{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,26]],"date-time":"2026-05-26T14:11:25Z","timestamp":1779804685065,"version":"3.53.1"},"reference-count":45,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2026,5,26]],"date-time":"2026-05-26T00:00:00Z","timestamp":1779753600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"ANID Grants FONDECYT Iniciacion","award":["11230178"],"award-info":[{"award-number":["11230178"]}]},{"name":"FONDECYT Iniciacion","award":["11241081"],"award-info":[{"award-number":["11241081"]}]},{"name":"Proyecto de EXPLORACION","award":["13240083"],"award-info":[{"award-number":["13240083"]}]},{"name":"Proyecto Nucleo UNAB","award":["DI-02-23\/NUC"],"award-info":[{"award-number":["DI-02-23\/NUC"]}]},{"name":"FONDECYT Regular","award":["1240695"],"award-info":[{"award-number":["1240695"]}]},{"name":"FONDEQUIP","award":["EQM230079"],"award-info":[{"award-number":["EQM230079"]}]},{"name":"FONDEQUIP","award":["FOVI230118"],"award-info":[{"award-number":["FOVI230118"]}]},{"name":"FONDEQUIP","award":["ICN2025_026"],"award-info":[{"award-number":["ICN2025_026"]}]},{"name":"Instituto Milenio Centro Interdisciplinario de Neurociencias de Valpara\u00edso"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The proton-activated chloride channel PAC\/TMEM206 is broadly expressed in mammalian tissues and contributes to acid-induced cell injury in pathological settings such as ischemia, inflammation, and tumor acidosis. In addition to its established proton sensitivity, PAC is strongly modulated by temperature: heating potentiates proton-evoked currents and shifts activation toward less acidic pH yet does not open the channel at neutral pH. How proton and thermal inputs are structurally integrated remains unclear. Here, we combined site-specific incorporation of the environmentally sensitive fluorescent amino acid ANAP with automated patch-clamp electrophysiology and molecular dynamics simulations to identify structural elements underlying PAC thermal modulation. ANAP reporters introduced across the extracellular domain and vestibule\u2013pore coupling region revealed residue-specific thermal- and proton-dependent spectral shifts, showing that heating and acidification remodel overlapping but non-identical local environments. The strongest ANAP-reported temperature-dependent changes were observed at R93, Y111, F196, R237, and F282, whereas proton-dependent changes prominently involved R93, Y111, H130, and R237. Several temperature-sensitive ANAP reporters mapped to an intersubunit region also highlighted by dynamic correlation analysis. Together, our results identify structural correlates of PAC thermal modulation consistent with a model in which protonation creates an activation-permissive landscape while heating reweights coupling across an intersubunit scaffold; this model generates testable predictions that should be addressed by targeted mutagenesis and thermodynamic characterization.<\/jats:p>","DOI":"10.3390\/ijms27114784","type":"journal-article","created":{"date-parts":[[2026,5,26]],"date-time":"2026-05-26T13:28:21Z","timestamp":1779802101000},"page":"4784","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Conformational Fingerprints Underlying Thermal Modulation of PAC\/TMEM206 Gating"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-7096-1530","authenticated-orcid":false,"given":"Rachel","family":"Reyes-Lizana","sequence":"first","affiliation":[{"name":"Center for Bioinformatics and Integrative Biology, Universidad Andres Bello, Santiago 8370146, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"German","family":"Fern\u00e1ndez","sequence":"additional","affiliation":[{"name":"Center for Bioinformatics and Integrative Biology, Universidad Andres Bello, Santiago 8370146, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Scarleth","family":"Duran-Morales","sequence":"additional","affiliation":[{"name":"Center for Bioinformatics and Integrative Biology, Universidad Andres Bello, Santiago 8370146, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-2518-6780","authenticated-orcid":false,"given":"Guillermo","family":"Carrasco-Faus","sequence":"additional","affiliation":[{"name":"Center for Bioinformatics and Integrative Biology, Universidad Andres Bello, Santiago 8370146, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yorley","family":"Duarte","sequence":"additional","affiliation":[{"name":"Center for Bioinformatics and Integrative Biology, Universidad Andres Bello, Santiago 