{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,28]],"date-time":"2026-07-28T08:02:08Z","timestamp":1785225728734,"version":"3.55.0"},"reference-count":51,"publisher":"Wiley","issue":"5","license":[{"start":{"date-parts":[[2026,1,29]],"date-time":"2026-01-29T00:00:00Z","timestamp":1769644800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,1,29]],"date-time":"2026-01-29T00:00:00Z","timestamp":1769644800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100004737","name":"National Health Research Institutes","doi-asserted-by":"publisher","award":["NHRI\u2010EX112\u201011236SI"],"award-info":[{"award-number":["NHRI\u2010EX112\u201011236SI"]}],"id":[{"id":"10.13039\/501100004737","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000897","name":"Cystic Fibrosis Foundation","doi-asserted-by":"publisher","award":["Hwang 22G0"],"award-info":[{"award-number":["Hwang 22G0"]}],"id":[{"id":"10.13039\/100000897","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000897","name":"Cystic Fibrosis Foundation","doi-asserted-by":"publisher","award":["Hwang 24G0"],"award-info":[{"award-number":["Hwang 24G0"]}],"id":[{"id":"10.13039\/100000897","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["physoc.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["The Journal of Physiology"],"published-print":{"date-parts":[[2026,3]]},"abstract":"\n Abstract<\/jats:title>\n \n GlyH\u2010101 is a commonly used pore blocker of the cystic fibrosis transmembrane conductance regulator (CFTR) to confirm the functional role of CFTR in model systems. Unlike most other anionic CFTR blockers GlyH\u2010101 blocks CFTR from the extracellular side, albeit previous studies suggesting an additional internal binding site. To explore the detailed mechanism of GlyH\u2010101 block we first examined GlyH\u2010101's effects on a hydrolysis\u2010deficient CFTR mutant (E1371S) whose open probability approaches unity. Whole\u2010cell recordings with extracellularly applied GlyH\u2010101 revealed two phases of current reduction: a rapid initial drop within seconds and a slower decay over tens of seconds. The fast phase blockade exhibited voltage dependence, consistent with previously reported external pore blocking. Single\u2010channel recordings in excised inside\u2010out patches with GlyH\u2010101 in the pipette solution showed a voltage\u2010dependent blockade with |z\u03b4| of 0.38, close to that reported previously. However intracellular application of GlyH\u2010101 also induced a similar voltage\u2010dependent block, suggesting membrane permeation and subsequent development of external block. To decrease the membrane permeability of GlyH\u2010101 we synthesised a hydrophilic analogue GlyH\u2010101\u20101. Application of GlyH\u2010101\u20101 from the cytoplasmic side of the membrane induced voltage\u2010independent CFTR current inhibition. Single\u2010channel recordings revealed two distinct shut states in the presence of cytoplasmic GlyH\u2010101\u20101, consistent with the two\u2010step blocking kinetics previously described for CFTRinh\u2010172. Single\u2010channel kinetics with a hydrophilic CFTRinh\u2010172 analogue further confirmed this inhibitory mechanism. Taken together our findings establish two distinct inhibitory mechanisms for GlyH\u2010101: a voltage\u2010dependent on\u2010off block through the external entrance and a two\u2010step voltage\u2010independent inhibition through the internal entrance.\n \n \n image<\/jats:alt-text>\n <\/jats:graphic>\n <\/jats:boxed-text>\n <\/jats:p>\n <\/jats:sec>\n \n Key points<\/jats:title>\n \n \n \n GlyH\u2010101, a hydrophobic cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor, can diffuse across cell membranes and plug CFTR's pore from both external and internal ends. Although the external block is voltage dependent, the internal block is not.<\/jats:p>\n <\/jats:list-item>\n \n A hydrophilic GlyH\u2010101 derivative is synthesized and shown to block CFTR's pore from its internal entrance in a voltage\u2010independent manner.<\/jats:p>\n <\/jats:list-item>\n \n At the single\u2010channel level our data support a two\u2010step inhibitory mechanism: a fast binding of the blocker in the pore and a slow conformational change step following binding \u2013 a mechanism first proposed for CFTRinh\u2010172.<\/jats:p>\n <\/jats:list-item>\n \n Although CFTRinh\u2010172 itself did not show two\u2010step inhibitory action, leveraging the cryo\u2010EM structure of CFTR\/CFTRinh\u2010172 complex, we synthesized an analogue that indeed blocks CFTR through the same two\u2010step mechanism.<\/jats:p>\n <\/jats:list-item>\n \n Our studies uncovered a common mechanism for CFTR inhibitors and underscore the potential role of structure\u2010based drug design in developing drugs for diseases caused by hyperactive CFTR.<\/jats:p>\n <\/jats:list-item>\n <\/jats:list>\n <\/jats:p>\n <\/jats:sec>","DOI":"10.1113\/jp288949","type":"journal-article","created":{"date-parts":[[2026,1,29]],"date-time":"2026-01-29T15:21:12Z","timestamp":1769700072000},"page":"1944-1970","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Two distinct mechanisms for GlyH\u2010101 block of the CFTR chloride channel"],"prefix":"10.1113","volume":"604","author":[{"given":"Yu\u2010Chen","family":"Chang","sequence":"first","affiliation":[{"name":"School of Medicine National Yang Ming Chiao Tung University Hsinchu City Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhiwei","family":"Ma","sequence":"additional","affiliation":[{"name":"Dalton Cardiovascular Research Center University of Missouri Columbia Missouri 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