Ion Channels and Respiratory Diseases

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Did You Know: Ion Channels and their function in respiratory disease targets

The SARS-CoV-2 pandemic which emerged in late 2019, coupled with continued seasonal and pandemic influenza infections, has caused a significant problem for global public health and health systems worldwide. Antivirals and vaccines effective against these viruses are desperately needed in order to stem the pandemic and save lives.

A number of viroporins and ion channels are involved in viral infections and respiratory disorders, a selection is provided below.

Viroporins

Viroporins are small, hydrophobic integral membrane ion channels essential in the life cycle of a diverse range of RNA and DNA viruses. They perform multiple functions in the virus life cycle including viral entry, particle production and virus spread.

Viroporins are potential drug targets for block of viral replication and the spread of infection.


Useful Reviews
Scott, C., & Griffin, S. 2015. Viroporins: structure, function and potential as antiviral targets. Journal of General Virology

J Gen Virol

Nieva, J.L., Madan, V., Carrasco, L. 2012. Viroporins: structure and biological functions. Nature Reviews Microbiology.

Nat Microbiol

Wang, K., Xie, S., Sun, B. 2011. Viral proteins function as ion channels. Biochim Biophys Acta Biomembr.

Biochim_Biophys_Acta


Viroporins of Sars-CoV

Sars-CoV 3a forms a potassium channel located on the surface of Sars-CoV infected cells and promotes virus release (Lu, W., et al., 2006). Additionally, the Sars-CoV 3a and E proteins have been shown to activate the NLRP3 inflammasome (Chen, I-Y., et al., 2019) and are involved in virus replication and pathogenesis (Castaño-Rodriguez et al, 2018).


Viroporins of Influenza virus

The M2 protein of influenza A is a proton-gated, proton selective ion channel that is essential for viral replication (Jalily, P.H., et al, 2020). Amantadine is a well-known blocker of the influenza A M2 proton channel, and has been used as an anti-influenza drug for many years. Unfortunately it is associated with a number of severe side effects and is no longer recommended for the treatment of influenza A due to resistance as a result of mutations in the M2 protein


Measuring viroporins

Viroporins can be measured using automated patch clamp when expressed in cell lines, or using the Orbit instruments when they are recombinantly expressed in bilayers.

SyncroPatch 384i
High throughput automated patch clamp

Orbit 16
Parallel bilayer recordings

CardioExcyte 96
Cytotoxicity measurements

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Ca2+-release activated calcium channel (CRAC)

Ca2+-release activated calcium channel (CRAC) is composed of Orai1, the pore forming subunit, and STIM1, the endoplasmic reticulum Ca2+ sensor. CRAC channels open in response to a depletion of intraluminal Ca2+ in the ER, allowing an influx of Ca2+ via a process known as store-operated calcium entry (SOCE). There is some evidence that CRAC channels inhibitors could be novel therapeutics for autoimmune of inflammatory conditions. The CRAC channel blocker, CM4620-IE, from CalciMedica has recently entered Phase II clinical trials to prevent a cytokine storm in COVID-19 patients.


Useful Reviews
Staudermann, T.A. 2018. CRAC channels as targets for drug discovery and development. Cell Calcium

Cell Calcium


Measuring CRAC Channels

CRAC channels can be measured using automated patch clamp when expressed in cell lines, or using the Orbit instruments when they are recombinantly expressed in bilayers.

SyncroPatch 384i
High throughput automated patch clamp

Orbit 16
Parallel bilayer recordings

CardioExcyte 96
Cytotoxicity measurements

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Volume-regulated anion channels (VRAC), LRRC8

Volume-regulated anion channels (VRAC) are widely expressed in mammalian cells. They transport chloride ions and other organic ions, e.g. taurine or glutamate and maybe even water, across the plasma membrane VRAC channels are composed of LRRC8 heteromers of which there are 5 known variations, LRRC8A-E. Cell-to-cell cGAMP transmission via LRRC8/VRAC channels has been shown to play a crucial role in anti-viral immunity.