8370146, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Valeria","family":"Marquez-Miranda","sequence":"additional","affiliation":[{"name":"Center for Bioinformatics and Integrative Biology, Universidad Andres Bello, Santiago 8370146, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5394-322X","authenticated-orcid":false,"given":"Ignacio","family":"D\u00edaz-Franulic","sequence":"additional","affiliation":[{"name":"Center for Bioinformatics and Integrative Biology, Universidad Andres Bello, Santiago 8370146, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"DiBartola, S.P. (2012). Chapter 9\u2014Introduction to Acid-Base Disorders. Fluid, Electrolyte, and Acid-Base Disorders in Small Animal Practice, W.B. Saunders. [4th ed.].","DOI":"10.1016\/B978-1-4377-0654-3.00016-0"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"12825","DOI":"10.1073\/pnas.202474099","article-title":"Hypoxia and acidosis activate cardiac myocyte death through the Bcl-2 family protein BNIP3","volume":"99","author":"Kubasiak","year":"2002","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"354","DOI":"10.1111\/imm.12917","article-title":"Hypoxia and acidosis: Immune suppressors and therapeutic targets","volume":"154","author":"Damgaci","year":"2018","journal-title":"Immunology"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1016\/j.tibs.2015.12.001","article-title":"The Warburg Effect: How Does it Benefit Cancer Cells?","volume":"41","author":"Liberti","year":"2016","journal-title":"Trends Biochem. Sci."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1007\/s10555-019-09786-5","article-title":"Pro-metastatic functions of lipoproteins and extracellular vesicles in the acidic tumor microenvironment","volume":"38","author":"Belting","year":"2019","journal-title":"Cancer Metastasis Rev."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1007\/s13402-024-00969-z","article-title":"Acidity and hypoxia of tumor microenvironment, a positive interplay in extracellular vesicle release by tumor cells","volume":"48","author":"Peppicelli","year":"2025","journal-title":"Cell. Oncol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"C699","DOI":"10.1152\/ajpcell.00188.2012","article-title":"Structure and activity of the acid-sensing ion channels","volume":"303","author":"Sherwood","year":"2012","journal-title":"Am. J. Physiol. Cell Physiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"295","DOI":"10.5483\/BMBRep.2013.46.6.121","article-title":"Acid-sensing ion channels (ASICs): Therapeutic targets for neurological diseases and their regulation","volume":"46","author":"Kweon","year":"2013","journal-title":"BMB Rep."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"20180108","DOI":"10.1098\/rsif.2018.0108","article-title":"Voltage and pH sensing by the voltage-gated proton channel, H(V)1","volume":"15","author":"DeCoursey","year":"2018","journal-title":"J. R. Soc. Interface"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"423","DOI":"10.1038\/s41422-025-01092-w","article-title":"Structural basis of stepwise proton sensing-mediated GPCR activation","volume":"35","author":"Yue","year":"2025","journal-title":"Cell Res."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"334","DOI":"10.1016\/j.semnephrol.2006.07.001","article-title":"Na+\/H+ exchangers in renal regulation of acid-base balance","volume":"26","author":"Bobulescu","year":"2006","journal-title":"Semin. Nephrol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"657","DOI":"10.1038\/ki.2013.138","article-title":"SLC26 Cl\u2212\/HCO3\u2212 exchangers in the kidney: Roles in health and disease","volume":"84","author":"Soleimani","year":"2013","journal-title":"Kidney Int."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Kittl, M., Helm, K., Beyreis, M., Mayr, C., Gaisberger, M., Winklmayr, M., Ritter, M., and Jakab, M. (2019). Acid- and Volume-Sensitive Chloride Currents in Microglial Cells. