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PAT1

Target Synonyms and Classification: The Proton-dependent Amino acid Transporter 1 (PAT1, LYAAT-1) belongs to the Amino Acid/Auxin Permease Family (AAAPF, TCDB: 2.A.18), which catalyze the H+ coupled uptake of auxins or amino acids. Since it is a human transporter, it’s also classified as an SLC transporter: PAT1 equals to SLC36A1 and belongs to the proton-coupled amino acid transporters (SLC36).

Function and Mechanism: The physiological role of PAT1 is manifold: In the small intestine it is responsible for the absorption of amino acids, while it serves as an efflux pathway in lysosomes after intralysosomal protein breakdown. It is also capable of regulating cell growth and sensing the availability of amino acids. H+/amino acid symport occurs with 1:1 stoichiometry. PAT1 is a low affinity / high capacity transporter and exhibits KM values in the range between 1 and 10 mM. Proline uptake in rPAT1 occurs with an KM of 2 mM and an pH optimum of 5.0. Proton affinity corresponds to an pK of 7.0 and is independent of substrate.

Organism and Localization: Members of the AAAP Family are only found in eukaryotes; PAT1 is a mammalian transporter. It is localized at the luminal surface of the small intestine and - in many cell types - also in lysosomes. In addition a nuclear localization was described. Diseases associated with defects of human PAT1 include Spinocerebellar Ataxia 45 and Iminoglycinuria. Moreover regulation of PAT1 by FLCN plays a crucial role in the Birt-Hogg-Dubé (BHD) syndrome.

Substrates and Inhibitors: PAT1 transports small neutral amino and imino acids: L-Gly, L-Ala and L-Pro are transported with similar rates; PAT1 does not discriminate between D- and L-isoforms of these amino acids. In additon β-Alanine is transported with similar affinity as α-Alanine. Other substrates include γ-amino butyrate, Hydroxyproline and taurine. PAT1 is also relevant in pharmacology and known to transport amino acid-based drugs to treat epilepsy, schizophrenia, bacterial infections, hyperglycemia and cancer. Serotonin, L-tryptophan, and tryptamine are not transported by, but effective inhibitors of PAT1 with IC50 values in the range of 5 – 10 mM.

Related Transporters: Within the SLC36 family are three other members with different tissue distributions and slightly different kinetic properties. PAT2 (SLC36a2) is more active at neutral and alkaline pH and has higher apparent affinites for its main substrates (KM < 1 mM).

Kdp ATPase

Target Synonyms and Classification: The K+ uptake ATPase (Kdp ATPase) belongs to the p-type ATPase Superfamily (TCDB: 3.A.3), which catalyze cation transport driven by ATP hydrolysis. It is rather a unique member of this superfamily, since it contains four subunits (KdpFABC).

Function and Mechanism: Kdp ATPase is an inducible high-affinity K+ transporter that is synthesized under conditions of severe K+ limitation or osmotic upshift. It maintains the desired concentration of internal K+ required for various physiological processes like turgor homeostasis and pH regulation. Kdp is composed of four protein subunits forming the KdpFABC complex. Here KdpA acts as a K+ transporter with high affinity (KD = 2 µM). It can maintain cytoplasmic potassium concentrations against gradients up to 10.000 fold. KdpB contains the ATPase domain and KdpC and KdpF may facilitate complex assembly and stabilize the complex. There is a very special mechanism in place which guarantees coupling between KdpA and KdpB: KdpA has a protein embedded tunnel and the cytoplasmic gate within KdpA is linked to the phosphorylation domain of KdpB. Here, a channel architecture was repurposed for active transport. Furthermore, KdpFABC acts as a functional dimer.

Organism and Localization: The Kdp system is widely dispersed among the different classes of bacteria including the cyanobacteria and was studied most in Escherichia coli. It localizes to the plasma membrane. Kdp activity is essential for cell growth at low potassium concentrations.

