurements on the same sensor allow for comparison of transport activity under different conditions. Here, we determine EC50 for PepT1-mediated glycylglycine transport and perform an inhibition experiment using the specific peptide inhibitor Lys[Z(NO2)]-Val.

Publication link
2021 – Selection of Transporter-Targeted Inhibitory Nanobodies by Solid-Supported-Membrane (SSM)-Based Electrophysiology
SURFE2R N1 Publication in Journal of Visualized Experiments (styled JoVE) (2021) Authors: Bärland N., Perez C.

Single domain antibodies (nanobodies) have been extensively used in mechanistic and structural studies of proteins and they pose an enormous potential as tools for developing clinical therapies, many of which depend on the inhibition of membrane proteins such as transporters. However, most of the methods used to determine the inhibition of transport activity are difficult to perform in high-throughput routines and depend on labeled substrates availability thereby complicating the screening of large nanobody libraries. Solid-supported membrane (SSM) electrophysiology is a high-throughput method, used for characterizing electrogenic transporters and measuring their transport kinetics and inhibition. Here we show the implementation of SSM-based electrophysiology to select inhibitory and non-inhibitory nanobodies targeting an electrogenic secondary transporter and to calculate nanobodies inhibitory constants. This technique may be especially useful for selecting inhibitory nanobodies targeting transporters for which labeled substrates are not available.

Publication link
2021 – Cryo-EM structures of excitatory amino acid transporter 3 visualize coupled substrate, sodium, and proton binding and transport
SURFE2R N1 Publication in Science Advances (2021) Authors: Qiu B., Matthies D., Fortea E., Yu Z., Boudker O.

Human excitatory amino acid transporter 3 (hEAAT3) mediates glutamate uptake in neurons, intestine, and kidney. Here, we report cryo-EM structures of hEAAT3 in several functional states where the transporter is empty, bound to coupled sodium ions only, or fully loaded with three sodium ions, a proton, and the substrate aspartate. The structures suggest that hEAAT3 operates by an elevator mechanism involving three functionally independent subunits. When the substrate-binding site is near the cytoplasm, it has a remarkably low affinity for the substrate, perhaps facilitating its release and allowing the rapid transport turnover. The mechanism of the coupled uptake of the sodium ions and the substrate is conserved across evolutionarily distant families and is augmented by coupling to protons in EAATs. The structures further suggest a mechanism by which a conserved glutamate residue mediates proton symport.

Publication link
2021 – Engineering and functional characterization of a proton-driven β-lactam antibiotic translocation module for bionanotechnological applications
SURFE2R N1 Publication in Nature Scientific Reports (2021) Authors: Stauffer M., Ucurum Z., Harder D., Fotiadis D.

Novel approaches in synthetic biology focus on the bottom-up modular assembly of natural, modified natural or artificial components into molecular systems with functionalities not found in nature. A possible application for such techniques is the bioremediation of natural water sources contaminated with small organic molecules (e.g., drugs and pesticides). A simple molecular system to actively accumulate and degrade pollutants could be a bionanoreactor composed of a liposome or polymersome scaffold combined with energizing- (e.g., light-driven proton pump), transporting- (e.g., proton-driven transporter) and degrading modules (e.g., enzyme). This work focuses on the engineering of a transport module specific for β-lactam antibiotics. We previously solved the crystal structure of a bacterial peptide transporter, which allowed us to improve the affinity for certain β-lactam antibiotics using structure-based mutagenesis combined with a bacterial uptake assay. We were able to identify specific mutations, which enhanced the affinity of the transporter for antibiotics containing certain structural features. Screening of potential compounds allowed for the identification of a β-lactam antibiotic ligand with relatively high affinity. Transport of antibiotics was evaluated using a solid-supported membrane electrophysiology assay. In summary, we have engineered a proton-driven β-lactam antibiotic translocation module, contributing to the growing toolset for bionanotechnological applications.

Publication link
2021 – A mechanistic switch from selective transporter to an ion channel impairs the filamentation signalling capability of ammonium transceptors in yeast
SURFE2R N1 pre-print Publication in bioRxiv (2021) Authors: Williamson G., Brito A. S., Bizior A., Tamburrino G., Mirandela G. D., Harris T., Hoskisson P. A., Zachariae U., Marini A. M., Boeckstaens M., Javelle A.

Ammonium translocation through biological membranes by the ubiquitous Amt-Mep-Rh family of transporters plays a key role in all domains of life. Two highly conserved histidine residues protrude into the lumen of these transporters, forming the family’s characteristic Twin-His motif. It has been hypothesized that the motif is essential to confer the selectivity of the transport mechanism. Here, using a combination of in vitro electrophysiology, in vivo yeast functional complementation and in silico molecular dynamics simulations, we demonstrate that variations in the Twin-His motif trigger a mechanistic switch between a specific transporter, depending on ammonium deprotonation, to an unspecific ion channel activity. We therefore propose that there is no selective filter that governs the specificity in Amt-Mep transporters but the inherent mechanism of translocation, dependent on the fragmentation of the substrate, ensures the high specificity of the translocation. We further show that both mechanisms coexist in fungal Mep2 Twin-His variants, disrupting the transceptor function and so inhibiting the filamentation process. These data strongly support a transport mechanism-mediated signalling process in the long-standing debate on the sensory function of Mep2-like transporters.

Publication link
2021 – A Solid Supported Membrane Electrophysiology Assay for Efficient Characterization of Ion-Coupled Transport
SURFE2R N1 Publication in Journal of Biological Chemistry (2021) Authors: Thomas N.E., Feng W., Henzler-Wildman K.A.

Transport stoichiometry determination can provide great insight into the mechanism and function of ion-coupled transporters. Traditional reversal potential assays are a reliable, general method for determining the transport stoichiometry of ion-coupled transporters, but the time and material costs of this technique hinder investigations of transporter behavior under multiple experimental conditions. Solid supported membrane electrophysiology (SSME) allows multiple recordings of liposomal or membrane samples adsorbed onto a sensor, and is sensitive enough to detect transport currents from moderate-flux transporters that are inaccessible to traditional electrophysiology techniques. Here, we use SSME to develop a new method for measuring transport stoichiometry with greatly improved throughput. Using this technique, we were able to verify the recent report of a fixed 2:1 stoichiometry for the proton:guanidinium antiporter Gdx, reproduce the 1H+:2Cl- antiport stoichiometry of CLC-ec1, and confirm loose proton:nitrate coupling for CLC-ec1. Furthermore, we were able to demonstrate quantitative exchange of internal contents of liposomes adsorbed onto SSME sensors to allow multiple experimental conditions to be tested on a single sample. Our SSME method provides a fast, easy, general method for measuring transport stoichiometry, which will facilitate future mechanistic and functional studies of ion-coupled transporters.

Publication link
2020 – The structural basis of promiscuity in small multidrug resistance transporters
SURFE2R N1 Publication in Nature Communications (2020) Authors: Kermani A.A., Macdonald C.B., Burata O.E., Koff B.B., Koide A., Denbaum E., Koide S., Stockbridge R.B.

By providing broad resistance to environmental biocides, transporters from the small multidrug resistance (SMR) family drive the spread of multidrug resistance cassettes among bacterial populations. A fundamental understanding of substrate selectivity by SMR transporters is needed to identify the types of selective pressures that contribute to this process. Using solid-supported membrane electrophysiology, we find that promiscuous transport of hydrophobic substituted cations is a general feature of SMR transporters. To understand the molecular basis for promiscuity, we solved X-ray crystal structures of a SMR transporter Gdx-Clo in complex with substrates to a maximum resolution of 2.3 Å. These structures confirm the family’s extremely rare dual topology architecture and reveal a cleft between two helices that provides accommodation in the membrane for the hydrophobic substituents of transported drug-like cations.

Publication link
2020 – Unlocking the Reversal Potential of Solid Supported Membrane Electrophysiology to Determine Transport Stoichiometry
SURFE2R N1 pre-Publication in bioRxiv (2020) Authors: Henzler-Wildman K.A., Thomas N.E.

Transport stoichiometry provides insight into the mechanism and function of ion-coupled transporters, but measuring transport stoichiometry is time-consuming and technically difficult. With the increasing evidence that many ion-coupled transporters employ multiple transport stoichiometries under different conditions, improved methods to determine transport stoichiometry are required to accurately characterize transporter activity. Reversal potential was previously shown to be a reliable, general method for determining the transport stoichiometry of ion-coupled transporters (Fitzgerald & Mindell, 2017). Here, we develop a new technique for measuring transport stoichiometry with greatly improved throughput using solid supported membrane electrophysiology (SSME). Using this technique, we are able to verify the recent report of a fixed 2:1 stoichiometry for the proton:guanidinium antiporter Gdx. Our SSME method requires only small amounts of transporter and provides a fast, easy, general method for measuring transport stoichiometry, which will facilitate future mechanistic and functional studies of ion-coupled transporters.

Publication link
2020 – Electrophysiology Measurements of Metal Transport by MntH2 from Enterococcus faecalis
SURFE2R N1 Publication in Membranes (2020) Authors: Gantner M., Laftsoglou T., Rong H., Postis V.L.G., Jueken L.J.C.

Transition metals are essential trace elements and their high-affinity uptake is required for many organisms. Metal transporters are often characterised using metal-sensitive fluorescent dyes, limiting the metals and experimental conditions that can be studied. Here, we have tested whether metal transport by Enterococcus faecalis MntH2 can be measured with an electrophysiology method that is based on the solid-supported membrane technology. E. faecalis MntH2 belongs to the Natural Resistance-Associated Macrophage Protein (Nramp) family of proton-coupled transporters, which transport divalent transition metals and do not transport the earth metals. Electrophysiology confirms transport of Mn(II), Co(II), Zn(II) and Cd(II) by MntH2. However, no uptake responses for Cu(II), Fe(II) and Ni(II) were observed, while the presence of these metals abolishes the uptake signals for Mn(II). Fluorescence assays confirm that Ni(II) is transported. The data are discussed with respect to properties and structures of Nramp-type family members and the ability of electrophysiology to measure charge transport and not directly substrate transport.

Publication link
2020 – Energy Coupling in Cation-Pumping Pyrophosphatase – Back to Mitchell
SURFE2R N1 Publication in Frontiers in Plant Science (2020) Authors: Baykov A.A.

Those of a certain age may remember (and their younger colleagues can read) accounts of the vivid debate in the 1970s surrounding the coupling mechanism involved in oxidative and photo phosphorylation. By that time, Mitchell's chemiosmotic hypothesis had already gained credence, and the debated issue was how a transmembrane H+ potential difference drives ATP synthesis by Ftype ATP synthases. The major mechanisms that were considered assumed that the membrane (Fo) and peripheral (F1) parts were functionally connected in different ways. Peter Mitchell proposed a “direct coupling” mechanism in which protons are translocated through Fo into the catalytic site of F1, where they participate directly in ADP phosphorylation and form water as the second product (Mitchell, 1974). Paul Boyer, the proponent of the main competing mechanism, advocated an “indirect coupling” mechanism (successively termed “alternating site”, “binding change”, or “rotational”) that implied that protons transfer their energy to the catalytic site indirectly, via distant conformational strain (Boyer, 1997). The debate was resolved in favor of Boyer's mechanism when it became clear that the alternative mechanism is inconsistent with H+ /ATP stoichiometry and, finally, when the three-dimensional structure of the F-ATPase was determined (Abrahams et al., 1994).

Publication link
2019 – Phosphatidylserine flipping by the P4-ATPase ATP8A2 is electrogenic
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in PNAS (2019) Authors: Tadini-Buoninsegni F., Mikkelsen S.A., Mogensen L.S., Molday R.S., and Andersen J.P.

Significance: Phospholipid flippases constitute the largest subfamily of P-type ATPases and have in eukaryotic organisms evolved as a central transport system for selective translocation of phospholipids across biological membranes to generate membrane lipid asymmetry, a property essential for numerous cellular processes. The importance of flippases is highlighted by severe neurological disorders and liver diseases caused by flippase dysfunction in humans. The electrogenicity of phospholipid transport by flippases has not previously been explored. We demonstrated that phosphatidylserine translocation by the flippase ATP8A2 generates electrical current, resulting from specific steps in the flippase reaction cycle moving the charged lipid head group between the membrane bilayer leaflets, and that no charged substrate is being countertransported. These findings unravel key features of phospholipid flippases. Abstract: Phospholipid flippases (P4-ATPases) utilize ATP to translocate specific phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of biological membranes, thus generating and maintaining transmembrane lipid asymmetry essential for a variety of cellular processes. P4-ATPases belong to the P-type ATPase protein family, which also encompasses the ion transporting P2-ATPases: Ca2+-ATPase, Na+/K+-ATPase, and H+,K+-ATPase. In comparison with the P2-ATPases, understanding of P4-ATPases is still very limited. The electrogenicity of P4-ATPases has not been explored, and it is not known whether lipid transfer between membrane bilayer leaflets can lead to displacement of charge across the membrane. A related question is whether P4-ATPases countertransport ions or other substrates in the opposite direction, similar to the P2-ATPases. Using an electrophysiological method based on solid supported membranes, we observed the generation of a transient electrical current by the mammalian P4-ATPase ATP8A2 in the presence of ATP and the negatively charged lipid substrate phosphatidylserine, whereas only a diminutive current was generated with the lipid substrate phosphatidylethanolamine, which carries no or little charge under the conditions of the measurement. The current transient seen with phosphatidylserine was abolished by the mutation E198Q, which blocks dephosphorylation. Likewise, mutation I364M, which causes the neurological disorder cerebellar ataxia, mental retardation, and disequilibrium (CAMRQ) syndrome, strongly interfered with the electrogenic lipid translocation. It is concluded that the electrogenicity is associated with a step in the ATPase reaction cycle directly involved in translocation of the lipid. These measurements also showed that no charged substrate is being countertransported, thereby distinguishing the P4-ATPase from P2-ATPases.

Publication link
2020 – A two-lane mechanism for selective biological ammonium transport
SURFE2R N1 Publication in eLife (2020) Authors: Williamson G., Tamburrino G., Bizior A., Boeckstaens M., Mirandela G.D., Bage M.G., Pisliakov A., Ives C.M., Terras E., Hoskisson P.A., Marini A.M., Zachariae U., Javelle A.

The transport of charged molecules across biological membranes faces the dual problem of accommodating charges in a highly hydrophobic environment while maintaining selective substrate translocation. This has been the subject of a particular controversy for the exchange of ammonium across cellular membranes, an essential process in all domains of life. Ammonium transport is mediated by the ubiquitous Amt/Mep/Rh transporters that includes the human Rhesus factors. Here, using a combination of electrophysiology, yeast functional complementation and extended molecular dynamics simulations, we reveal a unique two-lane pathway for electrogenic NH4+ transport in two archetypal members of the family, the transporters AmtB from Escherichia coli and Rh50 from Nitrosomonas europaea. The pathway underpins a mechanism by which charged H+ and neutral NH3 are carried separately across the membrane after NH4+ deprotonation. This mechanism defines a new principle of achieving transport selectivity against competing ions in a biological transport process.

Publication link
2018 – The lipid environment determines the activity of the E. coli ammonium transporter, AmtB
SURFE2R N1 Publication in Faseb J (2018) Authors: Mirandela G.D., Tamburrino G., Hoskisson P.A., Zachariae U., Javelle A.

The movement of ammonium across biological membranes is a fundamental process in all living organisms and is mediated by the ubiquitous Amt/Mep/Rh family of transporters. Recent structural analysis and coupled mass spectrometry studies have shown that the Escherichia coli ammonium transporter, AmtB, specifically binds phosphatidylglycerol (PG). Upon PG binding, several residues of AmtB undergo a small conformational change, which stabilizes the protein against unfolding. However, no studies have so far been conducted to explore if PG binding to AmtB has functional consequences. Here, we used an in vitro experimental assay with purified components together with molecular dynamics simulations to characterise the relation between PG binding and AmtB activity. Firstly, our results indicate that the function of Amt in archaebacteria and eubacteria may differ. Secondly, we show that PG is an essential cofactor for AmtB activity and that in the absence of PG AmtB cannot complete the full translocation cycle. Furthermore, our simulations reveal previously undiscovered PG binding sites on the intracellular side of the lipid bilayer between the AmtB subunits. The possible molecular mechanisms explaining the functional role of PG are discussed.

Publication link
2019 – Mutation of two key aspartate residues alters stoichiometry of the NhaB Na+/H+ exchanger from Klebsiella pneumoniae
SURFE2R N1 Publication in Nature Scientific Reports (2019) Authors: Patiño-Ruiz M., Fendler K., & Călinescu O.

Bacterial NhaB Na+/H+ exchangers belonging to the Ion Transporter superfamily are poorly characterized in contrast to Na+/H+ exchangers of the Cation Proton Antiporter superfamily which have NhaA from Escherichia coli as a prominent member. For a more detailed understanding of the intricacies of the exchanger’s transport mechanism, mutational studies are essential. Therefore, we mutated two protonatable residues present in the putative transmembrane region of NhaB from Klebsiella pneumoniae (KpNhaB), which could serve as substrate binding sites, Asp146 and Asp404, to either glutamate or alanine and analyzed transport function and stability of the mutants using electrophysiological and fluorimetric techniques. While mutation of either Asp residue to Glu only had slight to moderate effects on the transport activity of the exchanger, the mutations D404A and D146A, in particular, had more profound effects on the transport function. Furthermore, a double mutant, D146A/D404A, exhibited a remarkable behavior at alkaline pH, where recorded electrical currents changed polarity, showing steady-state transport with a stoichiometry of H+:Na+  1, as opposed to the H+:Na+ > 1 stoichiometry of the WT. Thus, we showed that Asp146 and Asp404 are part of the substrate binding site(s) of KpNhaB and engineered a Na+/H+ exchanger with a variable stoichiometry.

