{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,31]],"date-time":"2025-10-31T14:09:54Z","timestamp":1761919794966},"reference-count":31,"publisher":"Rockefeller University Press","issue":"5","content-domain":{"domain":["rupress.org"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2014,5,1]]},"abstract":"<jats:p>After endoplasmic reticulum (ER) Ca2+ store depletion, Orai channels in the plasma membrane (PM) are activated directly by ER-resident stromal interacting molecule (STIM) proteins to form the Ca2+-selective Ca2+ release-activated Ca2+ (CRAC) channel. Of the three human Orai channel homologues, only Orai3 can be activated by high concentrations (&amp;gt;50 \u00b5M) of 2-aminoethyl diphenylborinate (2-APB). 2-APB activation of Orai3 occurs without STIM1\u2013Orai3 interaction or store depletion, and results in a cationic, nonselective current characterized by biphasic inward and outward rectification. Here we use cysteine scanning mutagenesis, thiol-reactive reagents, and patch-clamp analysis to define the residues that assist in formation of the 2-APB\u2013activated Orai3 pore. Mutating transmembrane (TM) 1 residues Q83, V77, and L70 to cysteine results in potentiated block by cadmium ions (Cd2+). TM1 mutants E81C, G73A, G73C, and R66C form channels that are not sensitive to 2-APB activation. We also find that Orai3 mutant V77C is sensitive to block by 2-aminoethyl methanethiosulfonate (MTSEA), but not 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). Block induced by reaction with MTSEA is state dependent, as it occurs only when Orai3-V77C channels are opened by either 2-APB or by cotransfection with STIM1 and concurrent passive store depletion. We also analyzed TM3 residue E165. Mutation E165A in Orai3 results in diminished 2-APB\u2013activated currents. However, it has little effect on store-operated current density. Furthermore, mutation E165C results in Cd2+-induced block that is state dependent: Cd2+ only blocks 2-APB\u2013activated, not store-operated, mutant channels. Our data suggest that the dilated pore of 2-APB\u2013activated Orai3 is lined by TM1 residues, but also allows for TM3 E165 to approach the central axis of the channel that forms the conducting pathway, or pore.<\/jats:p>","DOI":"10.1085\/jgp.201411171","type":"journal-article","created":{"date-parts":[[2014,4,15]],"date-time":"2014-04-15T03:27:34Z","timestamp":1397532454000},"page":"621-631","update-policy":"http:\/\/dx.doi.org\/10.1085\/jgp.crossmarkpolicy","source":"Crossref","is-referenced-by-count":21,"title":["State-dependent block of Orai3 TM1 and TM3 cysteine mutants: Insights into 2-APB activation"],"prefix":"10.1085","volume":"143","author":[{"given":"Anna","family":"Amcheslavsky","sequence":"first","affiliation":[{"name":"Department of Physiology and Biophysics, University of California, Irvine, Irvine, CA 92697"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Olga","family":"Safrina","sequence":"additional","affiliation":[{"name":"Department of Physiology and Biophysics, University of California, Irvine, Irvine, CA 92697"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michael D.","family":"Cahalan","sequence":"additional","affiliation":[{"name":"Department of Physiology and Biophysics, University of California, Irvine, Irvine, CA 92697"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"291","published-online":{"date-parts":[[2014,4,14]]},"reference":[{"key":"2023072623193475300_bib1","doi-asserted-by":"publisher","first-page":"405","DOI":"10.1085\/jgp.201311030","article-title":"Orai3 TM3 point mutation G158C alters kinetics of 2-APB-induced gating by disulfide bridge formation with TM2 C101","volume":"142","author":"Amcheslavsky","year":"2013","journal-title":"J. Gen. Physiol."},{"key":"2023072623193475300_bib2","doi-asserted-by":"crossref","first-page":"2213","DOI":"10.1242\/jcs.114.12.2213","article-title":"The organisation and functions of local Ca2+ signals","volume":"114","author":"Bootman","year":"2001","journal-title":"J. Cell Sci."},{"key":"2023072623193475300_bib3","doi-asserted-by":"publisher","first-page":"669","DOI":"10.1038\/ncb0609-669","article-title":"STIMulating store-operated Ca2+ entry","volume":"11","author":"Cahalan","year":"2009","journal-title":"Nat. Cell Biol."},{"key":"2023072623193475300_bib4","doi-asserted-by":"publisher","first-page":"19265","DOI":"10.1074\/jbc.M801535200","article-title":"Complex actions of 2-aminoethyldiphenyl borate on store-operated calcium entry","volume":"283","author":"DeHaven","year":"2008","journal-title":"J. Biol. 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