Substrates and Inhibitors: KdpA has a very high specificity for potassium: Rb+, with a Pauling radius (1.49 Å) comparable to that of K+ (1.33 Å) is transported with much lower affinity (KM = 8 mM vs. KM = 2 µM). It was shown that KdpA depends on pH, showing highest K+ turnover rates at pH 7.4 – likely due to a proton leak pathway. The G232D mutant of KdpA instead transports various monovalent cations (K+ > Rb+ > Na+ > Li+ > H+). A common inhibitor for inhibition of p-type ATPase is orthovanadate. It inhibits the Kdp complex at 1 – 10 µM by trapping the protein in a transition state of phosphoenzyme hydrolysis.

Related Transporters: While KdpB is homologues to p-type ATPase a-subunits, KdpA is homologous to other K+ transporters like KcsA and KtrB. It contains a selectivity filter descenced from that of the bacterial channel KcsA.

 

CHT1

Target Synonyms and Classification: The high affinity choline transport 1 (CHT1) belongs to the Solute:Sodium Symporter Family (SSSF, TCDB: 2.A.21), which catalyze the cotransport of sodium and sugars, amino acids or other organo cations. Since it is a human transporter, it’s also classified as an SLC transporter: CHT1 equals to SLC5a7 and belongs to the sodium/glucose cotransport family (SLC5).

Function and Mechanism: CHT1 cotransports Na+, Cl- and choline+ in variable stoichiometry, depending on membrane voltage: 10 charges are translocated at – 80 mV; 3 at -20 mV. Leak currents in absence of choline have been described; CHT1 is classified as a high-affinity / low-capacity transporter with apparent affinity for choline in the range of KM = 2 µM and a maximum turnover rate of 14 molecules per second under physiological conditions; CHT1 has apparent affinities of KM(Na+) = 80 mM and KM(Cl-) = 50 mM. Internal sodium, but not internal chloride reduces choline induced transport, which lead to the conclusion that sodium release is the rate limiting step in the transport cycle. External chloride is required for choline transport, but Cl- may not be cotransported and rather plays a regulatory role. CHT1 also depends on external pH: CHT1 shows highest Vmax at pH 9.5, at pH 5.5 transport by CHT1 is completely abolished. Within this pH interval, the CHT1 transport activity follows a titration curve with pK = 7.4. Besides affecting Vmax, KM for Na+ decreases from 67 mM (pH 6.5) to 13 mM (pH 8.5) at – 60 mV.

Organism and Localization: CHT1 is mainly expressed in presynaptic cholinergic nerve terminals. Its predominant localization is in intracellular organelles, such as endosomal compartments and synaptic vesicles. CHT1 is translocated to the plasma membrane in response to neuronal activity and is then rapidly internalized by endocytosis. CHT1 actively transports extracellular choline into the presynaptic terminals, where it is used for synthesis of acetyl choline. The choline uptake is the rate limiting step in neuronal acetyl choline synthesis; mutations in CHT1 affecting surface transporter trafficking have been found to lead to hereditary motor neuropathy. Abnormal regulation of CHT contributes to cognitive imparimants and neuropsychiatric disorders, including Alzheimer’s disease.

Substrates and Inhibitors: CHT1 activity can be blocked by very low concentrations of hemicholinium-3 (HC-3) with an IC50 of 1 – 5 nM. Hence, CHT1 is also known as the Hemicholinium-3-sensitive choline transporter.

Related Transporters: There are two other mammalian families of choline transporters: The ubiquitously expressed, poly-specific, low-affinity organic cation transporters OCT1 and OCT2 are uniporters, belong to the SLC22 family and Major Facilitator Superfamily (TCDB: 2.A.1). The choline transporter-like (CTL) proteins (TCDB: 2.A.92) have intermediate affinity for choline and are expressed in the central nervous system. The 220 known SLC5 members – to which CHT1 belongs – have diverse function: These transporters transport glucose, myo-inositol and iodide, one is an anion transporter, and another is a glucose-activated ion channel. Members of this family also behave as uniporters, urea and water channels, and urea and water cotransporters.