Publication link
2017 – Insights into the mechanism of membrane pyrophosphatases by combining experiment and computer simulation
SURFE2R N1 Publication in Structural Dynamics (2017) Authors: Shah N.R., Wilkinson C., Harborne S.P.D., Turku A., Li K.-M., Sun Y.-J., Harris S., Goldman A.

Membrane-integral pyrophosphatases (mPPases) couple the hydrolysis of pyrophosphate (PPi) to the pumping of Na+, H+, or both these ions across a membrane. Recently solved structures of the Na+-pumping Thermotoga maritima mPPase (TmPPase) and H+-pumping Vigna radiata mPPase revealed the basis of ion selectivity between these enzymes and provided evidence for the mechanisms of substrate hydrolysis and ion-pumping. Our atomistic molecular dynamics (MD) simulations of TmPPase demonstrate that loop 5–6 is mobile in the absence of the substrate or substrate-analogue bound to the active site, explaining the lack of electron density for this loop in resting state structures. Furthermore, creating an apo model of TmPPase by removing ligands from the TmPPase:IDP:Na structure in MD simulations resulted in increased dynamics in loop 5–6, which results in this loop moving to uncover the active site, suggesting that interactions between loop 5–6 and the imidodiphosphate and its associated Mg2+ are important for holding a loop-closed conformation. We also provide further evidence for the transport-before-hydrolysis mechanism by showing that the non-hydrolyzable substrate analogue, methylene diphosphonate, induces low levels of proton pumping by VrPPase.

Publication link
2017 – Mechanisms of charge transfer in human copper ATPases ATP7A and ATP7B
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in IUBMB Life (2017) Authors: Tadini-Buoninsegni F., Smeazzetto S.

ATP7A and ATP7B are Cu+ -transporting ATPases of subclass IB and play a fundamental role in intracellular copper homeostasis. ATP7A/B transfer Cu+ ions across the membrane from delivery to acceptor proteins without establishing a free Cu+ gradient. Transfer of copper across the membrane is coupled to ATP hydrolysis. Current measurements on solid supported membranes (SSM) were performed to investigate the mechanism of copper-related charge transfer across ATP7A and ATP7B. SSM measurements demonstrated that electrogenic copper displacement occurs within ATP7A/B following addition of ATP and formation of the phosphorylated intermediate. Comparison of the time constants for cation displacement in ATP7A/B and sarcoplasmic reticulum Ca2+ -ATPase is consistent with the slower phosphoenzyme formation in copper ATPases. Moreover, ATP-dependent copper transfer in ATP7A/B is not affected by varying the pH, suggesting that net proton counter-transport may not occur in copper ATPases. Platinum anticancer drugs activate ATP7A/B and are subjected to ATP-dependent vectorial displacement with a mechanism analogous to that of copper.

Publication link
2017 – Discovery of Compounds that Positively Modulate the High Affinity Choline Transporter
SURFE2R N1 Publication in Frontiers in Molecular Neuroscience (2017) Authors: Choudhary P., Armstrong E.J., Jorgensen C.C., Piotrowski M., Barthmes M., Torella R., Johnston S.E., Maruyama Y., Janiszewski J.S., Storer R.I., Skerratt S.E., Benn C.L.

Cholinergic hypofunction is associated with decreased attention and cognitive deficits in the central nervous system in addition to compromised motor function. Consequently, stimulation of cholinergic neurotransmission is a rational therapeutic approach for the potential treatment of a variety of neurological conditions. High affinity choline uptake (HACU) into acetylcholine (ACh)-synthesizing neurons is critically mediated by the sodium- and pH-dependent high-affinity choline transporter (CHT, encoded by the SLC5A7 gene). This transporter is comparatively well-characterized but otherwise unexplored as a potential drug target. We therefore sought to identify small molecules that would enable testing of the hypothesis that positive modulation of CHT mediated transport would enhance activity-dependent cholinergic signaling. We utilized existing and novel screening techniques for their ability to reveal both positive and negative modulation of CHT using literature tools. A screening campaign was initiated with a bespoke compound library comprising both the Pfizer Chemogenomic Library (CGL) of 2,753 molecules designed specifically to help enable the elucidation of new mechanisms in phenotypic screens and 887 compounds from a virtual screening campaign to select molecules with field-based similarities to reported negative and positive allosteric modulators. We identified a number of previously unknown active and structurally distinct molecules that could be used as tools to further explore CHT biology or as a starting point for further medicinal chemistry.

Publication link
2017 – Effect of cisplatin on the transport activity of PII-type ATPases
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in Metallomics (2017) Authors: Tadini-Buoninsegni F., Sordi G, Smeazzetto S, Natile G, Arnesano F.

Cisplatin (cis-diamminedichlorido-Pt(ii)) is extensively used as a chemotherapeutic agent against various types of tumors. However, cisplatin administration causes serious side effects, including nephrotoxicity, ototoxicity and neurotoxicity. It has been shown that cisplatin can interact with P-type ATPases, e.g., Cu+-ATPases (ATP7A and ATP7B) and Na+/K+-ATPase. Cisplatin-induced inhibition of Na+/K+-ATPase has been related to the nephrotoxic effect of the drug. To investigate the inhibitory effects of cisplatin on the pumping activity of PII-type ATPases, electrical measurements were performed on sarcoplasmic reticulum Ca2+-ATPase (SERCA) and Na+/K+-ATPase embedded in vesicles/membrane fragments adsorbed on a solid-supported membrane. We found that cisplatin inhibits SERCA and Na+/K+-ATPase only when administered without a physiological reducing agent (GSH); in contrast, inhibition was also observed in the case of Cu+-ATPases in the presence of 1 mM GSH. Our results indicate that cisplatin is a much stronger inhibitor of SERCA (with an IC50 value of 1.3 μM) than of Na+/K+-ATPase (with an IC50 value of 11.1 μM); moreover, cisplatin inhibition of Na+/K+-ATPase is reversible, whereas it is irreversible in the case of SERCA. In the absence of a physiological substrate, while Cu+-ATPases are able to translocate cisplatin, SERCA and Na+/K+-ATPase do not perform ATP-dependent cisplatin displacement.

Publication link
2017 – Competition is the basis of the transport mechanism of the NhaB Na+/H+ exchanger from Klebsiella pneumoniae
SURFE2R-technology (custom-built system)  Publication in PLoS ONE (2017) Authors: Patiño-Ruiz M., Ganea C., Fendler K., Călinescu O.

Na+/H+ exchange is essential for survival of all organisms, having a role in the regulation of the intracellular Na+ concentration, pH and cell volume. Furthermore, Na+/H+ exchangers were shown to be involved in the virulence of the bacterium Yersinia pestis, indicating they might be potential targets for novel antibiotic treatments. The model system for Na+/H+ exchangers is the NhaA transporter from Escherichia coli, EcNhaA. Therefore, the general transport mechanism of NhaA exchangers is currently well characterized. However, much less is known about NhaB exchangers, with only a limited number of studies available. The pathogen Klebsiella pneumoniae, which is a major source of nosocomial infection, possesses three electrogenic Na+/H+ exchangers, KpNhaA1, KpNhaA2 and KpNhaB, none of which have been previously investigated. Our aim in this study was to functionally characterize KpNhaB using solid supported membrane-based electrophysiology as the main investigation technique, and thus provide the first electrophysiological investigation of an NhaB Na+/H+ exchanger. We found that NhaB can be described by the same competition-based mechanism that was shown to be valid for electrogenic NhaA and NapA, and for electroneutral NhaP Na+/H+ exchangers. For comparison we also characterized the activity of KpNhaA1 and KpNhaA2 and found that the three exchangers have complementary activity profiles, which is likely a survival advantage for K. pneumoniae when faced with environments of different salinity and pH. This underlines their importance as potential antibiotic drug targets.

Publication link
2017 – Conformational memory in the association of the transmembrane protein phospholamban with the sarcoplasmic reticulum calcium pump SERCA
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in Journal of Biological Chemistry (2017) Authors: Smeazzetto S., Armanious G.P., Moncelli M.R., Bak J.J., Lemieux M.J., Young H.S., Tadini-Buoninsegni F.

The sarcoplasmic reticulum Ca2+-ATPase SERCA promotes muscle relaxation by pumping calcium ions from the cytoplasm into the sarcoplasmic reticulum. SERCA activity is regulated by a variety of small transmembrane peptides, most notably by phospholamban in cardiac muscle and sarcolipin in skeletal muscle. However, how phospholamban and sarcolipin regulate SERCA is not fully understood. In the present study, we evaluated the effects of phospholamban and sarcolipin on calcium translocation and ATP hydrolysis by SERCA under conditions that mimic environments in sarcoplasmic reticulum membranes. For pre-steady-state current measurements, proteoliposomes containing SERCA and phospholamban or sarcolipin were adsorbed to a solid-supported membrane and activated by substrate concentration jumps. We observed that phospholamban altered ATP-dependent calcium translocation by SERCA within the first transport cycle, whereas sarcolipin did not. Using pre-steady-state charge (calcium) translocation and steady-state ATPase activity under substrate conditions (various calcium and/or ATP concentrations) promoting particular conformational states of SERCA, we found that the effect of phospholamban on SERCA depends on substrate preincubation conditions. Our results also indicated that phospholamban can establish an inhibitory interaction with multiple SERCA conformational states with distinct effects on SERCA's kinetic properties. Moreover, we noted multiple modes of interaction between SERCA and phospholamban and observed that once a particular mode of association is engaged it persists throughout the SERCA transport cycle and multiple turnover events. These observations are consistent with conformational memory in the interaction between SERCA and phospholamban, thus providing insights into the physiological role of phospholamban and its regulatory effect on SERCA transport activity.

Publication link
2016 – Membrane pyrophosphatases from Thermotoga maritima and Vigna radiata suggest a conserved coupling mechanism
SURFE2R N1 Publication in Nature Communications (2016) Authors: Li K., Wilkinson C., Kellosalo J., Tsai J., Kajander T, Jeuken L.J.C., Sun Y., Goldman A.

Membrane-bound pyrophosphatases (M-PPases), which couple proton/sodium ion transport to pyrophosphate synthesis/hydrolysis, are important in abiotic stress resistance and in the infectivity of protozoan parasites. Here, three M-PPase structures in different catalytic states show that closure of the substrate-binding pocket by helices 5–6 affects helix 13 in the dimer interface and causes helix 12 to move down. This springs a ‘molecular mousetrap’, repositioning a conserved aspartate and activating the nucleophilic water. Corkscrew motion at helices 6 and 16 rearranges the key ionic gate residues and leads to ion pumping. The pumped ion is above the ion gate in one of the ion-bound structures, but below it in the other. Electrometric measurements show a single-turnover event with a non-hydrolysable inhibitor, supporting our model that ion pumping precedes hydrolysis. We propose a complete catalytic cycle for both proton and sodium-pumping M-PPases, and one that also explains the basis for ion specificity.

Publication link
2016 – pH Regulation of Electrogenic Sugar/H+ Symport in MFS Sugar Permeases
SURFE2R-technology (custom-built system) Publication in PLoS ONE (2016) Authors: Bazzone A., Madej M.G., Kaback H.R., Fendler K.

Bacterial sugar symporters in the Major Facilitator Superfamily (MFS) use the H+ (and in a few cases Na+) electrochemical gradients to achieve active transport of sugar into the cell. Because a number of structures of MFS sugar symporters have been solved recently, molecular insight into the transport mechanism is possible from detailed functional analysis. We present here a comparative electrophysiological study of the lactose permease (LacY), the fucose permease (FucP) and the xylose permease (XylE), which reveals common mechanistic principles and differences. In all three symporters energetically downhill electrogenic sugar/H+ symport is observed. Comparison of the pH dependence of symport at symmetrical pH exhibits broad bell-shaped pH profiles extending over 3 to 6 pH units and a decrease at extremely alkaline pH ≥ 9.4 and at acidic to neutral pH = 4.6-7.5. The pH dependence can be described by an acidic to neutral apparent pK (pKapp) and an alkaline pKapp. Experimental evidence suggests that the alkaline pKapp is due to H+ depletion at the protonation site, while the acidic pKapp is due to inhibition of deprotonation. Since previous studies suggest that a single carboxyl group in LacY (Glu325) may be the only side chain directly involved in H+ translocation and a carboxyl side chain with similar properties has been identified in FucP (Asp46) and XylE (Asp27), the present results imply that the pK of this residue is switched during H+/sugar symport in all three symporters.

Publication link
2016 – Electrogenic Cation Binding in the Electroneutral Na+/H+ Antiporter of Pyrococcus abyssi
SURFE2R-technology (custom-built system) Publication in Journal of Biological Chemistry (2016) Authors: Călinescu O., Linder M., Wöhlert D., Yildiz Ö., Kühlbrandt W., Fendler K.

Na+/H+ antiporters in the CPA1 branch of the cation proton antiporter family drive the electroneutral exchange of H+ against Na+ ions and ensure pH homeostasis in eukaryotic and prokaryotic organisms. Although their transport cycle is overall electroneutral, specific partial reactions are electrogenic. Here, we present an electrophysiological study of the PaNhaP Na+/H+ antiporter from Pyrococcus abyssi reconstituted into liposomes. Positive transient currents were recorded upon addition of Na+ to PaNhaP proteoliposomes, indicating a reaction where positive charge is rapidly displaced into the proteoliposomes with a rate constant of k >200 s-1 We attribute the recorded currents to an electrogenic reaction that includes Na+ binding and possibly occlusion. Subsequently, positive charge is transported out of the cell associated with H+ binding, so that the overall reaction is electroneutral. We show that the differences in pH profile and Na+ affinity of PaNhaP and the related MjNhaP1 from Methanocaldococcus jannaschii can be attributed to an additional negatively charged glutamate residue in PaNhaP. The results are discussed in the context of the physiological function of PaNhaP and other microbial Na+/H+ exchangers. We propose that both, electroneutral and electrogenic Na+/H+ antiporters, represent a carefully tuned self-regulatory system, which drives the cytoplasmic pH back to neutral after any deviation.

Publication link
2016 – Functional characterization of solute carrier (SLC) 26/sulfate permease (SulP) proteins in membrane mimetic systems
SURFE2R-technology (custom-built system) Publication in Biochimica et Biophysica Acta (BBA) - Biomembranes (2016) Authors: Srinivasan L., Baars T.L., Fendler K., Michel H.

Solute carrier (SLC) 26 or sulfate permease (SulP) anion transporters, belong to a phylogenetically ancient family of secondary active transporters. Members of the family are involved in several human genetic diseases and cell physiological processes. Despite their importance, the substrates for transport by this family of proteins have been poorly characterized. In this study, recombinant StmYchM/DauA, a SulP from Salmonella typhimurium was purified to homogeneity and functionally characterized. StmYchM/DauA was found to be a dimer in solution as determined by size exclusion chromatography coupled to multiple angle light scattering. We report a functional characterization of the SulP proteins in two membrane mimetic systems and reveal a dual nature of anionic substrates for SulP. StmYchM/DauA functionally incorporated into nanodiscs could bind fumarate with millimolar affinities (KD = 4.6 ± 0.29 mM) as detected by intrinsic tryptophan fluorescence quench studies. In contrast, electrophysiological experiments performed in reconstituted liposomes indicate a strong bicarbonate transport in the presence of chloride but no detectable electrogenic fumarate transport. We hence suggest that while SulP acts as an electrogenic bicarbonate transporter, fumarate may serve as substrate under different conditions indicating multiple functions of SulP.

Publication link
2015 – Structural and Functional Studies of NirC from Salmonella typhimurium
SURFE2R-technology (custom-built system) Publication in Methods in Enzymology (2015) Authors: Rycovska-Blume A., Lü W., Andrade S., Fendler K., Einsle O.

NirC is a pentameric transport system for monovalent anions that is expressed in the context of assimilatory nitrite reductase NirBD in a wide variety of enterobacterial species. A NirC pentamer contains individual pores in each protomer that mediate the passage of at least the nitrite (NO2-) and nitrate (NO3-) anions. As a member of the formate/nitrite transporter family of membrane transport proteins, NirC shares a range of structural and functional features with the formate channel FocA and the hydrosulfide channel AsrD (HSC). NirC from the enteropathogen Salmonella typhimurium has been studied by X-ray crystallography, proton uptake assays, and different electrophysiological techniques, and the picture that has emerged shows a fast and versatile transport system for nitrite that doubles as a defense system during the enteric life of the bacterium. Structural and functional assays are described, which shed light on the transport mechanism of this important molecular machine. 

Publication link
2016 – Charge translocation by mitochondrial NADH:ubiquinone oxidoreductase (complex I) from Yarrowia lipolytica measured on solid-supported membranes
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in Biochemical and Biophysical Research Communications (2016) Authors: Siebels I., Dröse S.

The charge translocation by purified reconstituted mitochondrial complex I from the obligate aerobic yeast Yarrowia lipolytica was investigated after adsorption of proteoliposomes to solid-supported membranes. In presence of n-decylubiquinone (DBQ), pulses of NADH provided by rapid solution exchange induced charge transfer reflecting steady-state pumping activity of the reconstituted enzyme. The signal amplitude increased with time, indicating 'deactive→active' transition of the Yarrowia complex I. Furthermore, an increase of the membrane-conductivity after addition of 5-(N-ethyl-N-isopropyl)amiloride (EIPA) was detected which questiones the use of EIPA as an inhibitor of the Na+/H+-antiporter-like subunits of complex I. This investigation shows that electrical measurements on solid-supported membranes are a suitable method to analyze transport events and 'active/deactive' transition of mitochondrial complex I.

