Thermoring basis for the proton-driven heat activation of a sodium channel in myriapods

Thermoring basis for the proton-driven heat activation of a cation-selective channel in myriapods | 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 Research Article Thermoring basis for the proton-driven heat activation of a cation-selective channel in myriapods Guangyu Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7504001/v2 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Dec, 2025 Read the published version in Scientific Reports → Version 2 posted You are reading this latest preprint version Show more versions Abstract Thermo-gated ion channels undergo significant time-dependent structural rearrangements for activation. Although the tertiary noncovalent interaction networks can be constrained as thermorings to explain specific temperature thresholds and sensitivity, the timing of critical intra-subunit and inter-subunit noncovalent interactions unfolding remains poorly understood. Here the tertiary noncovalent interaction networks of the broad-range thermal receptor 1 (BRTNaC1) in myriapods were quantified by the thermoring model to estimate thermal thresholds and structural unfolding sequences. The results showed that the theoretical threshold of the weakest intra-subunit noncovalent interaction in the resting closed state of the D217N/E218Q mutant at low temperature matched the experimental activation threshold. Further, the strong inter-subunit swapping interactions at H352, although weakened before strong acid activation of the native channel below pH 4.5, were absent before weak acid-induced heat activation of the mutant at pH 7. Therefore, the different unfolding sequence from strong inter-subunit interactions to the weakest intra-subunit bridges may be required for proton-driven heat activation of BRTNaC1. Computational Biology Biophysics ion channel gating noncovalent thermoring structure thermosensitivity thermostability threshold unfolding sequence Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Full Text Additional Declarations The authors declare no competing interests. Supplementary Files SupportinginformationforBRTNaC1acidandheatcouplingR1.0.pdf Cite Share Download PDF Status: Published Journal Publication published 14 Dec, 2025 Read the published version in Scientific Reports → Version 2 posted You are reading this latest preprint version Show more versions 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. 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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-7504001","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":509581992,"identity":"50cbf7a6-96c6-49b4-9f5e-a06d0afd09f0","order_by":0,"name":"Guangyu 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18:10:37","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":146751,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7504001/v2/ec6a2a2fbb841dea70b22d9f.html"},{"id":96660404,"identity":"82fcd2cb-85ac-4ead-aa19-401640b23304","added_by":"auto","created_at":"2025-11-24 18:10:36","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":353340,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThermoring structures of closed\u003c/strong\u003e \u003cstrong\u003eBRTNaC1 at pH 8 and 4 °C. \u003c/strong\u003e(\u003cstrong\u003ea) \u003c/strong\u003eThe grid-like noncovalently interacting mesh network along the gating pathway of\u003cstrong\u003e \u003c/strong\u003eclosed BRTNaC1 based on cryo-EM data of a single subunit at pH 8 and 4 °C\u003cstrong\u003e \u003c/strong\u003e(PDB ID: 8YMR).\u0026nbsp; Along the gating pathway from R53 to F414, in addition to black disulfide bonds, salt bridges, pi interactions, and H-bonds between paired amino acid side chains are denoted in purple, green, and orange, respectively. The specific grid sizes necessary to regulate the least-stable noncovalent interactions in the grids are indicated with black numbers. \u0026nbsp;The weakest E84-R268 bridge in the biggest Grid\u003csub\u003e11\u003c/sub\u003e is highlighted in orange.