 

MntH2

Target Synonyms and Classification: The divalent metal cation transporter MntH2 belongs to the Natural Resistance-Associated Macrophage Protein family (NRAMP family, TCDB: 2.A.55), which catalyze the cotransport of protons and divalent cations.

Function and Mechanism: MntH2 is only poorly characterized; members of the NRAMP family are responsible for the uptake of divalent transition metals such as iron and manganese into cells. NRAMP proteins have been identified due to their role in resistance to intracellular bacterial pathogens. With KM values in the lower µM range for their main substrates they efficiently select against Ca2+ and Mg2+, which are several orders of magnitude more abundant. NRAMP family members usually act as H+ coupled symporters. One proposed mechanism involves individual translocation pathways for protons and metal ions.

Organism and Localization: MntH2 resides within the plasma membrane of Enterococcus faecalis. Eucaryotic homologues have been also found in endosomes and lysosomes.

Substrates and Inhibitors: NRAMP transporters transport a wide range of divalent metal cations. MntH2 transports divalent transition metals such as Mn(II), Co(II), Zn(II) and Cd(II) and does not transport the earth metals Cu(II), Fe(II) and Ni(II). However, these earth metals are able to inhibit Mn(II) transport.

Related Transporters: Members of the NRAMP family are found across all kingdoms. Humans and rodents possess two distinct NRAMPs: the broad specificity NRAMP2 (SLC11a2) transports Fe2+ and H+ in a 1:1 stoichiometry with apparent affinities of 6 µM and 1 µM, respectively. It transports in order of substrate preference: Fe2+> Zn2+> Mn2+> Co2+> Ca2+> Cu2+> Ni2+> Pb2+. NRAMP1 (SLC11a1) has preference for Mn2+ over Fe2+ and has been reported to function by metal:H+ antiport. Mutations in NRAMP1 have been associated with susceptibility to infectious diseases such as tuberculosis and leprosy, and inflammatory diseases such as rheumatoid arthritis and Crohn's disease.


Publications

 

Piezo1

Family:
Piezo channels; piezo-type mechanosensitive ion channel.

Members:
Piezo1 and Piezo2 in vertebrates, encoded by PIEZO1 and PIEZO2 genes, respectively.

Topology:
Piezo 1 is a trimeric mechanosensitive ion channel.Piezos are very large proteins with numerous (>14) predicted transmembrane (TM) domains per subunit.

Regulation and Function:
Piezo1 opens in response to mechanical stimuli such as shear stress and membrane stretch, allowing positively charged ions, including calcium, to flow into the cell.

Piezo1: Background information


Overview:

Piezo proteins are the pore-forming subunits of trimeric mechanosensitive ion channels that open in response to mechanical stimuli such as shear stress and membrane stretch, allowing positively charged ions, including calcium, to flow into the cell.


Data Sheet:

Gene:
PIEZO1

Human Protein:
UniProt Q92508

Tissue:
Expressed in tissues of hollow organs such as stomach, lungs, bladder, intestines, and endothelial cells lining the lumen of blood vessels. Also expressed red blood cells.

Function and pathology:
Gain-of-function (GOF) mutations in human Piezo1 cause hereditary xerocytosis (also known as dehydrated stomatocytosis), a familial anemia. Loss-of-function (LOF) mutations cause generalized lymphatic dysplasia characterized by varying degrees of anemia. Both channelopathies suggest a central role that Piezo1 plays in erythrocyte volume control.

Pathology:
Hereditary xerocytosis

Selective activators:
Yoda1; jedi1 (mouse); jedi1 (mouse)

Inhibitors:
Dooku1; ruthenium red; gadolinium

Assays:
Patch Clamp: whole cell, single channel, bilayers, mechanosensitive activation

Data and Applications

Webinars

Podcast

Introducing Professor Lars Kaestner (Saarland University) - Red Blood Cells

In this edition of the podcast - we speak to Prof. Lars Kaestner and his work with Red Blood Cells; specifically looking at advances in diagnostic tools for Neuroacanthocytosis.

Publications

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