Publication link
2015 – A universal mechanism for transport and regulation of CPA sodium proton exchangers
SURFE2R-technology (custom-built system) Publication in Biological Chemistry (2015) Authors: Călinescu O., Fendler K.

Recent studies performed on a series of Na+/H+ exchangers have led us to postulate a general mechanism for Na+/H+ exchange in the monovalent cation/proton antiporter superfamily. This simple mechanism employs a single binding site for which both substrates compete. The developed kinetic model is self-regulatory, ensuring down-regulation of transport activity at extreme pH, and elegantly explains the pH-dependent activity of Na+/H+ exchangers. The mechanism was experimentally verified and shown to describe both electrogenic and electroneutral exchangers. Using a small number of parameters, exchanger activity can be modeled under different conditions, providing insights into the physiological role of Na+/H+ exchangers.

Publication link
2015 – Hofmeister effect of anions on calcium translocation by sarcoplasmic reticulum Ca2+-ATPase
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in Nature Scientific Reports (2015) Authors: Tadini-Buoninsegni F., Moncelli M.R., Peruzzi N., Ninham B.W., Dei L., Lo Nostroa P.

The occurrence of Hofmeister (specific ion) effects in various membrane-related physiological processes is well documented. For example the effect of anions on the transport activity of the ion pump Na+, K+-ATPase has been investigated. Here we report on specific anion effects on the ATP-dependent Ca2+ translocation by the sarcoplasmic reticulum Ca2+-ATPase (SERCA). Current measurements following ATP concentration jumps on SERCA-containing vesicles adsorbed on solid supported membranes were carried out in the presence of different potassium salts. We found that monovalent anions strongly interfere with ATP-induced Ca2+ translocation by SERCA, according to their increasing chaotropicity in the Hofmeister series. On the contrary, a significant increase in Ca2+ translocation was observed in the presence of sulphate. We suggest that the anions can affect the conformational transition between the phosphorylated intermediates E1P and E2P of the SERCA cycle. In particular, the stabilization of the E1P conformation by chaotropic anions seems to be related to their adsorption at the enzyme/water and/or at the membrane/water interface, while the more kosmotropic species affect SERCA conformation and functionality by modifying the hydration layers of the enzyme.

Publication link
2014 – Substrate-bound outward-open state of the betaine transporter BetP provides insights into Na+ coupling
SURFE2R-technology (custom-built system) Publication in Nature Communications (2014) Authors: Perez C., Faust B., Mehdipour A.R., Francesconi K.A., Forrest L.R., Ziegler C.

The Na+-coupled betaine symporter BetP shares a highly conserved fold with other sequence unrelated secondary transporters, for example, with neurotransmitter symporters. Recently, we obtained atomic structures of BetP in distinct conformational states, which elucidated parts of its alternating-access mechanism. Here, we report a structure of BetP in a new outward-open state in complex with an anomalous scattering substrate, adding a fundamental piece to an unprecedented set of structural snapshots for a secondary transporter. In combination with molecular dynamics simulations these structural data highlight important features of the sequential formation of the substrate and sodium-binding sites, in which coordinating water molecules play a crucial role. We observe a strictly interdependent binding of betaine and sodium ions during the coupling process. All three sites undergo progressive reshaping and dehydration during the alternating-access cycle, with the most optimal coordination of all substrates found in the closed state.

Publication link
2015 – A sulfur‐based transport pathway in Cu+‐ATPases
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in EMBO Reports (2015) Authors: Mattle D., Zhang L., Sitsel O., Pedersen L.T., Moncelli M.R., Tadini-Buoninsegni F., Gourdon P., Rees D.C., Nissen P., Meloni G.

Cells regulate copper levels tightly to balance the biogenesis and integrity of copper centers in vital enzymes against toxic levels of copper. PIB‐type Cu+‐ATPases play a central role in copper homeostasis by catalyzing the selective translocation of Cu+ across cellular membranes. Crystal structures of a copper‐free Cu+‐ATPase are available, but the mechanism of Cu+ recognition, binding, and translocation remains elusive. Through X‐ray absorption spectroscopy, ATPase activity assays, and charge transfer measurements on solid‐supported membranes using wild‐type and mutant forms of the Legionella pneumophila Cu+‐ATPase (LpCopA), we identify a sulfur‐lined metal transport pathway. Structural analysis indicates that Cu+ is bound at a high‐affinity transmembrane‐binding site in a trigonal‐planar coordination with the Cys residues of the conserved CPC motif of transmembrane segment 4 (C382 and C384) and the conserved Met residue of transmembrane segment 6 (M717 of the MXXXS motif). These residues are also essential for transport. Additionally, the studies indicate essential roles of other conserved intramembranous polar residues in facilitating copper binding to the high‐affinity site and subsequent release through the exit pathway.

Publication link
2014 – NhaA Na+/H+ antiporter mutants that hardly react to the membrane potential
SURFE2R-technology (custom-built system) Publication in PLoS ONE (2014) Authors: Alkoby D., Rimon A., Budak M., Patino-Ruiz M., Călinescu O., Fendler K., Padan E..

pH and Na+ homeostasis in all cells requires Na+/H+ antiporters. The crystal structure, obtained at pH 4, of NhaA, the main antiporter of Escherichia coli, has provided general insights into an antiporter mechanism and its unique pH regulation. Here, we describe a general method to select various NhaA mutants from a library of randomly mutagenized NhaA. The selected mutants, A167P and F267C are described in detail. Both mutants are expressed in Escherichia coli EP432 cells at 70-95% of the wild type but grow on selective medium only at neutral pH, A167P on Li+ (0.1 M) and F267C on Na+ (0.6 M). Surprising for an electrogenic secondary transporter, and opposed to wild type NhaA, the rates of A167P and F267C are almost indifferent to membrane potential. Detailed kinetic analysis reveals that in both mutants the rate limiting step of the cation exchange cycle is changed from an electrogenic to an electroneutral reaction.

Publication link
2014 – Species differences in bacterial NhaA Na+/H+ exchangers
SURFE2R-technology (custom-built system) Publication in FEBS Letters (2014) Authors: Călinescu O., Danner E., Böhm M., Hunte C., Fendler K..

Bacteria have adapted their NhaA Na+/H+ exchangers responsible for salt homeostasis to their different habitats. We present an electrophysiological and kinetic analysis of NhaA from Helicobacter pylori and compare it to the previously investigated exchangers from Escherichia coli and Salmonella typhimurium. Properties of all three transporters are described by a simple model using a single binding site for H+ and Na+. We show that H. pylori NhaA only has a small acidic shift of its pH-dependent activity profile compared to the other transporters and discuss why a more drastic change in its pH activity profile is not physiologically required.

Publication link
2014 – Keeping it simple, transport mechanism and pH regulation in Na+/H+ exchangers
SURFE2R-technology (custom-built system) Publication in Journal of Biological Chemistry (2014) Authors: Călinescu O., Paulino C., Kühlbrandt W., Fendler K.

Na+/H+ exchangers are essential for regulation of intracellular proton and sodium concentrations in all living organisms. We examined and experimentally verified a kinetic model for Na+/H+ exchangers, where a single binding site is alternatively occupied by Na+ or one or two H+ ions. The proposed transport mechanism inherently down-regulates Na+/H+ exchangers at extreme pH, preventing excessive cytoplasmic acidification or alkalinization. As an experimental test system we present the first electrophysiological investigation of an electroneutral Na+/H+ exchanger, NhaP1 from Methanocaldococcus jannaschii (MjNhaP1), a close homologue of the medically important eukaryotic NHE Na+/H+ exchangers. The kinetic model describes the experimentally observed substrate dependences of MjNhaP1, and the transport mechanism explains alkaline down-regulation of MjNhaP1. Because this model also accounts for acidic down-regulation of the electrogenic NhaA Na+/H+ exchanger from Escherichia coli (EcNhaA, shown in a previous publication) we conclude that it applies generally to all Na+/H+ exchangers, electrogenic as well as electroneutral, and elegantly explains their pH regulation. Furthermore, the electrophysiological analysis allows insight into the electrostatic structure of the translocation complex in electroneutral and electrogenic Na+/H+ exchangers.

Publication link
2014 – Molecular characterization of the Na+/H+-antiporter NhaA from Salmonella Typhimurium
SURFE2R-technology (custom-built system) Publication in PLoS ONE (2014) Authors: Lentes C.J., Mir S.H., Boehm M., Ganea C., Fendler K., Hunte C.

Na+/H+ antiporters are integral membrane proteins that are present in almost every cell and in every kingdom of life. They are essential for the regulation of intracellular pH-value, Na+-concentration and cell volume. These secondary active transporters exchange sodium ions against protons via an alternating access mechanism, which is not understood in full detail. Na+/H+ antiporters show distinct species-specific transport characteristics and regulatory properties that correlate with respective physiological functions. Here we present the characterization of the Na+/H+ antiporter NhaA from Salmonella enterica serovar Thyphimurium LT2, the causing agent of food-born human gastroenteritis and typhoid like infections. The recombinant antiporter was functional in vivo and in vitro. Expression of its gene complemented the Na+-sensitive phenotype of an E. coli strain that lacks the main Na+/H+ antiporters. Purified to homogeneity, the antiporter was a dimer in solution as accurately determined by size-exclusion chromatography combined with multi-angle laser-light scattering and refractive index monitoring. The purified antiporter was fully capable of electrogenic Na+(Li+)/H+-antiport when reconstituted in proteoliposomes and assayed by solid-supported membrane-based electrophysiological measurements. Transport activity was inhibited by 2-aminoperimidine. The recorded negative currents were in agreement with a 1Na+(Li+)/2H+ stoichiometry. Transport activity was low at pH 7 and up-regulation above this pH value was accompanied by a nearly 10-fold decrease of KmNa (16 mM at pH 8.5) supporting a competitive substrate binding mechanism. K+ does not affect Na+ affinity or transport of substrate cations, indicating that selectivity of the antiport arises from the substrate binding step. In contrast to homologous E. coli NhaA, transport activity remains high at pH values above 8.5. The antiporter from S. Typhimurium is a promising candidate for combined structural and functional studies to contribute to the elucidation of the mechanism of pH-dependent Na+/H+ antiporters and to provide insights in the molecular basis of species-specific growth and survival strategies.

Publication link
2014 – Anticancer Ruthenium(III) Complex KP1019 Interferes with ATP-Dependent Ca2+ Translocation by Sarco-Endoplasmic Reticulum Ca2+-ATPase (SERCA)
SURFE2R N1 Publication in ChemMedChem (2014) Authors: Sadafi F.Z., Massai L., Bartolommei G., Moncelli M.R., Messori L., Tadini-Buoninsegni F.

Sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), a P-type ATPase that sustains Ca2+ transport and plays a major role in intracellular Ca2+ homeostasis, represents a therapeutic target for cancer therapy. Here, we investigated whether ruthenium-based anticancer drugs, namely KP1019 (indazolium [trans-tetrachlorobis(1H-indazole)ruthenate(III)]), NAMI-A (imidazolium [trans-tetrachloro(1H-imidazole)(S-dimethylsulfoxide)ruthenate(III)]) and RAPTA-C ([Ru(η6-p-cymene)dichloro(1,3,5-triaza-7-phosphaadamantane)]), and cisplatin (cis-diammineplatinum(II) dichloride) might act as inhibitors of SERCA. Charge displacement by SERCA adsorbed on a solid-supported membrane was measured after ATP or Ca2+ concentration jumps. Our results show that KP1019, in contrast tocancer the other metal compounds, is able to interfere with ATP-dependent translocation of Ca2+ ions. An IC50 value of 1 μM was determined for inhibition of calcium translocation by KP1019. Conversely, it appears that KP1019 does not significantly affect Ca2+ binding to the ATPase from the cytoplasmic side. Inhibition of SERCA at pharmacologically relevant concentrations may represent a crucial aspect in the overall pharmacological and toxicological profile of KP1019.

Publication link
2014 – Direct observation of electrogenic NH4(+) transport in ammonium transport (Amt) proteins
SURFE2R-technology (custom-built system) Publication in PNAS (2014) Authors: Wacker T., Garcia-Celma J.J., Lewe P., Andrade S.L.

Significance: We have detected and analyzed electrogenic transport of ammonium and methylammonium by members of the ammonium transport (Amt) family of membrane proteins using solid-supported membrane electrophysiology. Amt transport is pH-dependent and occurs at a rate of 30–300 ions per s per trimer, well in the range of other transport proteins. The study establishes, to our knowledge, the first in vitro assay system for Amt transport in a fully controlled setup and settles debate about whether Amt proteins function as passive ammonia channels or active ammonium transporters.Abstract:Significance: We have detected and analyzed electrogenic transport of ammonium and methylammonium by members of the ammonium transport (Amt) family of membrane proteins using solid-supported membrane electrophysiology. Amt transport is pH-dependent and occurs at a rate of 30–300 ions per s per trimer, well in the range of other transport proteins. The study establishes, to our knowledge, the first in vitro assay system for Amt transport in a fully controlled setup and settles debate about whether Amt proteins function as passive ammonia channels or active ammonium transporters. Abstract: Ammonium transport (Amt) proteins form a ubiquitous family of integral membrane proteins that specifically shuttle ammonium across membranes. In prokaryotes, archaea, and plants, Amts are used as environmental NH4+ sCaVengers for uptake and assimilation of nitrogen. In the eukaryotic homologs, the Rhesus proteins, NH4+/NH3 transport is used instead in acid–base and pH homeostasis in kidney or NH4+/NH3 (and eventually CO2) detoxification in erythrocytes. Crystal structures and variant proteins are available, but the inherent challenges associated with the unambiguous identification of substrate and monitoring of transport events severely inhibit further progress in the field. Here we report a reliable in vitro assay that allows us to quantify the electrogenic capacity of Amt proteins. Using solid-supported membrane (SSM)-based electrophysiology, we have investigated the three Amt orthologs from the euryarchaeon Archaeoglobus fulgidus. Af-Amt1 and Af-Amt3 are electrogenic and transport the ammonium and methylammonium cation with high specificity. Transport is pH-dependent, with a steep decline at pH values of ∼5.0. Despite significant sequence homologies, functional differences between the three proteins became apparent. SSM electrophysiology provides a long-sought-after functional assay for the ubiquitous ammonium transporters.

Publication link
2013 – Functional characterization of a ClC transporter by solid-supported membrane electrophysiology
SURFE2R-technology (custom-built system) Publication in Journal of General Physiology (2013) Authors: Garcia-Celma J., Szydelko A., Dutzler R.

EcClC, a prokaryotic member of the ClC family of chloride channels and transporters, works as coupled H+/Cl- exchanger. With a known structure and the possibility of investigating its behavior with different biochemical and biophysical techniques, the protein has become an important model system for the family. Although many aspects of its function have been previously characterized, it was difficult to measure transport on the same sample under different environmental conditions. To overcome this experimental limitation, we have studied EcClC by solid-supported membrane electrophysiology. The large transport-related transient currents and a simple way of relating transport rates to the measured signal have allowed a thorough investigation of ion selectivity, inhibition, and the dependence of transport on changes in ion concentration and pH. Our results confirm that the protein transports larger anions with about similar rates, whereas the smaller fluoride is not a substrate. We also show that 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), a known inhibitor of other anion transport protein, irreversibly inhibits EcClC from the intracellular side. The chloride dependence shows an apparent saturation at millimolar concentrations that resembles a similar behavior in eukaryotic ClC channels. Our experiments have also allowed us to quantify the pH dependence of transport. EcClC shows a strong activation at low pH with an apparent pKa of 4.6. The pronounced pH dependence is lost by the mutation of a conserved glutamate facing the extracellular solution that was previously shown to be an acceptor for transported protons, whereas it is largely retained by the mutation of an equivalent residue at the intracellular side. Our results have provided a quantitative basis for the transport behavior of EcClC, and they will serve as a reference for future investigations of novel electrogenic transporters with still-uncharacterized properties.

Publication link
2013 – Photocycle and vectorial proton transfer in a rhodopsin from the eukaryote Oxyrrhis marina
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in Biochemistry (2013) Authors: Janke C., Scholz F., Becker-Baldus J., Glaubitz C., Wood P.G., Bamberg E., Wachtveitl J., Bamann C.

Retinylidene photoreceptors are ubiquitously present in marine protists as first documented by the identification of green proteorhodopsin (GPR). We present a detailed investigation of a rhodopsin from the protist Oxyrrhis marina (OR1) with respect to its spectroscopic properties and to its vectorial proton transport. Despite its homology to GPR, OR1's features differ markedly in its pH dependence. Protonation of the proton acceptor starts at pH below 4 and is sensitive to the ionic conditions. The mutation of a conserved histidine H62 did not influence the pK(a) value in a similar manner as in other proteorhodopsins where the charged histidine interacts with the proton acceptor forming the so-called His-Asp cluster. Mutational and pH-induced effects were further reflected in the temporal behavior upon light excitation ranging from femtoseconds to seconds. The primary photodynamics exhibits a high sensitivity to the environment of the proton acceptor D100 that are correlated to the different initial states. The mutation of the H62 does not affect photoisomerization at neutral pH. This is in agreement with NMR data indicating the absence of the His-Asp cluster. The subsequent steps in the photocycle revealed protonation reactions at the Schiff base coupled to proton pumping even at low pH. The main electrogenic steps are associated with the reprotonation of the Schiff base and internal proton donor. Hence, OR1 shows a different theme of the His-Asp organization where the low pK(a) of the proton acceptor is not dominated by this interaction, but by other electrostatic factors.

