Subunit gating resulting from individual protonation events in Kir2 channels

Subunit gating resulting from individual protonation events in Kir2 channels | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Subunit gating resulting from individual protonation events in Kir2 channels Grigory Maksaev, Michael Bründl-Jirout, Anna Stary-Weinzinger, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2640647/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Jul, 2023 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Inwardly rectifying potassium (Kir) channels play a critical role in stabilizing the membrane potential, thus controlling numerous physiological phenomena in multiple tissues. Channel conductance is activated by cytoplasmic modulators that open the channel at the 'helix bundle crossing' (HBC), formed by the coming together of the M2 helices from each of the four subunits, at the cytoplasmic end of the transmembrane pore. We introduced a negative charge at the bundle crossing region (G178D) in classical inward rectifier Kir2.2 channel subunits that forces channel opening, allowing pore wetting and free movement of permeant ions between the cytoplasm and the inner cavity. Single-channel recordings reveal a striking pH-dependent subconductance behavior in G178D (or G178E and equivalent Kir2.1[G177E]) mutant channels that reflects individual subunit events. These subconductance levels are well resolved temporally and occur independently, with no evidence of cooperativity. Decreasing cytoplasmic pH shifts the probability towards lower conductance levels, and molecular dynamics simulations show how protonation of Kir2.2[G178D] and, additionally, the rectification controller (D173) pore-lining residues leads to changes in pore solvation, K+ ion occupancy, and ultimately K+ conductance. While subconductance gating has long been discussed, resolution and explanation have been lacking. The present data reveals how individual protonation events change the electrostatic microenvironment of the pore, resulting in distinct, uncoordinated, and relatively long-lasting conductance states, which depend on levels of ion pooling in the pore and the maintenance of pore wetting. Gating and conductance are classically understood as separate processes in ion channels. The remarkable sub-state gating behavior of these channels reveals how intimately connected ‘gating’ and ‘conductance’ are in reality. Biological sciences/Biophysics/Single-molecule biophysics Biological sciences/Chemical biology/Ion channels Kir2.1 Kir2.2 pH subconductance levels gating G178D force open molecular dynamics simulations single-channel recordings Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementarydataNCB.pdf MovieS1md16m844xD1734xG178Dcharged1usdf.mp4 Movie S1 MovieS2md16m842xD1732xG178Dcharged1usdf.mp4 Moivie S2 Cite Share Download PDF Status: Published Journal Publication published 28 Jul, 2023 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2640647","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":180330081,"identity":"59dfa46f-cd4b-48f6-97c9-a4c1ba74e74f","order_by":0,"name":"Grigory Maksaev","email":"","orcid":"https://orcid.org/0000-0001-6242-1634","institution":"Washington University in St. Louis","correspondingAuthor":false,"prefix":"","firstName":"Grigory","middleName":"","lastName":"Maksaev","suffix":""},{"id":180330082,"identity":"0053d498-88d1-4a03-b1cc-924e29f2cd95","order_by":1,"name":"Michael Bründl-Jirout","email":"","orcid":"","institution":"University of 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