Key Publications and Reviews

Zhou, C., Chen, X., Planells-Cases, R., et al. 2020. Transfer of cGAMP into Bystander Cells via LRRC8 Volume-Regulated Anion Channels Augments STING-Mediated Interferon Responses and Anti-viral Immunity. Immunity

Immunity


Strange, K., Yamada, T., & Denton, G. 2019. A 30-year journey from volume-regulated anion currents to molecular structure of the LRRC8 channel. Journal of General Physiology

J. Gen. Physiol.


Measuring VRAC Channels

VRAC Channels can be measured using automated patch clamp when expressed in cell lines, or using the Orbit instruments when they are recombinantly expressed in bilayers.

SyncroPatch 384i
High throughput automated patch clamp

Orbit 16
Parallel bilayer recordings

CardioExcyte 96
Cytotoxicity measurements

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VRAC Publications using Nanion Instruments

2016 - Human T cells in silico: Modelling their electrophysiological behaviour in health and disease

icon pl  Patchliner publication in Journal of Theoretical Biology (2016)

Authors: 
Ehling P., Meuth P., Eichinger P., Hermann A.M., Bittner S., Pawlowski M., Pankratz S., Herty M., Budde T., Meuth S.G.

Two pore channels (TPC)

Two pore channels (TPC) are calcium channels located on cellular endosomes. Activated by the second messenger NAADP and the phospholipid Phosphatidylinositol 3,5-bisphosphate, PtdIns(3,5)P2) the pore conducts primarily Ca2+ and Na+. It has been shown that they play a key role in Ebola virus infection and may provide a therapeutic target as an antiviral for Ebola virus and other filoviruses. In endosomal membranes they are responsible for entry of Ebola virus.

Key Publications

Sakurai, Y., Kolokoltsov, A.A., Chen, C-C., et al. 2015. Two-pore channels control Ebola virus host cell entry and are drug targets for disease treatment. Science

Science


Castonguay, J., Orth, J.H.C., Müller, T., et al. 2017. The two-pore channel TPC1 is required for efcient protein processing through early and recycling endosomes. Nature Scientific Reports

Science


Measuring Two Pore Channels

Two Pore Channels can be measured using automated patch clamp when expressed in cell lines, in lysosomes, or using the Orbit instruments when they are recombinantly expressed in bilayers.

Port-a-Patch
Automated patch clamp

Orbit 16
Parallel bilayer recordings

CardioExcyte 96
Cytotoxicity measurements

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Publications using Instruments from Nanion

2017 - Organelle membrane derived patches: reshaping classical methods for new targets

icon pap   Port-a-Patch and   icon vpp   Vesicle Prep Pro publication in Nature Scientific Reports (2017)

Authors:
Shapovalov G., Ritaine A., Bidaux G., Slomianny, C., Borowiec A-S., Gordienko D., Bultynck G., Skryma R., Prevarskaya N.

2014 - High susceptibility to fatty liver disease in two-pore channel 2-deficient mice

icon pap  Port-a-Patch publication in Nature Communications (2014)

Authors: 
Grimm C., Holdt L.M., Chen C.C., Hassan S., Müller C., Jörs S., Cuny H., Kissing S., Schröder B., Butz B., Northoff B., Castonguay J., Luber C.A., Moser M., Spahn S., Lüllmann-Rauch R., Fendel C., Klugbauer N., Griesbeck O., Haas A., Mann M., Bracher F., Teupser D., Saftig P., Biel M., Wahl-Schott C.

2010 - Planar Patch Clamp Approach to Characterize Ionic Currents from Intact Lysosomes

icon pap  Port-a-Patch publication in Science Signaling (2010)

Authors:
Schieder M., Rötzer K., Brüggemann A., Biel M., Wahl-Schott C.

2010 - Characterization of two pore channel 2 (TPCN2) -mediated Ca2+ currents in isolated lysosomes

icon pap  Port-a-Patch publication in The Journal of Biological Chemistry (2010)

Authors:
Schieder M., Rötzer K., Brüggemann A., Biel M., Wahl-Schott C.A.

Transporters - SLC6A19 in SARS-CoV-2

The sodium-dependent neutral amino acid transporter B0AT1 is a protein that in humans is encoded by the SLC6A19 gene. It mediates epithelial resorption of neutral amino acids across the apical membrane in the kidney and intestine. Recently it has been shown that the angiotensin-converting enzyme 2 (ACE2) and B0AT1 protein forms a complex for recognition of the SARS-CoV-2 virus, thereby playing a role in coronavirus recognition and infection.