Publication link
2013 – Electrophysiological characterization of uncoupled mutants of LacY
SURFE2R-technology (custom-built system) Publication in Biochemistry (2013) Authors: Gaiko O., Bazzone A., Fendler K., Kaback H.R.

In this study of the lactose permease of Escherichia coli (LacY), five functionally irreplaceable residues involved specifically in H+ translocation (Arg302 and Glu325) or in the coupling between protonation and sugar binding (Tyr236, Glu269, and His322) were mutated individually or together with mutant Glu325 → Ala. The wild type and each mutant were purified and reconstituted into proteoliposomes, which were then examined using solid-supported-membrane-based electrophysiology. Mutants Glu325 → Ala or Arg302 → Ala, in which H+ symport is abolished, exhibit a weakly electrogenic rapid reaction triggered by sugar binding. The reaction is essentially absent in mutant Tyr236 → Phe, Glu269 → Ala, and His322 → Ala, and each of these mutations blocks the electrogenic reaction observed in the Glu325 → Ala mutant. The findings are consistent with the interpretation that the electrogenic reaction induced by sugar binding is due to rearrangement of charged residues in LacY and that this reaction is blocked by mutation of each member of the Tyr236/Glu269/His322 triad. In addition, further support is provided for the conclusion that deprotonation is rate limiting for downhill lactose/H+ symport.

Publication link
2013 – Enhanced adsorption of Ca-ATPase containing vesicles on a negatively charged solid supported membrane for the investigation of membrane transporters
SURFE2R N1 Publication in Langmuir (2013) Authors: Sacconi A., Moncelli M.R., Mergheri G., Tadini-Buoninsegni F.

A convenient model system for a biological membrane is a solid-supported membrane (SSM), which consists of a gold-supported alkanethiol|phospholipid bilayer. In combination with a concentration jump method, SSMs have been used for the investigation of several membrane transporters. Vesicles incorporating sarcoplasmic reticulum Ca-ATPase (SERCA) were adsorbed on a negatively charged SSM (octadecanethiol|phosphatidylserine bilayer). The current signal generated by the adsorbed vesicles following an ATP concentration jump was compared to that produced by SERCA-containing vesicles adsorbed on a conventional SSM (octadecanethiol|phosphatidylcholine bilayer). A significantly higher current amplitude was recorded on the serine-based SSM. The adsorption of SERCA-incorporating vesicles on the SSM was then characterized by surface plasmon resonance (SPR). The SPR measurements clearly indicate that in the presence of Ca2+ and Mg2+, the amount of adsorbed vesicles on the serine-based SSM is about twice that obtained using the conventional SSM, thereby demonstrating that the higher current amplitude recorded on the negatively charged SSM is correlated with a greater quantity of adsorbed vesicles. The enhanced adsorption of membrane vesicles on the PS-based SSM may be useful to study membrane preparations with a low concentration of transport protein generating small current signals, as in the case of various recombinantly expressed proteins.

Publication link
2012 – The nitrite transport protein NirC from Salmonella typhimurium is a nitrite/proton antiporter
SURFE2R-technology (custom-built system) Publication in Biochimica et Biophysica Acta (BBA) - Biomembranes (2012) Authors: Rycovska A., Hatahet L., Fendler K., Michel H.

In anaerobically grown bacteria, transport of nitrite is catalyzed by an integral membrane protein of the form ate–nitrite transporter family, NirC, which in Salmonella typhimurium plays a critical role in intracellular virulence. We present a functional characterization of the S. typhimurium nitrite transporter StmNirC in native membrane vesicles as well as purified and reconstituted into proteoliposomes. Using an electrophysiological technique based on solid supported membranes, we show nitrite induced translocation of negative charges into proteoliposomes reconstituted with purified StmNirC. These data demonstrate the electrogenicity of StmNirC and its substrate specificity for nitrite. Monitoring changes in ΔpH on everted membrane vesicles containing overexpressed StmNirC using acridine orange as a pH indicator we demonstrate that StmNirC acts as a secondary active transporter. It promotes low affinity transport of nitrite coupled to H+ antiport with a pH independent profile in the pH range from 6 to 8. In addition to nitrite also nitrate is transported by StmNirC, but with reduced flux and complete absence of proton antiport activity. Taken together, these data suggest a bispecific anion selectivity of StmNirC with an ion specific transport mode. This may play a role in regulating nitrite transport under physiological conditions.

Publication link
2013 – Differential Effects of Mutations on the Transport Properties of the Na+/H+ Antiporter NhaA from Escherichia coli
SURFE2R-technology (custom-built system) Publication in Journal of Biological Chemistry (2013) Authors: Mager T., Braner M., Kubsch B., Hatahet L., Alkoby D., Rimon A., Padan E., Fendler K.

Na+/H+ antiporters show a marked pH dependence, which is important for their physiological function in eukaryotic and prokaryotic cells. In NhaA, the Escherichia coli Na+/H+ antiporter, specific single site mutations modulating the pH profile of the transporter have been described in the past. To clarify the mechanism by which these mutations influence the pH dependence of NhaA, the substrate dependence of the kinetics of selected NhaA variants was electrophysiologically investigated and analyzed with a kinetic model. It is shown that the mutations affect NhaA activity in quite different ways by changing the properties of the binding site or the dynamics of the transporter. In the first case, pK and/or KDNa are altered, and in the second case, the rate constants of the conformational transition between the inside and the outside open conformation are modified. It is shown that residues as far apart as 15–20 Å from the binding site can have a significant impact on the dynamics of the conformational transitions or on the binding properties of NhaA. The implications of these results for the pH regulation mechanism of NhaA are discussed.

Publication link
2012 – Distinctive features of catalytic and transport mechanisms in mammalian sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) and Cu+ (ATP7A/B) ATPases
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in Journal of Biological Chemistry (2012) Authors: Lewis D., Pilankatta R., Inesi G., Bartolommei G., Moncelli M.R., Tadini-Buoninsegni F.

Ca2+ (sarco-endoplasmic reticulum Ca2+ ATPase (SERCA)) and Cu+ (ATP7A/B) ATPases utilize ATP through formation of a phosphoenzyme intermediate (E-P) whereby phosphorylation potential affects affinity and orientation of bound cation. SERCA E-P formation is rate-limited by enzyme activation by Ca2+, demonstrated by the addition of ATP and Ca2+ to SERCA deprived of Ca2+ (E2) as compared with ATP to Ca2+-activated enzyme (E1·2Ca2+). Activation by Ca2+ is slower at low pH (2H+·E2 to E1·2Ca2+) and little sensitive to temperature-dependent activation energy. On the other hand, subsequent (forward or reverse) phosphoenzyme processing is sensitive to activation energy, which relieves conformational constraints limiting Ca2+ translocation. A “H+-gated pathway,” demonstrated by experiments on pH variations, charge transfer, and Glu-309 mutation allows luminal Ca2+ release by H+/Ca2+ exchange. As compared with SERCA, initial utilization of ATP by ATP7A/B is much slower and highly sensitive to temperature-dependent activation energy, suggesting conformational constraints of the headpiece domains. Contrary to SERCA, ATP7B phosphoenzyme cleavage shows much lower temperature dependence than EP formation. ATP-dependent charge transfer in ATP7A and -B is observed, with no variation of net charge upon pH changes and no evidence of Cu+/H+ exchange. As opposed to SERCA after Ca2+ chelation, ATP7A/B does not undergo reverse phosphorylation with Pi after copper chelation unless a large N-metal binding extension segment is deleted. This is attributed to the inactivating interaction of the copper-deprived N-metal binding extension with the headpiece domains. We conclude that in addition to common (P-type) phosphoenzyme intermediate formation, SERCA and ATP7A/B possess distinctive features of catalytic and transport mechanisms.

Publication link
2012 – Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP
SURFE2R-technology (custom-built system) Publication in PNAS (2012) Authors: Khafizov K., Perez C., Koshy C., Quick M., Fendler K., Ziegler C., Forrest L.R.

Sodium-coupled substrate transport plays a central role in many biological processes. However, despite knowledge of the structures of several sodium-coupled transporters, the location of the sodium-binding site(s) often remains unclear. Several of these structures have the five transmembrane-helix inverted-topology repeat, LeuT-like (FIRL) fold, whose pseudosymmetry has been proposed to facilitate the alternating-access mechanism required for transport. Here, we provide biophysical, biochemical, and computational evidence for the location of the two cation-binding sites in the sodium-coupled betaine symporter BetP. A recent X-ray structure of BetP in a sodium-bound closed state revealed that one of these sites, equivalent to the Na2 site in related transporters, is located between transmembrane helices 1 and 8 of the FIRL-fold; here, we confirm the location of this site by other means. Based on the pseudosymmetry of this fold, we hypothesized that the second site is located between the equivalent helices 6 and 3. Molecular dynamics simulations of the closed-state structure suggest this second sodium site involves two threonine sidechains and a backbone carbonyl from helix 3, a phenylalanine from helix 6, and a water molecule. Mutating the residues proposed to form the two binding sites increased the apparent Km and Kd for sodium, as measured by betaine uptake, tryptophan fluorescence, and 22Na+ binding, and also diminished the transient currents measured in proteoliposomes using solid supported membrane-based electrophysiology. Taken together, these results provide strong evidence for the identity of the residues forming the sodium-binding sites in BetP.

Publication link
2011 – Transport Mechanism and pH Regulation of the Na+/H+ Antiporter NhaA from Escherichia coli
SURFE2R-technology (custom-built system) Publication in Journal of Biological Chemistry (2011) Authors: Mager T., Rimon A., Padan E., Fendler K.

Using an electrophysiological assay the activity of NhaA was tested in a wide pH range from pH 5.0 to 9.5. Forward and reverse transport directions were investigated at zero membrane potential using preparations with inside-out and right side-out-oriented transporters with Na+ or H+ gradients as the driving force. Under symmetrical pH conditions with a Na+ gradient for activation, both the wt and the pH-shifted G338S variant exhibit highly symmetrical transport activity with bell-shaped pH dependences, but the optimal pH was shifted 1.8 pH units to the acidic range in the variant. In both strains the pH dependence was associated with a systematic increase of the Km for Na+ at acidic pH. Under symmetrical Na+ concentration with a pH gradient for NhaA activation, an unexpected novel characteristic of the antiporter was revealed; rather than being down-regulated, it remained active even at pH as low as 5. These data allowed a transport mechanism to advance based on competing Na+ and H+ binding to a common transport site and a kinetic model to develop quantitatively explaining the experimental results. In support of these results, both alkaline pH and Na+ induced the conformational change of NhaA associated with NhaA cation translocation as demonstrated here by trypsin digestion. Furthermore, Na+ translocation was found to be associated with the displacement of a negative charge. In conclusion, the electrophysiological assay allows the revelation of the mechanism of NhaA antiport and sheds new light on the concept of NhaA pH regulation.

Publication link
2012 – Assaying the proton transport and regulation of UCP1 using solid supported membranes
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in European Biophysics Journal (2012) Authors: Blesneac I., Ravaud S., Machillot P., Zoonens M., Masscheylen S., Miroux B., Vivaudou M., Pebay-Peyroula E.

The uncoupling protein 1 (UCP1) is a mitochondrial protein that carries protons across the inner mitochondrial membrane. It has an important role in non-shivering thermogenesis, and recent evidence suggests its role in human adult metabolism. Using rapid solution exchange on solid supported membranes, we succeeded in measuring electrical currents generated by the transport activity of UCP1. The protein was purified from mouse brown adipose tissue, reconstituted in liposomes and absorbed on solid supported membranes. A fast pH jump activated the ion transport, and electrical signals could be recorded. The currents were characterized by a fast rise and a slow decay, were stable over time, inhibited by purine nucleotides and activated by fatty acids. This new assay permits direct observation of UCP1 activity in controlled cell-free conditions, and opens up new possibilities for UCP1 functional characterization and drug screening because of its robustness and its potential for automation.

Publication link
2011 – G117C MelB, a mutant melibiose permease with a changed conformational equilibrium
SURFE2R-technology (custom-built system) Publication in Biochimica et Biophysica Acta (BBA) - Biomembranes (2011) Authors: Ganea C., Meyer-Lipp K., Lemonnier R., Krah A., Leblanc G., Fendler K.

Replacement of the glycine at position 117 by a cysteine in the melibiose permease creates an interesting phenotype: while the mutant transporter shows still transport activity comparable to the wild type its pre steady-state kinetic properties are drastically altered. The transient charge displacements after substrate concentration jumps are strongly reduced and the fluorescence changes disappear. Together with its maintained transport activity this indicates that substrate translocation in G117C melibiose permease is not impaired but that the initial conformation of the mutant transporter differs from that of the wild type permease. A kinetic model for the G117C melibiose permease based on a rapid dynamic equilibrium of the substrate free transporter is proposed. Implications of the kinetic model for the transport mechanism of the wild type permease are discussed.

Publication link
2011 – Robust Electrophysiological Assays using Solid Supported Membranes: the Organic Cation Transporter OCT2
SURFE2R ONE (a predecessor model of the SURFE2R N1) Publication in Australian Journal of Chemistry (2011) Authors: Gaiko O., Janausch I., Geibel S., Vollert H., Arndt P., Gonski S., Fendler K.

An electrophysiological assay platform based on solid supported membranes (SSM) for the organic cation transporter (OCT) is presented. Stable Chinese hamster ovary (CHO) cell lines overexpressing the human (hOCT2) and rat transporters (rOCT2) were generated and validated. Membrane preparations from the cell lines were investigated using SSM-based electrophysiology. Baculovirus transfected insect cells (HighFive and Mimic Sf9) were also tested with the same assay but yielded less than optimal results. The assays were validated by the determination of substrate affinities and inhibition by standard inhibitors. The study demonstrates the suitability of the SSM-based electrophysiological OCT assay for rapid and automatic screening of drug candidates.

Publication link
2011 – Characterisation of the purified human sodium/iodide symporter reveals that the protein is mainly present in a dimeric form and permits the detailed study of a native C-terminal fragment
SURFE2R-technology (custom-built system) Publication in Biochimica et Biophysica Acta (BBA) - Biomembranes (2011) Authors: Huc-Brandt S., Marcellin D., Graslin F., Averseng O., Bellanger L., Hivin P., Quemeneur E., Basquin C., NaVarro V., Pourcher T., Darrouzet E.

The sodium/iodide symporter is an intrinsic membrane protein that actively transports iodide into thyroid follicular cells. It is a key element in thyroid hormone biosynthesis and in the radiotherapy of thyroid tumours and their metastases. Sodium/iodide symporter is a very hydrophobic protein that belongs to the family of sodium/solute symporters. As for many other membrane proteins, particularly mammalian ones, little is known about its biochemistry and structure. It is predicted to contain 13 transmembrane helices, with an N-terminus oriented extracellularly. The C-terminal, cytosolic domain contains approximately one hundred amino acid residues and bears most of the transporter's putative regulatory sites (phosphorylation, sumoylation, di-acide, di-leucine or PDZ-binding motifs). In this study, we report the establishment of eukaryotic cell lines stably expressing various human sodium/iodide symporter recombinant proteins, and the development of a purification protocol which allowed us to purify milligram quantities of the human transporter. The quaternary structure of membrane transporters is considered to be essential for their function and regulation. Here, the oligomeric state of human sodium/iodide symporter was analysed for the first time using purified protein, by size exclusion chromatography and light scattering spectroscopy, revealing that the protein exists mainly as a dimer which is stabilised by a disulfide bridge. In addition, the existence of a sodium/iodide symporter C-terminal fragment interacting with the protein was also highlighted. We have shown that this fragment exists in various species and cell types, and demonstrated that it contains the amino-acids [512-643] from the human sodium/iodide symporter protein and, therefore, the last predicted transmembrane helix. Expression of either the [1-512] truncated domain or the [512-643] domain alone, as well as co-expression of the two fragments, was performed, and revealed that co-expression of [1-512] with [512-643] allowed the reconstitution of a functional protein. These findings constitute an important step towards an understanding of some of the post-translational mechanisms that finely tune iodide accumulation through human sodium/iodide symporter regulation.

Publication link
2011 – Development of an assay for Complex I/Complex III of the respiratory chain using solid supported membranes and its application in mitochondrial toxicity screening in drug discovery
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in ASSAY and Drug Development Technologies (2011) Authors: Preissl S., Bick I., Obrdlik P., Diekert K., Gul S., Gribbon P.

Membrane-bound transporter proteins are involved in cell signal transduction and metabolism as well as influencing key pharmacological properties such as drug bioavailability. The functional activity of transporters that belong to the group of electrically active membrane proteins can be directly monitored using the solid-supported membrane-based SURFE(2)R™ technology (SURFace Electrogenic Event Reader; Scientific Devices Heidelberg GmbH, Heidelberg, Germany). The method makes use of membrane fragments or vesicles containing transport proteins adsorbed onto solid-supported membrane-covered electrodes and allows the direct measurement of their activity. This technology has been used to develop a robust screening compatible assay for Complex I/Complex III, key components of the respiratory chain in 96-well microtiter plates. The assay was screened against 1,000 compounds from the ComGenex Lead-like small molecule library to ascertain whether mitochondrial liabilities might be an underlying, although undesirable feature of typical commercial screening libraries. Some 105 hits (compounds exhibiting >50% inhibition of Complex I/Complex III activity at 10 μM) were identified and their activities were subsequently confirmed in duplicate, yielding a confirmation rate of 68%. Analysis of the confirmed hits also provided evidence of structure-activity relationships and two compounds from one structural class were further evaluated in dose-response experiments. This study provides evidence that profiling of compounds for potential mitochondrial liabilities, even at an early stage of drug discovery, may be a necessary additional quality filter that should be considered during the compound screening and profiling cascade.