\u0026nbsp; The total grid sizes and the total grid size-controlled noncovalent interactions along the gating pathway from R53 to F414 are displayed in cyan and black circles, respectively. (\u003cstrong\u003eb\u003c/strong\u003e) The swapping interactions at H352. (\u003cstrong\u003ec) \u003c/strong\u003eThe location of the weakest E84-R268 bridges. (\u003cstrong\u003ed) \u003c/strong\u003eThe structure of the biggest Grid\u003csub\u003e11 \u003c/sub\u003ewith an 11-residue size to regulate the E84-R268 bridge. (\u003cstrong\u003ee)\u003c/strong\u003e The sequences of the biggest Grid\u003csub\u003e11\u003c/sub\u003e to control the highlighted E84-R268 bridge in the blue box.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7504001/v2/fce90dee5c2000674df4efb0.jpg"},{"id":96709931,"identity":"ca42f981-9893-4f9a-b91f-be4d37274fb0","added_by":"auto","created_at":"2025-11-25 10:09:48","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":311503,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThermoring structures of closed\u003c/strong\u003e \u003cstrong\u003eBRTNaC1/D217N/E218Q at pH 7 and 4 °C.\u003c/strong\u003e (\u003cstrong\u003ea) \u003c/strong\u003eThe grid-like noncovalently interacting mesh network along the gating pathway of\u003cstrong\u003e \u003c/strong\u003eclosed BRTNaC1/D217N/E218Q based on cryo-EM data of a single subunit at pH 7 and 4 °C\u003cstrong\u003e \u003c/strong\u003e(PDB ID: 8YMW).\u0026nbsp; Along the gating pathway from R56 to V411, in addition to black disulfide bonds, salt bridges, pi interactions, and H-bonds between paired amino acid side chains are denoted in purple, green, and orange, respectively. The specific grid sizes necessary to regulate the least-stable noncovalent interactions in the grids are indicated by black numbers.\u0026nbsp; The weakest I69-F397 bridge in the biggest Grid\u003csub\u003e14\u003c/sub\u003e is highlighted in orange.\u0026nbsp; The total grid sizes and the total grid size-controlled noncovalent interactions along the gating pathway from R56 to V411 are displayed in cyan and black circles, respectively. (\u003cstrong\u003eb\u003c/strong\u003e) The swapping interactions at H352. (c\u003cstrong\u003e) \u003c/strong\u003eThe location of the weakest I69-F397 bridges. (\u003cstrong\u003ed) \u003c/strong\u003eThe structure of the biggest Grid\u003csub\u003e14 \u003c/sub\u003ewith a 14-residue size to regulate the I69-F397 bridge. (\u003cstrong\u003ee)\u003c/strong\u003e The sequences of the biggest Grid\u003csub\u003e14\u003c/sub\u003e to control the highlighted I69-F397 bridge in the blue box.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7504001/v2/c04d5e02c962e6b6aec4b109.jpg"},{"id":96709876,"identity":"73b6b585-89dc-453e-8ee6-231eba73ca87","added_by":"auto","created_at":"2025-11-25 10:09:44","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":320611,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThermoring structures of open\u003c/strong\u003e \u003cstrong\u003eBRTNaC1/D217N/E218Q at pH 7 and 40 °C. \u003c/strong\u003e(\u003cstrong\u003ea) \u003c/strong\u003eThe grid-like noncovalently interacting mesh network along the gating pathway of\u003cstrong\u003e \u003c/strong\u003eopen BRTNaC1/D217N/E218Q based on cryo-EM data of a single subunit at pH 7 and 40 °C\u003cstrong\u003e \u003c/strong\u003e(PDB ID: 8YMX).\u0026nbsp; Along the gating pathway from V63 to I400, in addition to a black disulfide bond, salt bridges, pi interactions, and H-bonds between paired amino acid side chains are denoted in purple, green, and orange, respectively. The specific grid sizes necessary to regulate the least-stable noncovalent interactions in the grids are indicated with black numbers. \u0026nbsp;The first weakest R244-E332 bridge in the biggest Grid\u003csub\u003e7\u003c/sub\u003e is highlighted in orange while the second weakest P101-Y346 bridge is marked in yellow. The total grid sizes and the total grid size-controlled noncovalent interactions along the gating pathway from V63 to I400 are displayed in cyan and black circles, respectively. (\u003cstrong\u003eb\u003c/strong\u003e) The swapping interactions at H352. (\u003cstrong\u003ec) \u003c/strong\u003eThe location of the first weakest R244-E332 bridges. (\u003cstrong\u003ed) \u003c/strong\u003eThe structure of the biggest Grid\u003csub\u003e7 \u003c/sub\u003ewith a 7-residue size to regulate the R244-E332 bridge. (\u003cstrong\u003ee)\u003c/strong\u003e The sequences of the biggest Grid\u003csub\u003e7\u003c/sub\u003e to control the highlighted R244-E332 bridge in the blue box.