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Here, we used chimeric channels, targeted mutagenesis, and electrophysiology to define the determinants of DCPIB inhibition and elucidate how drug binding is coupled to channel gating. Effective inhibition required coordinated contributions from extracellular loop 1 (EL1) and transmembrane domain 2 (TM2), which together form a functional module governing access to and stabilization of DCPIB-bound states. Within TM2, a single hydrophobic residue strongly influenced inhibition, indicating that subtle differences in helix packing shape drug sensitivity. Using the homo-heptameric 8C-8A(IL125) channel as a defined model, we found that DCPIB acts exclusively from the extracellular side, inhibits cooperatively, and enhances voltage-dependent inactivation. Mutational analysis showed that DCPIB interacts permissively with R103 of the outer constriction site (OCS) but does not depend on it for inhibition, suggesting that the inhibitor intercalates between subunits within a hydrophobic cleft to achieve stable pore engagement. Charged substitutions within TM2 provided functional evidence that DCPIB penetrates beyond the OCS. Mutations at positions implicated in lipid interactions markedly altered DCPIB efficacy without affecting volume sensitivity, indicating that productive drug\u2013channel interactions, rather than lipid gating itself, are required for inhibition. Finally, charge neutralization on the conserved N-terminal constriction enhanced DCPIB sensitivity, supporting long-range coupling within the pore. Together, these results establish DCPIB as a state-dependent VRAC inhibitor that stabilizes an inactivated channel conformation and define mechanistic principles for the rational design of more potent and selective VRAC inhibitors.<\/jats:p>","DOI":"10.1085\/jgp.202614024","type":"journal-article","created":{"date-parts":[[2026,8,25]],"date-time":"2026-08-25T06:20:32Z","timestamp":1787638832000},"update-policy":"https:\/\/doi.org\/10.1085\/jgp.crossmarkpolicy","source":"Crossref","is-referenced-by-count":0,"title":["State-dependent inhibition of LRRC8 volume-regulated anion channels by DCPIB"],"prefix":"10.1085","volume":"158","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5808-2625","authenticated-orcid":true,"given":"Toshiki","family":"Yamada","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05dq2gs74","id-type":"ROR","asserted-by":"publisher"}],"name":"Vanderbilt University Medical Center 1 Department of Anesthesiology, , Nashville, TN,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6552-3185","authenticated-orcid":true,"given":"Erkan","family":"Karakas","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02vm5rt34","id-type":"ROR","asserted-by":"publisher"}],"name":"Vanderbilt University 2 Department of Molecular Physiology and Biophysics, , Nashville, TN,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0032-8586","authenticated-orcid":true,"given":"Jerod S.","family":"Denton","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05dq2gs74","id-type":"ROR","asserted-by":"publisher"}],"name":"Vanderbilt University Medical Center 1 Department of Anesthesiology, , Nashville, TN,","place":["USA"]},{"id":[{"id":"https:\/\/ror.org\/05dq2gs74","id-type":"ROR","asserted-by":"publisher"}],"name":"Vanderbilt University Medical Center 3 Department of Pharmacology, , Nashville, TN,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"291","published-online":{"date-parts":[[2026,9,22]]},"reference":[{"key":"2026092209403500300_bib1","doi-asserted-by":"publisher","DOI":"10.14814\/phy2.14303","article-title":"The LRRC8 volume-regulated anion channel inhibitor, DCPIB, inhibits mitochondrial respiration independently of the channel","volume":"7","author":"Afzal","year":"2019","journal-title":"Physiol. 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