Publication link
2010 – Solid-supported membrane technology for the investigation of the influenza A virus M2 channel activity
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in Pflügers Archiv - European Journal of Physiology (2010) Authors: Balannik V., Obrdlik P., Inayat S., Steensen C., Wang J., Rausch J.M., DeGrado W.F., Kelety B., Pinto L.H.

Influenza A virus encodes an integral membrane protein, A/M2, that forms a pH-gated proton channel that is essential for viral replication. The A/M2 channel is a target for the anti-influenza drug amantadine, although the effectiveness of this drug has been diminished by the appearance of naturally occurring point mutations in the channel pore. Thus, there is a great need to discover novel anti-influenza therapeutics, and, since the A/M2 channel is a proven target, approaches are needed to screen for new classes of inhibitors for the A/M2 channel. Prior in-depth studies of the activity and drug sensitivity of A/M2 channels have employed labor-intensive electrophysiology techniques. In this study, we tested the validity of electrophysiological measurements with solid-supported membranes (SSM) as a less labor-intensive alternative technique for the investigation of A/M2 ion channel properties and for drug screening. By comparing the SSM-based measurements of the activity and drug sensitivity of A/M2 wild-type and mutant channels with measurements made with conventional electrophysiology methods, we show that SSM-based electrophysiology is an efficient and reliable tool for functional studies of the A/M2 channel protein and for screening compounds for inhibitory activity against the channel.

Publication link
2010 – The G215R Mutation in the Cl−/H+-Antiporter ClC-7 Found in ADO II Osteopetrosis Does Not Abolish Function but Causes a Severe Trafficking Defect
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in PLoS ONE (2010) Authors: Schulz P., Werner J., Stauber T., Henriksen K., Fendler K.

BACKGROUND:ClC-7 is a ubiquitous transporter which is broadly expressed in mammalian tissues. It is implied in the pathogenesis of lysosomal storage disease and osteopetrosis. Because of its endosomal/lysosomal localization it is still poorly characterized.METHODOLOGY/PRINCIPAL FINDINGS:An electrophysiological characterization of rat ClC-7 using solid-supported membrane-based electrophysiology is presented. The measured currents show the characteristics of ClC-7 and confirm its function as a Cl-/H+-antiporter. We have used rat ClC-7 in CHO cells as a model system to investigate the functionality and cellular localization of the wt transporter and its variant G213R ClC-7 which is the analogue of human G215R ClC-7 responsible for autosomal dominant osteopetrosis type II. Our study shows that rat G213R ClC-7 is functional but has a localization defect in CHO cells which prevents it from being correctly targeted to the lysosomal membrane. The electrophysiological assay is tested as a tool for drug discovery. The assay is validated with a number of drug candidates. It is shown that ClC-7 is inhibited by DIDS, NPPB and NS5818 at micromolar concentrations.CONCLUSIONS/SIGNIFICANCE:It is suggested that the scenario found in the CHO model system also applies to the human transporter and that mislocalization rather than impaired functionality of G215R ClC-7 is the primary cause of the related autosomal dominant osteopetrosis type II. Furthermore, the robust solid-supported membrane-based electrophysiological assay is proposed for rapid screening for potential ClC-7 inhibitors which are discussed for treatment of osteoporosis.

Publication link
2010 – Electrophysiological characterization of ATPases in native synaptic vesicles and synaptic plasma membranes
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in Biochemical Journal (2010) Authors: Obrdlik P., Diekert K., Watzke N., Keipert C., Pehl U., Brosch C., Boehm N., Bick I., Ruitenberg M., Volknandt W., Kelety B.

Vesicular V-ATPase (V-type H+-ATPase) and the plasma membrane-bound Na+/K+-ATPase are essential for the cycling of neurotransmitters at the synapse, but direct functional studies on their action in native surroundings are limited due to the poor accessibility via standard electrophysiological equipment. We performed SSM (solid supported membrane)-based electrophysiological analyses of synaptic vesicles and plasma membranes prepared from rat brains by sucrose-gradient fractionation. Acidification experiments revealed V-ATPase activity in fractions containing the vesicles but not in the plasma membrane fractions. For the SSM-based electrical measurements, the ATPases were activated by ATP concentration jumps. In vesicles, ATP-induced currents were inhibited by the V-ATPase-specific inhibitor BafA1 (bafilomycin A1) and by DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate). In plasma membranes, the currents were inhibited by the Na+/K+-ATPase inhibitor digitoxigenin. The distribution of the V-ATPase- and Na+/K+-ATPase-specific currents correlated with the distribution of vesicles and plasma membranes in the sucrose gradient. V-ATPase-specific currents depended on ATP with a K0.5 of 51+/-7 microM and were inhibited by ADP in a negatively co-operative manner with an IC50 of 1.2+/-0.6 microM. Activation of V-ATPase had stimulating effects on the chloride conductance in the vesicles. Low micromolar concentrations of DIDS fully inhibited the V-ATPase activity, whereas the chloride conductance was only partially affected. In contrast, NPPB [5-nitro-2-(3-phenylpropylamino)-benzoic acid] inhibited the chloride conductance but not the V-ATPase. The results presented describe electrical characteristics of synaptic V-ATPase and Na+/K+-ATPase in their native surroundings, and demonstrate the feasibility of the method for electrophysiological studies of transport proteins in native intracellular compartments and plasma membranes.

Publication link
2010 – Electrophysiology of respiratory chain complexes and the ADP-ATP exchanger in native mitochondrial membranes
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in Biochemistry (2010) Authors: Watzke N., Diekert K., Obrdlik P.

Transport of protons and solutes across mitochondrial membranes is essential for many physiological processes. However, neither the proton-pumping respiratory chain complexes nor the mitochondrial secondary active solute transport proteins have been characterized electrophysiologically in their native environment. In this study, solid-supported membrane (SSM) technology was applied for electrical measurements of respiratory chain complexes CI, CII, CIII, and CIV, the F(O)F(1)-ATPase/synthase (CV), and the adenine nucleotide translocase (ANT) in inner membranes of pig heart mitochondria. Specific substrates and inhibitors were used to validate the different assays, and the corresponding K(0.5) and IC(50) values were in good agreement with previously published results obtained with other methods. In combined measurements of CI-CV, it was possible to detect oxidative phosphorylation (OXPHOS), to measure differential effects of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) on the respective protein activities, and to determine the corresponding IC(50) values. Moreover, the measurements revealed a tight functional coupling of CI and CIII. Coenzyme Q (CoQ) analogues decylubiquinone (DBQ) and idebenone (Ide) stimulated the CII- and CIII-specific electrical currents but had inverse effects on CI-CIII activity. In summary, the results describe the electrophysiological and pharmacological properties of respiratory chain complexes, OXPHOS, and ANT in native mitochondrial membranes and demonstrate that SSM-based electrophysiology provides new insights into a complex molecular mechanism of the respiratory chain and the associated transport proteins. Besides, the SSM-based approach is suited for highly sensitive and specific testing of diverse respiratory chain modulators such as inhibitors, CoQ analogues, and uncoupling agents.

Publication link
2010 – ATP dependent charge movement in ATP7B Cu+-ATPase is demonstrated by pre-steady state electrical measurements
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in FEBS Letters (2010) Authors: Tadini-Buoninsegni F., Bartolommei G., Moncelli M.R., Pilankatta R., Lewis D., Inesi G.

ATP7B is a copper dependent P-type ATPase, required for copper homeostasis. Taking advantage of high yield heterologous expression of recombinant protein, we investigated charge transfer in ATP7B. We detected charge displacement within a single catalytic cycle upon ATP addition and formation of phosphoenzyme intermediate. We attribute this charge displacement to movement of bound copper within ATP7B. Based on specific mutations, we demonstrate that enzyme activation by copper requires occupancy of a site in the N-terminus extension which is not present in other transport ATPases, as well as of a transmembrane site corresponding to the cation binding site of other ATPases.

Publication link
2010 – Delineating electrogenic reactions during lactose/H+ symport
SURFE2R-technology (custom-built system) Publication in Biochemistry (2010) Authors: Garcia-Celma J.J., Ploch J., Smirnova I., Kaback H.R., Fendler K.

Electrogenic reactions accompanying downhill lactose/H+ symport catalyzed by the lactose permease of Escherichia coli (LacY) have been assessed using solid-supported membrane-based electrophysiology with improved time resolution. Rates of charge translocation generated by purified LacY reconstituted into proteoliposomes were analyzed over a pH range from 5.2 to 8.5, which allows characterization of two electrogenic steps in the transport mechanism: (i) a weak electrogenic reaction triggered by sugar binding and observed under conditions where H+ translocation is abolished either by acidic pH or by a Glu325 → Ala mutation in the H+ binding site (this step with a rate constant of ∼200 s−1 for wild-type LacY leads to an intermediate proposed to represent an “occluded” state) and (ii) a major electrogenic reaction corresponding to 94% of the total charge translocated at pH 8, which is pH-dependent with a maximum rate of ∼30 s−1 and a pK of 7.5. This partial reaction is assigned to rate-limiting H+ release on the cytoplasmic side of LacY during turnover. These findings together with previous electrophysiological results and biochemical−biophysical studies are included in an overall kinetic mechanism that allows delineation of the electrogenic steps in the reaction pathway.

Publication link
2009 – Inhibitory effect of Pb2+ on the transport cycle of the Na+/K+-ATPase
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in Chemical Research in Toxicology (2009) Authors: Gramigni E., Tadini-Buoninsegni F., Bartolommei G., Santini G., Chelazzi G., Moncelli M.R.

The effect of Pb2+ on the transport cycle of the Na+/K+-ATPase was characterized in detail at a molecular level by combining electrical and biochemical measurements. Electrical measurements were performed by adsorbing purified membrane fragments containing Na+/K+-ATPase on a solid-supported membrane. Upon adsorption, the Na+/K+-ATPase was activated by carrying out concentration jumps of different activating substrates, for example, Na+ and ATP. Charge movements following Na+/K+-ATPase activation were measured in the presence of various Pb2+ concentrations to investigate the effect of Pb2+ on different ion translocating steps of the pump cycle. These charge measurements were then compared to biochemical measurements of ATPase activity in the presence of increasing Pb2+ concentration. Our results indicate that Pb2+ inhibits cycling of the enzyme, but it does not affect cytoplasmic Na+ binding and release of Na+ ions at the extracellular side at concentrations below 10 μM. To explain the inhibitory effect of Pb2+ on the Na+/K+-ATPase, we propose that Pb2+ may interfere with the hydrolytic cleavage of the phosphorylated intermediate E2P, which occurs in the K+-related branch of the pump cycle.

Publication link
2009 – Measuring Ion Channels on Solid Supported Membranes
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in Biophysical Journal (2009) Authors: Schulz P., Dueck B., Mourot A., Hatahet L., Fendler K.

Application of solid supported membranes (SSMs) for the functional investigation of ion channels is presented. SSM-based electrophysiology, which has been introduced previously for the investigation of active transport systems, is expanded for the analysis of ion channels. Membranes or liposomes containing ion channels are adsorbed to an SSM and a concentration gradient of a permeant ion is applied. Transient currents representing ion channel transport activity are recorded via capacitive coupling. We demonstrate the application of the technique to liposomes reconstituted with the peptide cation channel gramicidin, vesicles from native tissue containing the nicotinic acetylcholine receptor, and membranes from a recombinant cell line expressing the ionotropic P2X2 receptor. It is shown that stable ion gradients, both inside as well as outside directed, can be applied and currents are recorded with an excellent signal/noise ratio. For the nicotinic acetylcholine receptor and the P2X2 receptor excellent assay quality factors of Z′ = 0.55 and Z′ = 0.67, respectively, are obtained. This technique opens up new possibilities in cases where conventional electrophysiology fails like the functional characterization of ion channels from intracellular compartments. It also allows for robust fully automatic assays for drug screening.

Publication link
2009 – Electrogenic ion pumps investigated on a solid supported membrane: comparison of current and voltage measurements
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in Langmuir (2009) Authors: Bartolommei G., Moncelli M.R., Rispoli G., Kelety B., Tadini-Buoninsegni F.

Current and voltage measurements were performed on Na,K-ATPase and sarcoplasmic reticulum (SR) Ca-ATPase. Measurements of current transients under short-circuit conditions and of voltage transients under open-circuit conditions were carried out by employing a solid supported membrane (SSM). Purified membrane fragments containing Na,K-ATPase or native SR vesicles were adsorbed on a SSM and were activated by performing substrate concentration jumps. Current and voltage transients were recorded in the external circuit. They are related to pump activity and can be attributed to electrogenic events in the reaction cycles of the two enzymes. While current transients of very small amplitude are difficult to detect, the corresponding voltage transients can be measured with higher accuracy because of a much more favorable signal-to-noise ratio. Therefore, voltage measurements are preferable for the investigation of slow processes generating low current signals, e.g., for the analysis of low turnover transporters.

Publication link
2009 – Electrophysiological characterization of LacY
SURFE2R-technology (custom-built system) Publication in PNAS (2009) Authors: Garcia-Celma J.J., Smirnova I.N., Kaback H.R., Fendler K.

Electrogenic events due to the activity of wild-type lactose permease from Escherichia coli (LacY) were investigated with proteoliposomes containing purified LacY adsorbed on a solid-supported membrane electrode. Downhill sugar/H+ symport into the proteoliposomes generates transient currents. Studies at different lipid-to-protein ratios and at different pH values, as well as inactivation by N-ethylmaleimide, show that the currents are due specifically to the activity of LacY. From analysis of the currents under different conditions and comparison with biochemical data, it is suggested that the predominant electrogenic event in downhill sugar/H+ symport is H+ release. In contrast, LacY mutants Glu-325→Ala and Cys-154→Gly, which bind ligand normally, but are severely defective with respect to lactose/H+ symport, exhibit only a small electrogenic event on addition of LacY-specific substrates, representing 6% of the total charge displacement of the wild-type. This activity is due either to substrate binding per se or to a conformational transition after substrate binding, and is not due to sugar/H+ symport. We propose that turnover of LacY involves at least 2 electrogenic reactions: (i) a minor electrogenic step that occurs on sugar binding and is due to a conformational transition in LacY; and (ii) a major electrogenic step probably due to cytoplasmic release of H+ during downhill sugar/H+ symport, which is the limiting step for this mode of transport.

Publication link
2009 – An automatic electrophysiological assay for the neuronal glutamate transporter mEAAC1
SURFE2R-technology (custom-built system) Publication in Journal of Neuroscience Methods (2009) Authors: Krause R., Watzke N., Kelety B., Dörner W., Fendler K.

A rapid and robust electrophysiological assay based on solid supported membranes (SSM) for the murine neuronal glutamate transporter mEAAC1 is presented. Measurements at different concentrations revealed the EAAC1 specific affinities for l-glutamate (Km = 24 μM), l-aspartate (Km = 5 μM) and Na+ (Km = 33 mM) and an inhibition constant Ki for dl-threo-β-benzyloxyaspartic acid (TBOA) of 1 μM. Inhibition by 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid (HIP-B) was not purely competitive with an IC50 of 13 μM. Experiments using SCN− concentration jumps yielded large transient currents in the presence of l-glutamate showing the characteristics of the glutamate-gated anion conductance of EAAC1. Thus, SSM-based electrophysiology allows the analysis of all relevant transport modes of the glutamate transporter on the same sample.K+ and Na+ gradients could be applied to the transporter. Experiments in the presence and absence of Na+ and K+ gradients demonstrated that the protein is still able to produce a charge translocation when no internal K+ is present. In this case, the signal amplitude is smaller and a lower apparent affinity for l-glutamate of 144 μM is found.Finally the assay was adapted to a commercial fully automatic system for SSM-based electrophysiology and was validated by determining the substrate affinities and inhibition constants as for the laboratory setup. The combination of automatic function and its ability to monitor all transport modes of EAAC1 make this system an universal tool for industrial drug discovery.

Publication link
2009 – DtpB (YhiP) and DtpA (TppB, YdgR) are prototypical proton-dependent peptide transporters of Escherichia coli
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in The FEBS Journal (2009) Authors: Harder D., Stolz J., Casagrande F., Obrdlik P., Weitz D., Fotiadis D., Daniel H.

The genome of Escherichia coli contains four genes assigned to the peptide transporter (PTR) family. Of these, only tppB (ydgR) has been characterized, and named tripeptide permease, whereas protein functions encoded by the yhiP, ybgH and yjdL genes have remained unknown. Here we describe the overexpression of yhiP as a His-tagged fusion protein in E. coli and show saturable transport of glycyl-sarcosine (Gly-Sar) with an apparent affinity constant of 6.5 mm. Overexpression of the gene also increased the susceptibility of cells to the toxic dipeptide alafosfalin. Transport was strongly decreased in the presence of a protonophore but unaffected by sodium depletion, suggesting H+-dependence. This was confirmed by purification of YhiP and TppB by nickel affinity chromatography and reconstitution into liposomes. Both transporters showed Gly-Sar influx in the presence of an artificial proton gradient and generated transport currents on a chip-based sensor. Competition experiments established that YhiP transported dipeptides and tripeptides. Western blot analysis revealed an apparent mass of YhiP of 40 kDa. Taken together, these findings show that yhiP encodes a protein that mediates proton-dependent electrogenic transport of dipeptides and tripeptides with similarities to mammalian PEPT1. On the basis of our results, we propose to rename YhiP as DtpB (dipeptide and tripeptide permease B), by analogy with the nomenclature in other bacteria. We also propose to rename TppB as DtpA, to better describe its function as the first protein of the PTR family characterized in E. coli.