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20143475"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1007\/s00424-006-0193-z","article-title":"Role of acid-sensitive outwardly rectifying anion channels in acidosis-induced cell death in human epithelial cells","volume":"454","author":"Wang","year":"2007","journal-title":"Pflug. Arch. Eur. J. Physiol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"19230","DOI":"10.1074\/jbc.M301096200","article-title":"A Novel Voltage-dependent Chloride Current Activated by Extracellular Acidic pH in Cultured Rat Sertoli Cells","volume":"278","author":"Auzanneau","year":"2003","journal-title":"J. Biol. Chem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"e49187","DOI":"10.7554\/eLife.49187","article-title":"Identification of TMEM206 proteins as pore of PAORAC\/ASOR acid-sensitive chloride channels","volume":"8","author":"Ullrich","year":"2019","journal-title":"eLife"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"395","DOI":"10.1126\/science.aav9739","article-title":"PAC, an evolutionarily conserved membrane protein, is a proton-activated chloride channel","volume":"364","author":"Yang","year":"2019","journal-title":"Science"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"108683","DOI":"10.1016\/j.celrep.2020.108683","article-title":"Proton-activated chloride channel PAC regulates endosomal acidification and transferrin receptor-mediated endocytosis","volume":"34","author":"Yang","year":"2021","journal-title":"Cell Rep."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"181","DOI":"10.1007\/164_2023_673","article-title":"Physiological Functions of the Volume-Regulated Anion Channel VRAC\/LRRC8 and the Proton-Activated Chloride Channel ASOR\/TMEM206","volume":"283","author":"Kostritskaia","year":"2024","journal-title":"Handb. Exp. Pharmacol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"923","DOI":"10.1007\/s00424-024-02964-7","article-title":"Functional expression of the proton sensors ASIC1a, TMEM206, and OGR1 together with BK(Ca) channels is associated with cell volume changes and cell death under strongly acidic conditions in DAOY medulloblastoma cells","volume":"476","author":"Pissas","year":"2024","journal-title":"Pflug. Arch. Eur. J. Physiol."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Melek, K., Hauert, B., and Kappel, S. (2024). TMEM206 Contributes to Cancer Hallmark Functions in Colorectal Cancer Cells and Is Regulated by p53 in a p21-Dependent Manner. Cells, 13.","DOI":"10.3390\/cells13221825"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"278","DOI":"10.4161\/chan.27748","article-title":"Temperature sensitivity of acid-sensitive outwardly rectifying (ASOR) anion channels in cortical neurons is involved in hypothermic neuroprotection against acidotoxic necrosis","volume":"8","author":"Numata","year":"2014","journal-title":"Channels"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1535","DOI":"10.1007\/s00424-013-1296-y","article-title":"Acid-sensitive outwardly rectifying (ASOR) anion channels in human epithelial cells are highly sensitive to temperature and independent of ClC-3","volume":"465","author":"Numata","year":"2013","journal-title":"Pflug. Arch. Eur. J. Physiol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/B978-0-12-800181-3.00002-6","article-title":"Temperature sensing by thermal TRP channels: Thermodynamic basis and molecular insights","volume":"74","author":"Feng","year":"2014","journal-title":"Curr. Top. Membr."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2211","DOI":"10.1042\/BST20210301","article-title":"Thermodynamic and structural basis of temperature-dependent gating in TRP channels","volume":"49","author":"Verdugo","year":"2021","journal-title":"Biochem. Soc. Trans."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"19492","DOI":"10.1073\/pnas.1117485108","article-title":"A thermodynamic framework for understanding temperature sensing by transient receptor potential (TRP) channels","volume":"108","author":"Clapham","year":"2011","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"20298","DOI":"10.1073\/pnas.2004303117","article-title":"A folding reaction at the C-terminal domain drives temperature sensing in TRPM8 channels","volume":"117","author":"Raddatz","year":"2020","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1113\/jphysiol.2005.088377","article-title":"Gating of TRP channels: A voltage connection?","volume":"567","author":"Nilius","year":"2005","journal-title":"J. Physiol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"35438","DOI":"10.1074\/jbc.M114.612713","article-title":"Temperature and voltage coupling to channel opening in transient receptor potential melastatin 8 (TRPM8)","volume":"289","author":"Raddatz","year":"2014","journal-title":"J. Biol. Chem."