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7504001/v2/b7b5d8e0ea838dc73f1eec7d.jpg"},{"id":96660407,"identity":"c895707f-9c91-467d-a511-ef1f337c858c","added_by":"auto","created_at":"2025-11-24 18:10:36","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":315319,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThermoring structures of closed\u003c/strong\u003e \u003cstrong\u003eBRTNaC1 at pH 4 and 4 °C.\u003c/strong\u003e (\u003cstrong\u003ea) \u003c/strong\u003eThe grid-like noncovalently interacting mesh network along the gating pathway of\u003cstrong\u003e \u003c/strong\u003eclosed BRTNaC1 based on cryo-EM data of a single subunit at pH 4 and 4 °C\u003cstrong\u003e \u003c/strong\u003e(PDB ID: 8YMS).\u0026nbsp; Along the gating pathway from R53 to F414, in addition to black disulfide bonds, salt bridges, pi interactions, and H-bonds between paired amino acid side chains are denoted in purple, green, and orange, respectively. The specific grid sizes necessary to regulate the least-stable noncovalent interactions in the grids are indicated with black numbers. \u0026nbsp;The weakest H77-D389 bridge in the biggest Grid\u003csub\u003e9\u003c/sub\u003e is highlighted in orange.\u0026nbsp; The total grid sizes and the total grid size-controlled noncovalent interactions along the gating pathway from R56 to V411 are displayed in cyan and black circles, respectively. (\u003cstrong\u003eb\u003c/strong\u003e) The swapping interactions at H352. (\u003cstrong\u003ec) \u003c/strong\u003eThe location of the weakest H77-D389 bridges. (\u003cstrong\u003ed) \u003c/strong\u003eThe structure of the biggest Grid\u003csub\u003e9 \u003c/sub\u003ewith a 9-residue size to regulate the H77-D389 bridge. (\u003cstrong\u003ee)\u003c/strong\u003e The sequences of the biggest Grid\u003csub\u003e9\u003c/sub\u003e to control the highlighted H77-D389 bridge in the blue box.\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7504001/v2/eb2bea32775b280d26560329.jpg"},{"id":96660415,"identity":"dd4d7276-09d7-463a-b272-b11e86e27523","added_by":"auto","created_at":"2025-11-24 18:10:36","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":322406,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThermoring structures of open\u003c/strong\u003e \u003cstrong\u003eBRTNaC1 at pH 4 and 4 °C.\u003c/strong\u003e (\u003cstrong\u003ea) \u003c/strong\u003eThe grid-like noncovalently interacting mesh network along the gating pathway of\u003cstrong\u003e \u003c/strong\u003eopen BRTNaC1 based on cryo-EM data of a single subunit at pH 4 and 4 °C\u003cstrong\u003e \u003c/strong\u003e(PDB ID: 8YMU).\u0026nbsp; Along the gating pathway from R53 to F414, in addition to a black disulfide bond, salt bridges, pi interactions, and H-bonds between paired amino acid side chains are denoted in purple, green, and orange, respectively. The specific grid sizes necessary to regulate the least-stable noncovalent interactions in the grids are indicated with black numbers.\u0026nbsp; The weakest K122-E218 bridge in the biggest Grid\u003csub\u003e7’ \u003c/sub\u003eis highlighted in orange.\u0026nbsp; The total grid sizes and the total grid size-controlled noncovalent interactions along the gating pathway from R53 to F414 are displayed in cyan and black circles, respectively. (\u003cstrong\u003eb\u003c/strong\u003e) The swapping interactions at H352. (\u003cstrong\u003ec) \u003c/strong\u003eThe location of the weakest K122-E218 bridges. (\u003cstrong\u003ed) \u003c/strong\u003eThe structure of the biggest Grid\u003csub\u003e7’ \u003c/sub\u003ewith a 7-residue size to regulate the K122-E218 bridge. (\u003cstrong\u003ee)\u003c/strong\u003e The sequences of the biggest Grid\u003csub\u003e7’\u003c/sub\u003e to control the highlighted K122-E218 bridge in the blue box.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7504001/v2/bc74896941c0951399da80e6.jpg"},{"id":96709445,"identity":"638835e4-7105-4e37-b499-794e3544dc71","added_by":"auto","created_at":"2025-11-25 10:09:01","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":137268,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTentative model on the proton-switched heat activation of BRTNaC1.\u003c/strong\u003e Cryo-EM structures of closed BRTNaC1 at pH 8 and 4 °C (PDB ID: 8YMR), closed BRTNaC1/D217N/E218Q at pH 7 and 4 °C (PDB ID: 8YMW), activated BRTNaC1 at pH 4 and 4 °C (PDB ID: 8YMU), and open BRTNaC1/D217N/E218Q at pH 7 and 40 °C (PDB ID: 8YMX) were used to create this model. The strong swapping p interactions at H352 close the channel above pH 6.5. However, primary protonation of H352 below pH 6.5 disrupts these interactions, leading to a series of conformational rearrangements from Q212 to E218 via S360, resulting in channel opening above 33 °C.