Publication link
2008 – Identification of the arginine/ornithine antiporter ArcD from Halobacterium salinarum
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in FEBS Letters (2008) Authors: Wimmer F., Oberwinkler T., Bisle B., Tittor J., Oesterhelt D.

This paper identifies the first arginine/ornithine antiporter ArcD from the domain of archea. The functional role of ArcD is demonstrated by transport assays with radioactive labelled arginine, by its necessity to enable arginine fermentation under anaerobic growth conditions and by the consumption of arginine from the medium during growth. All three experimentally observables are severely disturbed when the deletion strain ΔArcD is used. The isolated protein is verified by mass spectrometry and reconstituted in vesicles. The proteoliposomes are attached to a membrane and capacitive currents are recorded which appear upon initiation of the transport process by change from arginine‐free to arginine‐containing buffer. This clearly demonstrates that the purified 34 kD protein is the functional unit.

Publication link
2008 – Rapid activation of the melibiose permease MelB immobilized on a solid-supported membrane
SURFE2R-technology (custom-built system) Publication in Langmuir (2008) Authors: Garcia-Celma J.J., Dueck B., Stein M., Schlueter M., Meyer-Lipp K., Leblanc G., Fendler K.

Rapid solution exchange on a solid-supported membrane (SSM) is investigated using fluidic structures and a solid-supported membrane of 1 mm diameter in wall jet geometry. The flow is analyzed with a new technique based on specific ion interactions with the surface combined with an electrical measurement. The critical parameters affecting the time course of the solution exchange and the transfer function describing the time resolution of the SSM system are determined. The experimental data indicate that solution transport represents an intermediate situation between the plug flow and the Hagen−Poiseuille laminar flow regime. However, to a good approximation the rise of the surface concentration can be described by Hagen−Poiseuille flow with ideal mixing at the surface of the SSM. Using an improved cuvette design, solution exchange as fast as 2 ms was achieved at the surface of a solid-supported membrane. As an application of the technique, the rate constant of a fast electrogenic reaction in the melibiose permease MelB, a bacterial (Escherichia coli) sugar transporter, is determined. For comparison, the kinetics of a conformational transition of the same transporter was measured using stopped-flow tryptophan fluorescence spectroscopy. The relaxation time constant obtained for the charge displacement agrees with that determined in the stopped-flow experiments. This demonstrates that upon sugar binding MelB undergoes an electrogenic conformational transition with a rate constant of k ≈ 250 s−1.

Publication link
2007 – Functional and structural characterization of a prokaryotic peptide transporter with features similar to mammalian PEPT1
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in Journal of Biological Chemistry (2007) Authors: Weitz D., Harder D., Casagrande F., Fotiadis D., Obrdlik P., Kelety B., Daniel H.

The ydgR gene of Escherichia coli encodes a protein of the proton-dependent oligopeptide transporter (POT) family. We cloned YdgR and overexpressed the His-tagged fusion protein in E. coli BL21 cells. Bacterial growth inhibition in the presence of the toxic phosphonopeptide alafosfalin established YgdR functionality. Transport was abolished in the presence of the proton ionophore carbonyl cyanide p-chlorophenylhydrazone, suggesting a proton-coupled transport mechanism. YdgR transports selectively only di- and tripeptides and structurally related peptidomimetics (such as aminocephalosporins) with a substrate recognition pattern almost identical to the mammalian peptide transporter PEPT1. The YdgR protein was purified to homogeneity from E. coli membranes. Blue native-polyacrylamide gel electrophoresis and transmission electron microscopy of detergent-solubilized YdgR suggest that it exists in monomeric form. Transmission electron microscopy revealed a crown-like structure with a diameter of approximately 8 nm and a central density. These are the first structural data obtained from a proton-dependent peptide transporter, and the YgdR protein seems an excellent model for studies on substrate and inhibitor interactions as well as on the molecular architecture of cell membrane peptide transporters.

Publication link
2008 – Effect of Clotrimazole on the Pump Cycle of the Na,K-ATPase
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in Biophysical Journal (2008) Authors: Bartolommei G., Devaux N., Tadini-Buoninsegni F., Moncelli M., Apell H.-J.

The effect of the antimycotic drug clotrimazole (CLT) on the Na,K-ATPase was investigated using fluorescence and electrical measurements. The results obtained by steady-state fluorescence experiments with the electrochromic styryl dye RH421 were combined with those achieved by a pre-steady-state method based on fast solution exchange on a solid supported membrane that adsorbs the protein. Both techniques are suitable for monitoring the electrogenic steps of the pump cycle and are in general complementary, yielding distinct kinetic information. The experiments show clearly that CLT affects specific partial reactions of the pump cycle of the Na,K-ATPase with an affinity in the low micromolar range and in a reversible manner. All results can be consistently explained by proposing the CLT-promoted formation of an ion-occluded-CLT-bound conformational E2 state E2CLT(X2), that acts as a “dead-end” side track of the pump cycle, where X stands for H+ or K+. Na+ binding, enzyme phosphorylation, and Na+ transport were not affected by CLT, and at high CLT concentrations ~1/3 of the enzyme remained active in the physiological transport mode. The presence of Na+ and K+ destabilized the inactivated form of the Na,K-ATPase.

Publication link
2006 – The inner interhelix loop 4-5 of the melibiose permease from Escherichia coli takes part in conformational changes after sugar binding
SURFE2R-technology (custom-built system) Publication in Journal of Biological Chemistry (2006) Authors: Meyer-Lipp K., Séry N., Ganea C., Basquin C., Fendler K., Leblanc G.

Cytoplasmic loop 4-5 of the melibiose permease from Escherichia coli is essential for the process of Na+-sugar translocation (Abdel-Dayem, M., Basquin, C., Pourcher, T., Cordat, E., and Leblanc, G. (2003) J. Biol. Chem. 278, 1518-1524). In the present report, we analyze functional consequences of mutating each of the three acidic amino acids in this loop into cysteines. Among the mutants, only the E142C substitution impairs selectively Na+-sugar translocation. Because R141C has a similar defect, we investigated these two mutants in more detail. Liposomes containing purified mutated melibiose permease were adsorbed onto a solid supported lipid membrane, and transient electrical currents resulting from different substrate concentration jumps were recorded. The currents evoked by a melibiose concentration jump in the presence of Na+, previously assigned to an electrogenic conformational transition (Meyer-Lipp, K., Ganea, C., Pourcher, T., Leblanc, G., and Fendler, K. (2004) Biochemistry 43, 12606-12613), were much smaller for the two mutants than the corresponding signals in cysteineless MelB. Furthermore, in R141C the stimulating effect of melibiose on Na+ affinity was lost. Finally, whereas tryptophan fluorescence spectroscopy revealed impaired conformational changes upon melibiose binding in the mutants, fluorescence resonance energy transfer measurements indicated that the mutants still show cooperative modification of their sugar binding sites by Na+. These data suggest that: 1) loop 4-5 contributes to the coordinated interactions between the ion and sugar binding sites; 2) it participates in an electrogenic conformational transition after melibiose binding that is essential for the subsequent obligatory coupled translocation of substrates. A two-step mechanism for substrate translocation in the melibiose permease is suggested.

Publication link
2006 – Transporter assays using solid supported membranes: a novel screening platform for drug discovery
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in ASSAY and Drug Development Technologies (2006) Authors: Kelety B., Diekert K., Tobien J., Watzke N., Dörner W., Obrdlik P., Fendler K.

Transporters are important targets in drug discovery. However, high throughput-capable assays for this class of membrane proteins are still missing. Here we present a novel drug discovery platform technology based on solid supported membranes. The functional principles of the technology are described, and a sample selection of transporter assays is discussed: the H+-dependent peptide transporter PepT1, the gastric proton pump, and the Na+/Ca2+ exchanger. This technology promises to have an important impact on the drug discovery process.

Publication link
2006 – Establishment of Cell-Free Electrophysiology for Ion Transporters: Application for Pharmacological Profiling
SURFE2R ONE (a predecessor model of SURFE2R N1) Publication in Journal of Biomolecular Screening (2006) Authors: Geibel S., Flores-Herr N., Licher T., Vollert H.

Ion transporters are emerging targets of increasing importance to the pharmaceutical industry because of their relevance to a wide range of numerous indications of cardiovascular, metabolic, and inflammatory diseases. However, traditional iontransporter assay technologies using radioactive or fluorescent ligands and substrates or manual patch clamping suffer from several problems: limited sensitivity and robustness, significant numbers of false positives and false negatives, and cost. The authors describe a novel method for the measurement of ion transporters using cell-free electrophysiology based on the SURFE2R (surface electrogenic event reader) technology platform. The main advantages of the method described here are high sensitivity and simple handling. Material for assays is mainly a simplemembrane preparation, which can be stored over weeks and months. Thus, the application of the method does not depend on a permanently running cell-culture lab. The application of the technology itself uses a bench-top system and chips loaded with membrane fragments. The SURFE2R technology was used to establish an Na+/Ca2+-exchanger assay. The assay performance, as judged by the Z' value of 0.73 and the signal-to-background ratio of 7.6, suggests that this is a reliable and robust assay. The authors compared the technology with patch-clamp experiments: Themeasurement of activity of 17 different inhibitors and the determination of an IC 50value indicated a good correlation between SURFE2R technology and patch clamp results. Using the SURFE2R technology, results were obtainedwith 20 times higher throughput and required less-qualified personnel compared with manual patch clamping.

Publication link
2006 – Structure and function of prokaryotic glutamate transporters from Escherichia coli and Pyrococcus horikoshii
SURFE2R-technology (custom-built system) Publication in Biochemistry (2006) Authors: Raunser S., Appel M., Ganea C., Geldmacher-Kaufer U., Fendler K., Kühlbrandt W.

The glutamate transporters GltPEc from Escherichia coli and GltPPh from Pyrococcus horikoshii were overexpressed in E. coli and purified to homogeneity with a yield of 1-2 mg/L of culture. Single-particle analysis and electron microscopy indicate that GltP(Ph) is a trimer in detergent solution. Electron microscopy of negatively stained GltPPh two-dimensional crystals shows that the transporter is a trimer also in the membrane. Gel filtration of GltPEc indicates a reversible equilibrium of two oligomeric states in detergent solution that we identified as a trimer and hexamer by blue-native gel electrophoresis and cross-linking. The purified transporters were fully active upon reconstitution into liposomes, as demonstrated by the uptake of radioactively labeled L-aspartate or L-glutamate. L-aspartate/L-glutamate transport of GltPEc involves the cotransport of protons and depends only on pH, whereas GltP(Ph) catalyzes L-glutamate transport with a cotransport of H+ or Na+. L-glutamate induces a fast transient current in GltP(Ph) proteoliposomes coupled to a solid supported membrane (SSM). We show that the electric signal depends on the concentration of Na+ or H+ outside the proteoliposomes and that GltP(Ph) does not require K+ inside the proteoliposomes. In addition, the electrical currents are inhibited by TBOA and HIP-B. The half-saturation concentration for activation of GltPPh glutamate transport (K0.5glut) is 194 µM.

Publication link
2004 – Time-resolved charge translocation by sarcoplasmic reticulum Ca-ATPase measured on a solid supported membrane
SURFE2R-technology (custom-built system) Publication in Biophysical Journal (2004) Authors: Tadini Buoninsegni F., Bartolommei G., Moncelli M.R., Inesi G., Guidelli R.

Sarcoplasmic reticulum vesicles were adsorbed on an octadecanethiol/phosphatidylcholine mixed bilayer anchored to a gold electrode, and the Ca-ATPase contained in the vesicles was activated by ATP concentration jumps both in the absence and in the presence of K(+) ions and at different pH values. Ca2+ concentration jumps in the absence of ATP were also carried out. The resulting capacitive current transients were analyzed together with the charge under the transients. The relaxation time constants of the current transients were interpreted on the basis of an equivalent circuit. The current transient after ATP concentration jumps and the charge after Ca2+ concentration jumps in the absence of ATP exhibit almost the same dependence upon the Ca2+ concentration, with a half-saturating value of approximately 1.5 µM. The pH dependence of the charge after Ca2+ translocation demonstrates the occurrence of one H+ per one Ca2+ countertransport at pH 7 by direct charge-transfer measurements. The presence of K+ decreases the magnitude of the current transients without altering their shape; this decrease is explained by K+ binding to the cytoplasmic side of the pump in the E1 conformation and being released to the same side during the E1-E2 transition.

Publication link
2005 – Kinetics of charge translocation in the passive downhill uptake mode of the Na+/H+ antiporter NhaA of Escherichia coli
SURFE2R 500 (a predecessor model of SURFE2R N1) Publication in Biochimica et Biophysica Acta (BBA) - Bioenergetics (2005) Authors: Zuber D., Krause R., Venturi M., Padan E., Bamberg E., Fendler K.

The Na+/H+ antiporter NhaA is the main Na+ extrusion system in E. coli. Using direct current measurements combined with a solid supported membrane (SSM), we obtained electrical data of the function of NhaA purified and reconstituted in liposomes. These measurements demonstrate NhaA's electrogenicity, its specificity for Li+ and Na+ and its pronounced pH dependence in the range pH 6.5-8.5. The mutant G338S, in contrast, presents a pH independent profile, as reported previously. A complete right-side-out orientation of the NhaA antiporter within the proteoliposomal membrane was determined using a NhaA-specific antibody based ELISA assay. This allowed for the first time the investigation of NhaA in the passive downhill uptake mode corresponding to the transport of Na+ from the periplasmic to the cytoplasmic side of the membrane. In this mode, the transporter has kinetic properties differing significantly from those of the previously investigated efflux mode. The apparent Km values were 11 mM for Na+ and 7.3 mM for Li+ at basic pH and 180 mM for Na+ and 50 mM for Li+ at neutral pH. The data demonstrate that in the passive downhill uptake mode pH regulation of the carrier affects both apparent Km as well as turnover (Vmax).

Publication link
2004 – Calcium transport by sarcoplasmic reticulum Ca-ATPase can be investigated on a solid-supported membrane
SURFE2R-technology (custom-built system) Publication in Bioelectrochemistry (2004) Authors: Bartolommei G., Buoninsegni F.T., Moncelli M.R.

Sarcoplasmic reticulum (SR) native vesicles incorporating Ca-ATPase are adsorbed on a solid-supported lipid membrane (SSM). Upon adsorption, the ion pumps are chemically activated by concentration jumps of ATP and the capacitive current transients generated by SR Ca-ATPase are measured under potentiostatic conditions. The Michaelis-Menten constant, KM, for ATP is evaluated by varying the concentration of ATP in the activating solution. This preliminary result shows that ion transport by SR Ca-ATPase can be suitably investigated by a technique based on concentration jumps on an SSM.

Publication link
2004 – Charge translocation during cosubstrate binding in the Na+/proline transporter of E.coli
SURFE2R-technology (custom-built system) Publication in Journal of Molecular Biology (2004) Authors: Zhou A., Wozniak A., Meyer-Lipp K., Nietschke M., Jung H., Fendler K.

Charge translocation associated with the activity of the Na+/proline cotransporter PutP of Escherichia coli was analyzed for the first time. Using a rapid solution exchange technique combined with a solid-supported membrane (SSM), it was demonstrated that Na+ and/or proline individually or together induce a displacement of charge. This was assigned to an electrogenic Na+ and/or proline binding process at the cytoplasmic face of the enzyme with a rate constant of k>50 s−1 which preceeds the rate-limiting step. Based on the kinetic analysis of our electrical signals, the following characteristics are proposed for substrate binding in PutP. (1) Substrate binding is electrogenic not only for Na+, but also for the uncharged cosubstrate proline. The charge displacement associated with the binding of both substrates is of comparable size and independent of the presence of the respective cosubstrate. (2) Both substrates can bind individually to the transporter. Under physiological conditions, an ordered binding mechanism prevails, while at sufficiently high concentrations, each substrate can bind in the absence of the other. (3) Both substrate binding sites interact cooperatively with each other by increasing the affinity and/or the speed of binding of the respective cosubstrate. (4) Proline binding proceeds in a two-step process: low affinity (∼1 mM) electroneutral substrate binding followed by a nearly irreversible electrogenic conformational transition.

Publication link
2002 – Photocurrents Generated by Bacteriorhodopsin Adsorbed on Thiol/Lipid Bilayers Supported by Mercury
SURFE2R-technology (custom-built system) Publication in Langmuir (2002) Authors: Dolfi A., Tadini-Buoninsegni F., Moncelli M.R., Guidelli R.