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"8633","DOI":"10.1073\/pnas.1922714117","article-title":"A paradigm of thermal adaptation in penguins and elephants by tuning cold activation in TRPM8","volume":"117","author":"Yang","year":"2020","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/bs.mie.2021.01.048","article-title":"ANAP: A versatile, fluorescent probe of ion channel gating and regulation","volume":"654","author":"Puljung","year":"2021","journal-title":"Methods Enzymol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"eabm3942","DOI":"10.1126\/sciadv.abm3942","article-title":"Gating choreography and mechanism of the human proton-activated chloride channel ASOR","volume":"8","author":"Wang","year":"2022","journal-title":"Sci. Adv."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"4764","DOI":"10.1021\/cr400355w","article-title":"Cellular Incorporation of Unnatural Amino Acids and Bioorthogonal Labeling of Proteins","volume":"114","author":"Lang","year":"2014","journal-title":"Chem. Rev."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"3790","DOI":"10.1038\/s41467-020-17582-x","article-title":"Molecular mechanisms underlying menthol binding and activation of TRPM8 ion channel","volume":"11","author":"Xu","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"103395","DOI":"10.1016\/j.isci.2021.103395","article-title":"Ion permeation controlled by hydrophobic residues and proton binding in the proton-activated chloride channel","volume":"24","author":"Cai","year":"2021","journal-title":"iScience"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"e2200727119","DOI":"10.1073\/pnas.2200727119","article-title":"Molecular determinants of pH sensing in the proton-activated chloride channel","volume":"119","author":"Kots","year":"2022","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"e82955","DOI":"10.7554\/eLife.82955","article-title":"Molecular mechanism underlying desensitization of the proton-activated chloride channel PAC","volume":"11","author":"Ruan","year":"2022","journal-title":"eLife"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"350","DOI":"10.1038\/s41586-020-2875-7","article-title":"Structures and pH-sensing mechanism of the proton-activated chloride channel","volume":"588","author":"Ruan","year":"2020","journal-title":"Nature"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"2160","DOI":"10.1016\/j.bpj.2013.03.055","article-title":"The role of allosteric coupling on thermal activation of thermo-TRP channels","volume":"104","author":"Islas","year":"2013","journal-title":"Biophys. J."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"12540","DOI":"10.1021\/ja4059553","article-title":"A genetically encoded fluorescent probe in mammalian cells","volume":"135","author":"Chatterjee","year":"2013","journal-title":"J. Am. Chem. Soc."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"779","DOI":"10.1006\/jmbi.1993.1626","article-title":"Comparative protein modelling by satisfaction of spatial restraints","volume":"234","author":"Sali","year":"1993","journal-title":"J. Mol. Biol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/0263-7855(96)00018-5","article-title":"VMD: Visual molecular dynamics","volume":"14","author":"Humphrey","year":"1996","journal-title":"J. Mol. Graph."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Gowers, R., Linke, M., Barnoud, J., Reddy, T., Melo, M., Seyler, S.L., Doma\u0144ski, J., Dotson, D., Buchoux, S., and Kenney, I. (2016). MDAnalysis: A Python Package for the Rapid Analysis of Molecular Dynamics Simulations, Los Alamos National Laboratory.","DOI":"10.25080\/Majora-629e541a-00e"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1147","DOI":"10.1016\/S0006-3495(97)78147-5","article-title":"Molecular dynamics study of time-correlated protein domain motions and molecular flexibility: Cytochrome P450BM-3","volume":"73","author":"Arnold","year":"1997","journal-title":"Biophys. J."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"2695","DOI":"10.1093\/bioinformatics\/btl461","article-title":"Bio3d: An R package for the comparative analysis of protein structures","volume":"22","author":"Grant","year":"2006","journal-title":"Bioinformatics"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/11\/4784\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,26]],"date-time":"2026-05-26T13:28:47Z","timestamp":1779802127000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/11\/4784"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,26]]},"references-count":45,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2026,6]]}},"alternative-id":["ijms27114784"],"URL":"https:\/\/doi.org\/10.3390\/ijms27114784","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,26]]}}}