Key Publications

Yan, R., Zhang, Y., Li, Y., et al. 2020. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science

Science


Measuring SLC19A and other Transporters

SLC19A can be measured using the SSM-electrophysiology technique. It may also be measured in bilayers if reconstituted and the effect of drugs acting on transporters can be measured using impedance.

SURFE2R
Transporter measurements

Orbit 16
Parallel bilayer recordings

CardioExcyte 96
Cytotoxicity measurements

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TMEM16A

TMEM16A is a calcium-activated chloride channel expressed in surface epithelium of the airway as well as submucosal glands and goblet cells. It is upregulated during inflammatory conditions. Recently a postitive modulator of TMEM16A has been shown to enhance anion and fluid secretion from cystic fibrosis patients. This type of approach presents a novel approach tot he treatment of cystic fibrosis and other respiratory disorders.

Key Publication

Danahay, H.L., Lilley, S., Fox, R., et al. 2019. TMEM16A Potentiation: A Novel Therapeutic Approach for the Treatment of Cystic Fibrosis. American Journal of Respiratory and Critical Care Medicine

AJRCCM


Measuring TMEM16A

TMEM16A Channels can be measured using automated patch clamp when expressed in cell lines, and can be activated using the internal perfusion feature of the SyncroPatch 384i or using the Orbit instruments when they are recombinantly expressed in bilayers.

SyncroPatch 384i
High throughput automated patch clamp

Orbit 16
Parallel bilayer recordings

CardioExcyte 96
Cytotoxicity measurements

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Application Notes

TMEM16A (ANO1) - "Internal perfusion of Ca2+ to activate TMEM16A/ANO1 on the SyncroPatch 384PE"

icon sp96   SyncroPatch 384PE (a predecessor model of SyncroPatch 384i) application note   logo pdf   (1.1 MB)
Cells were kindly provided by SB Drug Discovery.

P2X3 in chronic cough

P2X receptors are trimeric ligand-gated ion channels which are activated by extracellular ATP. When activated, small monovalent cations pass through the pore. P2X chanenls have been shown to play a role in inflammatory, visceral, and neuropathic pain states, and in addition to this, in airways hyperreactivity, migraine, itch, and cancer pain. More recently, the antagonist of P2X2/3 and P2X3 receptors, MK‐7264 (gefapixant), has entered Phase III clinical trials for refractory and unexplained chronic cough.

Key Publications

Ford, A.P. & Undem, B.J. 2013. The therapeutic promise of ATP antagonism at P2X3 receptors in respiratory and urological disorders. Frontiers in Cellular Neuroscience

Frontiers in Cellular Neuroscience


Ford, A.P. 2012. In pursuit of P2X3 antagonists: novel therapeutics for chronic pain and afferent sensitization. Purinergic Signalling

Purinergic Signalling


Measuring P2X3 Channels

P2X3 Channels can be measured using automated patch clamp when expressed in cell lines, or using the Orbit instruments when they are recombinantly expressed in bilayers.

SyncroPatch 384i
High throughput automated patch clamp

Orbit 16
Parallel bilayer recordings

CardioExcyte 96
Cytotoxicity measurements

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Application Notes

P2X3 - "Activation and inhibition of P2X3 channels recorded on the SyncroPatch 384PE"

icon sp96   SyncroPatch 384PE (a predecessor model of SyncroPatch 384i) application note:   logo pdf   (1.5 MB)
Cells were engineered and kindly provided by Axxam S.p.A., Milan.  

P2X3 - "Activation and inhibition of P2X3 channels recorded on the Patchliner"

icon sp96   Patchliner application note:   logo pdf   (0.6 MB)
Cells were engineered and kindly provided by Axxam S.p.A., Milan.  

P2X2 / P2X3 - "Pharmacology of P2X2/3 channels recorded on the SyncroPatch 384PE"

icon sp96   SyncroPatch 384PE (a predecessor model of SyncroPatch 384i) application note:   logo pdf   (5 MB)
Cells were engineered and kindly provided by Axxam S.p.A., Milan.  

