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Nonetheless, their mechanisms of molecular permeation and selectivity remain mostly unexplored. Combining fluorescence microscopy and electrophysiology, we developed a novel technique that allows kinetic analysis of molecular permeation through connexin and CALHM1 channels in Xenopus oocytes rendered translucent. Using this methodology, we found that (1) molecular flux through these channels saturates at low micromolar concentrations, (2) kinetic parameters of molecular transport are sensitive to modulators of channel gating, (3) molecular transport and ionic currents can be differentially affected by mutation and gating, and (4) N-terminal regions of these channels control transport kinetics and permselectivity. Our methodology allows analysis of how human disease\u2013causing mutations affect kinetic properties and permselectivity of molecular signaling and enables the study of molecular mechanisms, including selectivity and saturability, of molecular transport in other large-pore channels.<\/jats:p>","DOI":"10.1085\/jgp.202012607","type":"journal-article","created":{"date-parts":[[2020,9,24]],"date-time":"2020-09-24T02:21:01Z","timestamp":1600914061000},"update-policy":"https:\/\/doi.org\/10.1085\/jgp.crossmarkpolicy","source":"Crossref","is-referenced-by-count":21,"title":["A novel voltage-clamp\/dye uptake assay reveals saturable transport of molecules through CALHM1 and connexin channels"],"prefix":"10.1085","volume":"152","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3373-9138","authenticated-orcid":true,"given":"Pablo S.","family":"Gaete","sequence":"first","affiliation":[{"name":"Department of Pharmacology, Physiology, and Neuroscience, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ 1"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2823-6619","authenticated-orcid":true,"given":"Mauricio A.","family":"Lillo","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Physiology, and Neuroscience, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ 1"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"William","family":"L\u00f3pez","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Physiology, and Neuroscience, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ 1"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7307-1438","authenticated-orcid":true,"given":"Yu","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Physiology, and Neuroscience, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ 1"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6360-3875","authenticated-orcid":true,"given":"Wenjuan","family":"Jiang","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA 2"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3581-754X","authenticated-orcid":true,"given":"Yun","family":"Luo","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA 2"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3782-0550","authenticated-orcid":true,"given":"Andrew L.","family":"Harris","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Physiology, and Neuroscience, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ 1"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9203-1602","authenticated-orcid":true,"given":"Jorge E.","family":"Contreras","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Physiology, and Neuroscience, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ 1"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"291","published-online":{"date-parts":[[2020,10,19]]},"reference":[{"key":"2023072821443742300_bib1","doi-asserted-by":"publisher","first-page":"3257","DOI":"10.1021\/ct300400x","article-title":"Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone \u03c6, \u03c8 and side-chain \u03c7(1) and \u03c7(2) dihedral angles","volume":"8","author":"Best","year":"2012","journal-title":"J. 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