The kinetics of light-driven proton transport by bacteriorhodopsin (bR) were investigated over a broad pH range upon adsorbing purple membrane (PM) fragments on a mercury-supported mixed alkanethiol/phospholipid bilayer. The light-on and light-off capacitive photocurrents were measured under short-circuit conditions in the absence of photoartifacts. Using dioleoylphosphatidylcholine as the lipid monolayer, a bell-shaped curve of the peak current versus pH, with a maximum in the proximity of 6, was obtained. The analysis of the biphasic decay kinetics of the light-on and light-off currents allows an estimate of the pKa values for the steps releasing protons to, and taking up protons from, the bathing solution. In particular, the pKa values obtained from the light-off current (pK1 = 3.5, pK2 = 5.3, pK3 = 7.5, and pK4 = 9.0) suggest a mechanism similar to that proposed by Balashov et al. for dark adaptation, albeit in the opposite direction (Balashov, S. P.; Imasheva, E. S.; Govindjee, R.; Sheves, M.; Ebrey, T. G. Biophys. J. 1996, 70, 473). The time dependence of the light-on and light-off currents in the proximity of pH 6 is interpreted on the basis of both a simple equivalent circuit and a kinetic model making use of spectroscopic data available in the literature. When using dioleoylphosphatidylserine (DOPS) as the lipid monolayer, an inversion in the sign of both light-on and light-off currents, as well as a change in their shape and magnitude, was observed by increasing the pH above 9 and then, at all pH values from 9 to 1, by subsequently decreasing the pH on the same mercury-supported mixed alkanethiol/DOPS bilayer. The normal situation was restored only by adding sodium azide. This inversion in current and the notable hysteresis observed under these conditions are critically discussed.

Publication link
2003 – Charge displacements during ATP-hydrolysis and synthesis of the Na+-transporting FoF1-ATPase of Ilyobacter tartaricus
SURFE2R-technology (custom-built system) Publication in Biophysical Journal (2003) Authors: Burzik C., Kaim G., Dimroth P., Bamberg E., Fendler K.

Transient electrical currents generated by the Na+-transporting FoF1-ATPase of Ilyobacter tartaricus were observed in the hydrolytic and synthetic mode of the enzyme. Two techniques were applied: a photochemical ATP concentration jump on a planar lipid membrane and a rapid solution exchange on a solid supported membrane. We have identified an electrogenic reaction in the reaction cycle of the FoF1-ATPase that is related to the translocation of the cation through the membrane bound Fo subcomplex of the ATPase. In addition, we have determined rate constants for the process: For ATP hydrolysis this reaction has a rate constant of 15–30 s−1 if H+ is transported and 30–60 s−1 if Na+ is transported. For ATP synthesis the rate constant is 50–70 s−1.

Publication link
1999 – Kinetics of electrogenic transport by the ADP/ATP carrier
SURFE2R-technology (custom-built system) Publication in Biophysical Journal (1999) Authors: Gropp T., Brustovetsky N., Klingenberg M., Müller V., Fendler K., Bamberg E.

The electrogenic transport of ATP and ADP by the mitochondrial ADP/ATP carrier (AAC) was investigated by recording transient currents with two different techniques for performing concentration jump experiments: 1) the fast fluid injection method: AAC-containing proteoliposomes were adsorbed to a solid supported membrane (SSM), and the carrier was activated via ATP or ADP concentration jumps. 2) BLM (black lipid membrane) technique: proteoliposomes were adsorbed to a planar lipid bilayer, while the carrier was activated via the photolysis of caged ATP or caged ADP with a UV laser pulse. Two transport modes of the AAC were investigated, ATPex-0in and ADPex-0in. Liposomes not loaded with nucleotides allowed half-cycles of the ADP/ATP exchange to be studied. Under these conditions the AAC transports ADP and ATP electrogenically. Mg2+ inhibits the nucleotide transport, and the specific inhibitors carboxyatractylate (CAT) and bongkrekate (BKA) prevent the binding of the substrate. The evaluation of the transient currents yielded rate constants of 160 s−1 for ATP and ≥400 s−1 for ADP translocation. The function of the carrier is approximately symmetrical, i.e., the kinetic properties are similar in the inside-out and right-side-out orientations. The assumption from previous investigations, that the deprotonated nucleotides are exclusively transported by the AAC, is supported by further experimental evidence. In addition, caged ATP and caged ADP bind to the carrier with similar affinities as the free nucleotides. An inhibitory effect of anions (200–300 mM) was observed, which can be explained as a competitive effect at the binding site. The results are summarized in a transport model.

Publication link
2001 – Evidence for intraprotein charge transfer during the transport activity of the melibiose permease from Escherichia coli
SURFE2R-technology (custom-built system) Publication in Biochemistry (2001) Authors: Ganea C., Pourcher T., Leblanc G., Fendler K.

Electrogenic activity associated with the activity of the melibiose permease (MelB) of Escherichia coli was investigated by using proteoliposomes containing purified MelB adsorbed onto a solid-supported membrane. Transient currents were selectively recorded by applying concentration jumps of Na+ ions (or Li+) and/or of different sugar substrates of MelB (melibiose, thio-methyl galactoside, raffinose) using a fast-flow solution exchange system. Characteristically, the transient current response was fast, including a single decay exponential component (τ ≈ 15 ms) on applying a Na+ (or Li+) concentration jump in the absence of sugar. On imposing a Na+ (or Li+) jump on proteoliposomes preincubated with the sugar, a sugar jump in a preparation preincubated with the cation, or a simultaneous jump of the cation and sugar substrates, the electrical transients were biphasic and comprised both the fast and an additional slow (τ ≈ 350 ms) decay components. Finally, selective inactivation of the cosubstrate translocation step by acylation of MelB cysteins with N-ethyl maleimide suppressed the slow response components and had no effect on the fast transient one. We suggest that the fast transient response reflects charge transfer within MelB during cosubstrate binding while the slow component is associated with charge transfer across the proteoliposome membrane. From the time course of the transient currents, we estimate a rate constant for Na+ binding in the absence and presence of melibiose of k > 50 s-1 and one for melibiose binding in the absence of Na+ of k ≈ 10 s-1.

Publication link
1999 – Charge Translocation by the Na/K-ATPase Investigated on Solid Supported Membranes: Rapid Solution Exchange with a New Technique
SURFE2R-technology (custom-built system) Publication in Biophysical Journal (1999) Authors: Pintschovius J., Fendler K.

Adsorption of Na+/K+-ATPase containing membrane fragments from pig kidney to lipid membranes allows the detection of electrogenic events during the Na+/K+-ATPase reaction cycle with high sensitivity and time resolution. High stability preparations can be obtained using solid supported membranes (SSM) as carrier electrodes for the membrane fragments. The SSMs are prepared using an alkanethiol monolayer covalently linked to a gold surface on a glass substrate. The hydrophobic surface is covered with a lipid monolayer (SAM, self-assembled monolayer) to obtain a double layer system having electrical properties similar to those of unsupported bilayer membranes (BLM). As we have previously shown (, Biophys. J. 64:384-391), the Na+/K+-ATPase on a SSM can be activated by photolytic release of ATP from caged ATP. In this publication we show the first results of a new technique which allows rapid solution exchange at the membrane surface making use of the high mechanical stability of SSM preparations. Especially for substrates, which are not available as a caged substance-such as Na+ and K+-this technique is shown to be capable of yielding new results. The Na+/K+-ATPase was activated by rapid concentration jumps of ATP and Na+ (in the presence of ATP). A time resolution of up to 10 ms was obtained in these experiments. The aim of this paper is to present the new technique together with the first results obtained from the investigation of the Na+/K+-ATPase. A comparison with data taken from the literature shows considerable agreement with our experiments.

Publication link
1999 – Charge Translocation by the Na+/K+-ATPase Investigated on Solid Supported Membranes: Cytoplasmic Cation Binding and Release
SURFE2R-technology (custom-built system) Publication in Biophysical Journal (1999) Authors: Pintschovius J., Fendler K., Bamberg E.

In the preceding publication (Pintschovius and Fendler, 1999. Biophys. J. 76:000–000) a new technique was described that was able to produce concentration jumps of arbitrary ion species at the surface of a solid supported membrane (SSM). This technique can be used to investigate the kinetics of ion translocating proteins adsorbed to the SSM. Charge translocation of the Na+/K+-ATPase in the presence of ATP was investigated. Here we describe experiments carried out with membrane fragments containing Na+/K+-ATPase from pig kidney and in the absence of ATP. Electrical currents are measured after rapid addition of Na+. We demonstrate that these currents can be explained only by a cation binding process on the cytoplasmic side, most probably to the cytoplasmic cation binding site of the Na+/K+-ATPase. An electrogenic reaction of the protein was observed only with Na+, but not with other monovalent cations (K+, Li+, Rb+, Cs+). Using Na+ activation of the enzyme after preincubation with K+ we also investigated the K+-dependent half-cycle of the Na+/K+-ATPase. A rate constant for K+ translocation in the absence of ATP of 0.2–0.3 s−1 was determined. In addition, these experiments show that K+ deocclusion, and cytoplasmic K+ release are electroneutral.

Publication link
1993 – Charge transport by ion translocating membrane proteins on solid supported membranes
SURFE2R-technology (custom-built system) Publication in Biophysical Journal (1993) Authors: Seifert K., Fendler K., Bamberg E.

A new method for the investigation of ion translocating membrane proteins is presented. Protein containing membrane fragments or vesicles are adsorbed to a solid supported membrane. The solid supported membrane consists of a lipid monolayer on a gold evaporated or gold sputtered glass substrate which is coated with a long chained mercaptan (CH3(CH2)mSH, m = 15, 17). Specific conductance and specific capacitance of the solid supported membrane are comparable to those of a black lipid membrane. However, the solid supported membrane has the advantage of a much higher mechanical stability. The electrical activity of bacteriorhodopsin, Na,K-ATPase, H,K-ATPase, and Ca-ATPase on the solid supported membrane is measured and compared to signals obtained on a conventionally prepared black lipid membrane. It is shown that both methods yield similar results. The solid supported membrane therefore represents an alternative method for the investigation of electrical properties of ion translocating transmembrane proteins.

Presentation PDF
2020 – Unlocking the (Reversal) Potential of SSM Electrophysiology: Transporter Stoichiometry with the SURFE2R N1
Presenter: Nathan Thomas 5th Year PhD student at University of Wisconsin-Madison, USA (Dr. Henzler-Wildman's lab) Source:
The 64th Annual Meeting of the Biophysical Society, San Diego, CA (USA), Feb 15-19. 2020
Webinar
19.05.2020 | Webinar: Insights and New Approaches in Transporter Characterization – SURFE2R N1
Speakers:
Dr. Maria Barthmes (Product Manager, Nanion Technologies)
Nathan Thomas (5th Year PhD, University of Wisconsin-Madison [Dr. Henzler-Wildman's lab])
Prof. Dr. Camilo Perez (Head of Research Group - BIOZENTRUM, University of Basel, Switzerland)

Maria will introduce SSM-based Electrophysiology going over basic features and principles of the method and have a look at experimental workflows.

Nathan Thomas
"Unlocking the (Reversal) Potential of SSM Electrophysiology: Transporter Stoichiometry with the SURFE2R N1”

Camillo Perez:
"Characterization of a choline uniporter by SSM-based electrophysiology" disclaimer: due to a pending manuscript submission, this presentation will be made available in full in the coming weeks. Please stay tuned.

Webinar
28.06.2022 | Webinar: Structures and mechanism of the plant PIN-FORMED auxin transporter
Speakers:
Associate Professor Bjørn Panyella Pedersen
(Aarhus University, Denmark, Dept of Molecular Biology and Genetics)
Associate Professor Ulrich Hammes
(Technical University of Munich, Germany, Plant Systems Biology)

Auxins are central hormones in all plants and controls virtually all aspects of growth and development. The PIN-FORMED (PIN) protein family is a key player in this process. Here we present biophysical analysis and structures of Arabidopsis thaliana PIN8 at 2.9-3.4 Å resolution; two outward facing conformations with and without auxin bound, and one inward facing conformation with the known inhibitor and herbicide naphthylphthalamic acid (NPA) bound. The structure forms a homo-dimer with each monomer divided into a transport and scaffold domain with a clearly defined auxin binding site. Next to the binding site, a proline-proline crossover is a pivot point for structural changes associated with transport, which we show by biophysical analysis to be independent of proton and ion gradients and likely driven by the negative charge of the auxin. Our results provide the first comprehensive molecular model for auxin recognition and transport by PINs.

Webinar
16.06.2020 | Webinar: Functional studies on membrane transporters using the SURFE2R N1
Speakers:

Dr. Maria Barthmes (Product Manager, Nanion Technologies)
Dr. Andre Bazzone (Application Scientist, Nanion Technologies)

SSM (solid supported membrane)-based electrophysiology employed by the SURFE2R instruments is a capacitive sensor-based method to detect membrane currents generated by low turnover proteins such as transporters and membrane pumps. By resolving protein activity in real time and label free it introduces the advantages of electrophysiology to the field of membrane transporters. During this webinar you will learn all about SSM-based electrophysiology and its applications.

First, we discuss the basics and principles behind this technology in depth. Then we focus on practical topics, like preparation of samples and experimental workflows, and finally introduce some datasets to highlight the potential and possibilities for membrane transporter studies.aria will introduce SSM-based Electrophysiology going over basic features and principles of the method and have a look at experimental workflows.

Webinar
18.03.2021 | Webinar: Understanding Drug Efflux – Small Multidrug Resistance Family – Stoichiometry and Specificity
Speakers:
Randy Stockbridge
(Assistant Professor, Department of Molecular, Cellular, and Developmental Biology - University of Michigan)
Katherine Henzler-Wildman
(Professor Biochemistry, Department of Biochemistry - University of Wisconsin-Madison)

Title: Determining transport stoichiometry using SSME

Randy Stockbridge
(Assistant Professor, Department of Molecular, Cellular, and Developmental Biology - University of Michigan)

Abstract: Transporters from the small multidrug resistance (SMR) family provide broad resistance to environmental biocides, driving the spread of multidrug resistance cassettes among bacterial populations. Understanding substrate specificity is essential to understand this process. Using solid-supported membrane electrophysiology, we measure the transport of different substrates by SMR family members, and show that promiscuous transport of hydrophobic substituted cations is a general feature of all SMR transporters, including those whose primary physiological role is in bacterial nitrogen metabolism.

Title: Basis of promiscuity in small multidrug resistance transporters

Katherine Henzler-Wildman
(Professor Biochemistry, Department of Biochemistry - University of Wisconsin-Madison)

Abstract: Transport stoichiometry can provide great insight into the mechanism and function of ion-coupled transporters. Traditional reversal potential assays are a reliable, general method for determining the transport stoichiometry of ion-coupled transporters, but the time and material costs of this technique hinder investigations of transporter behavior under multiple experimental conditions. Our prior work on EmrE has demonstrated that it is not a tightly coupled transporter and that the net transport stoichiometry is likely to vary with pH and substrate identity. This has motivated us to develop an SSME-based assay for assessing transport stoichiometry that is rapid and easily adaptable to different substrates and pH conditions. Here we present results for Gdx and CLC-Ec1, two well-characterized transporters that demonstrate the success of our approach. Our SSME-based method reproduces the fixed 2H+:1 guanidinium+ antiport stoichiometry of Gdx, the 1H+:2Cl- antiport stoichiometry of CLC-ec1, and loose proton:nitrate coupling for CLC-ec1. This method requires only small amounts of transporter and provides a fast, easy method to characterize transport stoichiometry under varied conditions, which will facilitate future mechanistic and functional studies of ion-coupled transporters.

Webinar
06.05.2021 | Webinar: Secondary transport inhibition and regulation by SSM-electrophysiology
Professor Dr. Christine Ziegler
(University of Regensburg, Faculty of Biology and Pre-Clinics, Institute of Biophysics and physical Biochemistry, Structural Biology-Biophysics II)

Professor Dr. Camilo Perez
(University of Basel, Biozentrum, Center for Molecular Life Sciences)

Title: The K+ switch in BetP: from coupling to regulation

Professor Dr. Christine Ziegler
(University of Regensburg, Faculty of Biology and Pre-Clinics, Institute of Biophysics and physical Biochemistry, Structural Biology-Biophysics II)

Abstract: The bacterial betaine transporter BetP is a prime example for an efficient osmotic stress sensor and regulated secondary transporter, respectively. BetP’s full activation depends on the presence of 300mM internal K+, however, K+ is not transported. Several K+ binding sites were identified at the osmo-sensor, but also close to a Na+ site. We introduced a point mutation based in one of the two sodium binding sites in order to switch BetP from Na+ to K+ coupling. Using cryoEM/X-ray crystallography combined with SSM/Stopped-Flow Trp-fluorescence we discovered an intriguing competition between Na+ and K+ binding in BetP, which hints towards a change in the functional role of K+ in LeuT-fold transporter during evolution. BetP shares its overall fold with SLC6 neurotransmitter transporters, which also show differences in their ability to facilitate K+-coupled antiport. Therefore, our structure-function study provides new insights into an evolutionary switching of K+ between coupling to regulatory ion.

Title: Selection of transporter-targeted inhibitory nanobodies by SSM-based electrophysiology

Professor Dr. Camilo Perez
(University of Basel, Biozentrum, Center for Molecular Life Sciences)

Abstract: Single domain antibodies (nanobodies) have been extensively used in machanistic and structural studies of protein and pose an enormous potential as tools for developing clinical therapies, many of which depend on inhibition of membrane proteins such as transporters.However, most of the methods used to determine inhibition of transport activity are difficult to perform in high-throughput routines and depend on labeled substrates availability. This complicates the screening of large nanobody libraries. Solid-supported membrane (SSM) electrophysiology to select inhibitory and non-inhibitory nanobodies targeting an electrogenic secondary transporter and to calculate nanobodies inhibitory constants. This technique may be especially useful for selecting inhibitory nanobodies targeting transporters for which labeled substrates are not available.

