Ion Channels and Respiratory Diseases

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.

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Useful Reviews

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

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

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

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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).

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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

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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.

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.

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Useful Reviews

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

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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.

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.

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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

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

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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.

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VRAC Publications using Nanion Instruments

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.

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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

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

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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.

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Publications using Instruments from Nanion

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.

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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

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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.

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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.

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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

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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.

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Application Notes

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 P2X_2/3 and P2X₃ receptors, MK‐7264 (gefapixant), has entered Phase III clinical trials for refractory and unexplained chronic cough.

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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

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

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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.

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Application Notes

Publications using Instruments from Nanion

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.

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Useful Reviews

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

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

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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.

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Application Notes

Publications using Instruments from Nanion