P2X2 / P2X3 - "P2X2/3 receptors recorded on Nanion's Patchliner"

icon pl   Patchliner application note:   logo pdf   (0.4 MB)
Cells were kindly provided by Evotec.  

Publications using Instruments from Nanion

2019 - Action of MK‐7264 (Gefapixant) at human P2X3 and P2X2/3 receptors and in vivo efficacy in models of sensitisation

icon pap   Port-a-Patch publication in British Journal of Pharmacology (2019)

Authors:
Richards D., Gever J.R., Ford A.P., Fountain S.J.

2011 - State-of-the-art automated patch clamp devices: heat activation, action potentials, and high throughput in ion channel screening

icon pap   Port-a-Patch,   icon pl  Patchliner and   icon sp96   SyncroPatch 96 (a predecessor model of SyncroPatch 384PE) publication in Frontiers in Pharmacology (2011)

Authors: 
Stoelzle S., Obergrussberger A., Brüggemann A., Haarmann C., George M., Kettenhofen R., Fertig N.

CFTR

Cystic fibrosis (CF) is a fatal hereditary lung disease. Mutations in the cystic fibrosis conductance regulator (CFTR) channel have been shown to cause CF. CFTR is a regulated epithelial chloride channel and mutations cause a reduction in activity of the channel via a variety of mechanisms. CFTR dysfunction causes chronic mucus obstruction, neutrophilic inflammation and bacterial infection in CF airways. This results in defective electrolyte transport in airway epithelia and thereby, chronic lung infection and premature mortality. Therefore, compounds which increase activity of CFTR have therapeutic potential for treating CF and other respiratory disorders, such as chronic obstructive pulmonary disease (COPD) or asthma.

Useful Reviews

Noone, P.G. & Knowles, M.R. 2001. 'CFTR-opathies': disease phenotypes associated with cystic fibrosis transmembrane regulator gene mutations. Respiratory Research

Respiratory Research


Mall, M.A., & Hartl, D. 2014. CFTR: cystic fibrosis and beyond. European Respiratory Journal

Respiratory research


Measuring CFTR Channels

CFTR Channels can be measured using automated patch clamp when expressed in cell lines, or using the Orbit instruments when they are recombinantly expressed in bilayers. The CFTR channel is an ABC transporter and can also be measured using the SSM-electrophysiology technique.

SyncroPatch 384i
High throughput automated patch clamp

Orbit 16
Parallel bilayer recordings

SURFE2R
Transporter measurements

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Application Notes
Publications using Instruments from Nanion

2020 - Targeting different binding sites in the CFTR structures allows to synergistically potentiate channel activity

icon pl  Patchliner publication in European Journal of Medicinal Chemistry (2020)

Authors: 
Froux L., Elbahnsi A., Boucherle B., Billet A., Baatallah N., Hoffmann B., Alliot J., Zelli R., Zeinyeh W., Haudecoeur R., Chevalier B., Fortuné A., Mirval S., Simard C., Lehn P., Mornon J-P., Hinzpeter A., Becq F., Callebaut I., Décout J-L.

2017 - Development of Automated Patch Clamp Technique to Investigate CFTR Chloride Channel Function

icon pl  Patchliner publication in Frontiers in Pharmacology (2017)

Authors: 
Billet A.,Froux L., Hanrahan J.W., Becq F.

2017 - Buserelin alleviates chloride transport defect in human cystic fibrosis nasal epithelial cells

icon pap  Port-a-Patch publication in PLoS ONE (2017)

Authors: 
Calvez M.L., Benz N., Huguet F., Saint-Pierre A., Rouillé E., Coraux C., Férec C., Kerbiriou M., Trouvé P.

2014 - Improvement of Chloride Transport Defect by Gonadotropin-Releasing Hormone (GnRH) in Cystic Fibrosis Epithelial Cells

icon pap  Port-a-Patch publication in PLoS One (2014)

Authors: 
Benz N., Le Hir S., Norez C., Kerbiriou M., Calvez M.-L., Becq F., Trouvé P. , Férec C.


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