Luminal autoinhibitory loop and binding-site protonation regulate VAChT substrate release

Luminal autoinhibitory loop and binding-site protonation regulate VAChT substrate release | 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 Luminal autoinhibitory loop and binding-site protonation regulate VAChT substrate release Yanqing Zhang, Feiwen Wei, Wei Zhang, Jing Nan, Jufang Wang, Yuan Xiao, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8816900/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The vesicular acetylcholine transporter (VAChT) packages acetylcholine (ACh) into synaptic vesicles to drive cholinergic neurotransmission, which underpins cognition, movement, and neuronal survival. Its dysfunction is implicated in disorders such as Alzheimer’s disease and myasthenia gravis. Here, we present cryo-EM structures of human VAChT in apo forms at both neutral and acidic pH, as well as in complexes with its substrate and inhibitor vesamicol (VSM). Our structures are captured in a lumen-facing, occluded conformation, wherein a unique luminal loop seals the transport pathway, a feature distinct from other SLC18 family members. Combined with molecular dynamics (MD) simulations and mutagenesis analyses, we identified three key protonation sites critical for transport and demonstrated the luminal loop acts as an intrinsic gate, controlling substrate release through autoinhibitory mechanism. Our findings elucidate the transport and inhibition mechanisms of VAChT and provide a structural framework for developing therapeutics against cholinergic dysfunction. Biological sciences/Biochemistry/Proteins/Membrane proteins Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Full Text Additional Declarations There is NO Competing Interest. Supplementary Files VAChTapo.txt Related Manuscript File VAChTVSM.txt Related Manuscript File VAChTACh.txt Related Manuscript File VAChTprotonated.txt Related Manuscript File VAChTVSM.mrc Related Manuscript File VAChTapo.mrc Related Manuscript File VAChTACh.mrc Related Manuscript File VAChTprotonated.mrc Related Manuscript File Cite Share Download PDF Status: Under Review 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-8816900","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":588246773,"identity":"4265a951-b29f-4b92-ab3d-b38be7a5fcd8","order_by":0,"name":"Yanqing 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