\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7504001/v2/912a4870f00713a2dfed780b.jpg"},{"id":98341565,"identity":"dd2930db-dcd4-4581-95fe-7167c0b7e19c","added_by":"auto","created_at":"2025-12-16 17:46:17","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3455025,"visible":true,"origin":"","legend":"","description":"","filename":"ThermoringbasisforacidinducedheatactivationofBRTNaC1R1.6clear.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7504001/v2_covered_78d6715c-4905-470b-a2cc-e3fef3969acd.pdf"},{"id":96660409,"identity":"21092ae2-15b1-4a06-8942-27317ec15966","added_by":"auto","created_at":"2025-11-24 18:10:36","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":229490,"visible":true,"origin":"","legend":"","description":"","filename":"SupportinginformationforBRTNaC1acidandheatcouplingR1.0.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7504001/v2/120b65200844860970e86648.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eThermoring basis for the proton-driven heat activation of a cation-selective channel in myriapods\u003c/p\u003e","fulltext":[],"fulltextSource":"","fullText":"","funders":[{"identity":"833b8e07-ec4f-421c-9f6c-c9c05f9751e8","identifier":"10.13039/100000968","name":"American Heart Association","awardNumber":"10SDG4120011","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"University of Dalifornia Davis","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ion channel gating, noncovalent thermoring structure, thermosensitivity, thermostability, threshold, unfolding sequence","lastPublishedDoi":"10.21203/rs.3.rs-7504001/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7504001/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThermo-gated ion channels undergo significant time-dependent structural rearrangements for activation. Although the tertiary noncovalent interaction networks can be constrained as thermorings to explain specific temperature thresholds and sensitivity, the timing of critical intra-subunit and inter-subunit noncovalent interactions unfolding remains poorly understood. Here the tertiary noncovalent interaction networks of the broad-range thermal receptor 1 (BRTNaC1) in myriapods were quantified by the thermoring model to estimate thermal thresholds and structural unfolding sequences. The results showed that the theoretical threshold of the weakest intra-subunit noncovalent interaction in the resting closed state of the D217N/E218Q mutant at low temperature matched the experimental activation threshold. Further, the strong inter-subunit swapping interactions at H352, although weakened before strong acid activation of the native channel below pH 4.5, were absent before weak acid-induced heat activation of the mutant at pH 7. Therefore, the different unfolding sequence from strong inter-subunit interactions to the weakest intra-subunit bridges may be required for proton-driven heat activation of BRTNaC1.\u003c/p\u003e","manuscriptTitle":"Thermoring basis for the proton-driven heat activation of a cation-selective channel in myriapods","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2025-11-24 18:10:31","doi":"10.21203/rs.3.rs-7504001/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2025-09-03 09:26:37","doi":"10.21203/rs.3.rs-7504001/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"43ee4efc-79ce-40f4-ace3-56b2fcffba09","owner":[],"postedDate":"November 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":57884425,"name":"Computational Biology"},{"id":57884426,"name":"Biophysics"}],"tags":[],"updatedAt":"2025-12-16T17:46:10+00:00","versionOfRecord":{"articleIdentity":"rs-7504001","link":"https://doi.org/10.1038/s41598-025-31732-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-15 00:00:00","publishedOnDateReadable":"December 15th, 2025"},"versionCreatedAt":"2025-11-24 18:10:31","video":"","vorDoi":"10.1038/s41598-025-31732-5","vorDoiUrl":"https://doi.org/10.1038/s41598-025-31732-5","workflowStages":[]},"version":"v2","identity":"rs-7504001","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7504001","identity":"rs-7504001","version":["v2"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}