Webinar
13.10.2020 | Webinar: Employing SSM electrophysiology to capture electrogenic fluxes in membrane proteins
Speakers:
Dr. Matthias Quick (Columbia University; NY, USA)
This is an on-demand webinar from Nan]i[on and Friends 2020.

My lab has been focusing on the study of ion-dependent transporters with special emphasis on Na+ or H+-coupled symporters. Whereas flux studies with radiolabeled solutes using the target protein reconstituted in proteoliposomes provided a wealth of information, the determination of the thermodynamically-coupled solute transport-associated flux of H+ or Na+ has been challenging. This can be attributed in part to the low transport turnover numbers of these transporters and difficulties associated with their functional expression in suitable model systems that allow for their characterization with traditional electrophysiological methods (e.g., two-electrode voltage clamp or patch-clamp methods).

By using the SURFE2R N1 SSM platform, our team was able to quickly collect data of solute transport-associated flux of co-transported ions across the membrane of proteoliposomes containing different target proteins. With this technology it is possible to collect data for a full kinetic characterization of a target protein such as its dependence on substrate and ion concentrations, pH, and potential essential additives, as well as its substrate recognition profile. The SURFE2R system also enables the use of a wide range of substrates that are readily commercially available, avoiding the use of radiolabeled compounds.

Webinar
01.07.2021 | Webinar: SSM-electrophysiology as a Tool for Uncovering Transporter Function
Speakers:
Olga Boudker, Professor of Physiology and Biophysics
(Weill Cornell Medical College)
Lars Jeuken, Professor of Molecular Biophysics
(Faculty of Biological Sciences, University of Leeds)

Olga Boudker, Professor of Physiology and Biophysics
(Weill Cornell Medical College)

Abstract:

Human excitatory amino acid transporter 3 (hEAAT3) mediates glutamate uptake in neurons, intestine, and kidney. We have determined cryo-EM structures of hEAAT3 in several functional states where the transporter is empty, bound to coupled sodium ions only, or fully loaded with three sodium ions, a proton, and the substrate aspartate. The structures suggest that hEAAT3 operates by an elevator mechanism involving three functionally independent subunits. When the substrate-binding site is near the cytoplasm, it has a remarkably low affinity for the substrate, perhaps facilitating its release and allowing the rapid transport turnover. The mechanism of the coupled uptake of the sodium ions and the substrate is conserved across evolutionarily distant families and is augmented by coupling to protons in EAATs. The structures further suggest a mechanism by which a conserved glutamate residue mediates proton symport.

Title: SSM-based electrophysiological characterization of a metal transporter

Lars Jeuken, Professor of Molecular Biophysics,
(Faculty of Biological Sciences, University of Leeds)

Abstract:

Transition metals are essential trace elements and their high-affinity uptake is required for many organisms. Metal transporters are often characterized using metal-sensitive fluorescent dyes encapsulated in proteoliposomes, limiting the metals and experimental conditions that can be studied. Here, we have tested whether metal transport by Enterococcus faecalis MntH2 can be measured with the solid-supported membrane (SSM) technology. SSM uptake assays confirm transport of Mn(II), Co(II), Zn(II), and Cd(II) by MntH2. However, no uptake responses for Cu(II), Fe(II) nor Ni(II) were observed, while the presence of these metals abolishes the uptake signals for Mn(II). Although E. faecalis MntH2 is hypothesized to be a proton-metal symporter, no proton symport could be detected with either SSM or fluorescence assays. These data are discussed with respect to fluorescence uptake assays with Mn(II) and Ni(II), where transport was measured on the time scale of minutes, in sharp contrast to the sub-second timescale of the SSM technology.

Webinar
02.12.2021 | Webinar: Structure and Function of Na+ and H+ dependent transporters
Speakers: Professor David Drew
(Stockholm University, Professor in Biochemistry)
Associate Professor Dr. Matthias Quick
(Columbia University Irving Medical Center, Departments of Psychiatry and Physiology and Cellular Biophysics, Associate Professor of Neurobiology)

Title: Structure and mechanism of the Na+/H+ exchanger NHA2

Professor David Drew
(Stockholm University, Professor in Biochemistry)

Abstract: SLC9B2, also known as NHA2, correlates with the long-sought after sodium/lithium (Na+/Li+) exchanger linked to the pathogenesis of diabetes mellitus and essential hypertension in humans. Despite its functional importance, structural information and the molecular basis of its ion-exchange mechanism have been lacking. Here, we I briefly present the cryo EM structures of bison NHA2 in detergent and in nanodiscs at 3.0 and 3.5 Å resolution, respectively. I will then show how SSM-based electrophysiology has enabled us to conclude that NHA2 catalyses the electroneutral rather than electrogenic exchange of ions. The ion-binding site is quite distinctive, with a tryptophan-arginine-glutamate triad separated from the well-established ion-binding aspartates. These triad residues fine-tune ion binding specificity, as demonstrated by a salt-bridge swap mutant that converts NHA2 into a Li+-specific transporter.

Title: The molecular mechanism of the Na+/H+ antiporter NhaA – from binding to flux

Associate Professor Dr. Matthias Quick
(Columbia University Irving Medical Center, Departments of Psychiatry and Physiology and Cellular Biophysics, Associate Professor of Neurobiology)

Abstract: The Na+/H+ antiporter NhaA represents the archetype of Na+/H+ exchangers, evolutionarily conserved proteins in all kingdoms of life that are essential in cellular ion homeostasis. While structural information has provided excellent starting points in developing mechanistic models of NhaA-mediated transport, the correlation between the correlation of Na+ and H+ binding and flux remains still enigmatic. Since structural information about the composition of the Na+ and H+ sites in NhaA is missing, functional assays are required to gain insight into the molecular events that regulate NhaA activity. By using the SURFE2R N1 SSM platform, our team was able to collect data of NhaA-mediated ion flux across the membrane of NhaA-containing proteoliposomes. Direct Na+ binding studies in conjunction with flux studies reveal that, whereas Na+ transport is impaired at low pH, NhaA can bind Na+ in pH-independent fashion, providing new insight into the interplay of the two cations during transport.

Product Sheet PDF
SURFE2R N1 – Product Sheet
Product video
2020 – SURFE2R N1 Product Video
Label free, real time measurements - electrophysiological approach. Record the action of billion transporters simultaneously.

Label free, real time measurements - electrophysiological approach. Record the action of billion transporters simultaneously.

Poster PDF
2018 – Transported by light: optogenetic control of NCX1
SURFE2R N1 poster, Biophysics Annual Meeting 2018
Poster PDF
2022 – Functional investigation of GABA and Glutamate re-uptake transporters EAAT3 and GAT1 using SSM-based electrophysiology
Publication link
2021 – An Overview of Cell-Based Assay Platforms for the Solute Carrier Family of Transporters
SURFE2R N1 and SURFE2R 96SE review Publication in Frontiers in Pharmacology (2021) Authors: Dvorak V., Wiedmer T., Ingles-Prieto A., Altermatt P., Batoulis H., Bärenz F., Bender E., Digles D., Dürrenberger F., Heitman L.H., IJzerman A.P., Kell D.B., Kickinger S., Körzö D., Leippe P., Licher T., Manolova V., Rizzetto R., Sassone F., Scarabottolo L., Schlessinger A., Schneider V., Sijben H.J., Steck A-L., Sundström H., Tremolada S., Wilhelm M., Wright Muelas M., Zindel D., Steppan C-M., Superti-Furga G.

The solute carrier (SLC) superfamily represents the biggest family of transporters with important roles in health and disease. Despite being attractive and druggable targets, the majority of SLCs remains understudied. One major hurdle in research on SLCs is the lack of tools, such as cell-based assays to investigate their biological role and for drug discovery. Another challenge is the disperse and anecdotal information on assay strategies that are suitable for SLCs. This review provides a comprehensive overview of state-of-the-art cellular assay technologies for SLC research and discusses relevant SLC characteristics enabling the choice of an optimal assay technology. The Innovative Medicines Initiative consortium RESOLUTE intends to accelerate research on SLCs by providing the scientific community with high-quality reagents, assay technologies and data sets, and to ultimately unlock SLCs for drug discovery.

Poster PDF
2015 – Functional Characterization of Prokaryotic NCX by Solid Supported Membrane Technology
SURFE2R N1 poster, Gordon Research Conference 2015
Publication link
2020 – Protein Adsorption on Solid Supported Membranes: Monitoring the Transport Activity of P-Type ATPases
SURFE2R 96SE and SURFE2R N1 Review in Molecules (2020) Authors: Tadini-Buoninsegni F.

P-type ATPases are a large family of membrane transporters that are found in all forms of life. These enzymes couple ATP hydrolysis to the transport of various ions or phospholipids across cellular membranes, thereby generating and maintaining crucial electrochemical potential gradients. P-type ATPases have been studied by a variety of methods that have provided a wealth of information about the structure, function, and regulation of this class of enzymes. Among the many techniques used to investigate P-type ATPases, the electrical method based on solid supported membranes (SSM) was employed to investigate the transport mechanism of various ion pumps. In particular, the SSM method allows the direct measurement of charge movements generated by the ATPase following adsorption of the membrane-bound enzyme on the SSM surface and chemical activation by a substrate concentration jump. This kind of measurement was useful to identify electrogenic partial reactions and localize ion translocation in the reaction cycle of the membrane transporter. In the present review, we discuss how the SSM method has contributed to investigate some key features of the transport mechanism of P-type ATPases, with a special focus on sarcoplasmic reticulum Ca2+-ATPase, mammalian Cu+-ATPases (ATP7A and ATP7B), and phospholipid flippase ATP8A2.

Publication link
2021 – A Solid Supported Membrane-Based Technology for Electrophysical Screening of B0AT1-Modulating Compounds
SURFE2R 96SE Publication in SLAS DISCOVERY: Advancing the Science of Drug Discovery. (2021) Authors: Gerbeth-Kreul C., Pommereau A., Ruf S., Kane Jr J.L., Kuntzweiler T., Hessler G., Engel C.K., Shum P., Wei L., Czech J., Licher T.

Classical high-throughput screening (HTS) technologies for the analysis of ionic currents across biological membranes can be performed using fluorescence-based, radioactive, and mass spectrometry (MS)-based uptake assays. These assays provide rapid results for pharmacological HTS, but the underlying, indirect analytical character of these assays can be linked to high false-positive hit rates. Thus, orthogonal and secondary assays using more biological target-based technologies are indispensable for further compound validation and optimization. Direct assay technologies for transporter proteins are electrophysiology-based, but are also complex, time-consuming, and not well applicable for automated profiling purposes. In contrast to conventional patch clamp systems, solid supported membrane (SSM)-based electrophysiology is a sensitive, membrane-based method for transporter analysis, and current technical developments target the demand for automated, accelerated, and sensitive assays for transporter-directed compound screening. In this study, the suitability of the SSM based technique for pharmacological compound identification and optimization was evaluated performing cell-free SSMbased measurements with the electrogenic amino acid transporter B0AT1 (SLC6A19). Electrophysiological characterization of leucine-induced currents demonstrated that the observed signals were specific to B0AT1. Moreover, B0 AT1-dependent responses were successfully inhibited using an established in-house tool compound. Evaluation of current stability and data reproducibility verified the robustness and reliability of the applied assay. Active compounds from primary screens of large compound libraries were validated, and false-positive hits were identified. These results clearly demonstrate the suitability of the SSM-based technique as a direct electrophysiological method for rapid and automated identification of small molecules that can inhibit B0AT1 activity

Publication link
2020 – Label-Free Bioelectrochemical Methods for Evaluation of Anticancer Drug Effects at a Molecular Level
SURFE2R 96SE and SURFE2R N1 Review article in Frontiers in Sensors (2020) Authors: Tadini-Buoninsegni F.,Palchetti I.

Cancer is a multifactorial family of diseases that is still a leading cause of death worldwide. More than 100 different types of cancer affecting over 60 human organs are known. Chemotherapy plays a central role for treating cancer. The development of new anticancer drugs or new uses for existing drugs is an exciting and increasing research area. This is particularly important since drug resistance and side effects can limit the efficacy of the chemotherapy. Thus, there is a need for multiplexed, cost-effective, rapid, and novel screening methods that can help to elucidate the mechanism of the action of anticancer drugs and the identification of novel drug candidates. This review focuses on different label-free bioelectrochemical approaches, in particular, impedance-based methods, the solid supported membranes technique, and the DNA-based electrochemical sensor, that can be used to evaluate the effects of anticancer drugs on nucleic acids, membrane transporters, and living cells. Some relevant examples of anticancer drug interactions are presented which demonstrate the usefulness of such methods for the characterization of the mechanism of action of anticancer drugs that are targeted against various biomolecules.

Publication link
2017 – SSM-Based Electrophysiology for Transporter Research
SURFE2R N1 and SURFE2R 96SE book chapter in Methody in Enzymology Authors: Bazzone A., Barthmes M., Fendler K.

Functional characterization of transport proteins using conventional electrophysiology can be challenging, especially for low turnover transporters or transporters from bacteria and intracellular compartments. Solid-supported membrane (SSM)-based electrophysiology is a sensitive and cell-free assay technique for the characterization of electrogenic membrane proteins. Purified proteins reconstituted into proteoliposomes or membrane vesicles from cell culture or native tissues are adsorbed to the sensor holding an SSM. A substrate or a ligand is applied via rapid solution exchange. The electrogenic transporter activity charges the sensor, which is recorded as a transient current. The high stability of the SSM allows cumulative measurements on the same sensor using different experimental conditions. This allows the determination of kinetic properties including EC50, IC50, Km, KD, and rate constants of electrogenic reactions. About 100 different transporters have been measured so far using this technique, among them symporters, exchangers, uniporters, ATP-, redox-, and light-driven ion pumps, as well as receptors and ion channels. Different instruments apply this technique: the laboratory setups use a closed flow-through arrangement, while the commercially available SURFE2R N1 resembles a pipetting robot. For drug screening purposes high-throughput systems, such as the SURFE2R 96SE enable the simultaneous measurement of up to 96 sensors.

Publication link
2014 – Industrializing Electrophysiology: HT Automated Patch Clamp on SyncroPatch 96 Using Instant Frozen Cells
SURFE2R N96 (predecessor model of SURFE2R 96SE) book chapter in Patch-Clamp Methods and Protocols Authors: Polonchuk L.

Patch-clamping is a powerful technique for investigating the ion channel function and regulation. However, its low throughput hampered profiling of large compound series in early drug development. Fortunately, automation has revolutionized the area of experimental electrophysiology over the past decade. Whereas the first automated patch-clamp instruments using the planar patch-clamp technology demonstrated rather a moderate throughput, few second-generation automated platforms recently launched by various companies have significantly increased ability to form a high number of high-resistance seals. Among them is SyncroPatch 96 (Nanion Technologies GmbH, Munich, Germany), a fully automated giga-seal patch-clamp system with the highest throughput on the market. By recording from up to 96 cells simultaneously, the SyncroPatch 96 allows to substantially increase throughput without compromising data quality. This chapter describes features of the innovative automated electrophysiology system and protocols used for a successful transfer of the established hERG assay to this high-throughput automated platform.

Publication link
2019 – Functional Reconstitution of Membrane Proteins Derived From Eukaryotic Cell-Free Systems
SURFE2R N1 and Orbit 16 Publication in Frontiers in Pharmacology (2019) Authors: Dondapati S.K., Lübberding H., Zemella A., Thoring L., Wüstenhagen D.A., Kubick S.

Cell-free protein synthesis (CFPS) based on eukaryotic Sf21 lysate is gaining interest among researchers due to its ability to handle the synthesis of complex human membrane proteins (MPs). Additionally Sf21 cell-free systems contain endogenous microsomal vesicles originally derived from the endoplasmic reticulum (ER). After CFPS, MPs will be translocated into the microsomal vesicles membranes present in the lysates. Thus microsomal membranes offer a natural environment for de novo synthesized MPs. Despite the advantage of synthesizing complex MPs with post translational modifications directly into the microsomal membranes without any additional solubilization supplements, batch based Sf21 cell-free synthesis suffers from low yields. The bottleneck for MPs in particular after the synthesis and incorporation into the microsomal membranes is to analyze their functionality. Apart from low yields of the synthesized MPs with batch based cell-free synthesis, the challenges arise in the form of cytoskeleton elements and peripheral endogenous proteins surrounding the microsomes which may impede the functional analysis of the synthesized proteins. So careful sample processing after the synthesis is particularly important for developing the appropriate functional assays. Here we demonstrate how MPs (native and batch synthesized) from ER derived microsomes can be processed for functional analysis by electrophysiology and radioactive uptake assay methods. Treatment of the microsomal membranes either with a sucrose washing step in the case of human serotonin transporter (hSERT) and sarco/endoplasmic reticulum Ca2+/ATPase (SERCA) pump or with mild detergents followed by the preparation of proteoliposomes in the case of the human voltage dependent anionic channel (hVDAC1) helps to analyze the functional properties of MPs.

Poster PDF
2021 – Drug Effects on human Na+/Ca2+ Exchanger and Implications for Drug Development
FLEXcyte 96, SURFE2R N1 and SURFE2R 96SE poster, Biophysical Society Meeting 2021

How can we help you?

Contact our specialist Dr. Maria Barthmes (Product Manager of the SURFE2R product family). Maria is delighted to help you:

Maria.Barthmes@nanion.de
or call: +49 89 2190 95-068
or connect via LinkedIn