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The mechanism of inhibition and the structural basis of 4AP binding to Kv channels remain unclear. Here, we determined the structure of the Shaker V369I-I372L-S376T (ILT) mutant bound to 4AP at 3.3\u00c5, demonstrating that 4AP binds to the closed state of the channel. This structure is inconsistent with an open channel block mechanism. Electrophysiology experiments show that 4AP binds even when intracellular pore access is constitutively blocked, suggesting that 4AP enters the pore through membrane-facing fenestrations. MD simulations and mutational analysis agree with the proposed fenestration pathway and suggest that 4AP binds in its neutral form. These results support a mechanism where 4AP binds to a partially activated closed state that prevents complete activation of Kv channels, explaining its pharmacological activity.<\/jats:p>","DOI":"10.64898\/2026.09.12.751153","type":"posted-content","created":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T04:20:17Z","timestamp":1789532417000},"source":"Crossref","is-referenced-by-count":0,"title":["Mechanistical and structural basis of Kv channel inhibition by 4-aminopyridine"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0200-1069","authenticated-orcid":false,"given":"Bernardo I","family":"Pinto-Anwandter","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Richa","family":"Agrawal","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA"},{"name":"Center for Mechanical Excitability, The University of Chicago, Chicago, IL 60637, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Trayder","family":"Thomas","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eduardo","family":"Perozo","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA"},{"name":"Center for Mechanical Excitability, The University of Chicago, Chicago, IL 60637, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Beno\u00eet","family":"Roux","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA"},{"name":"Department of Chemistry, The University of Chicago, Chicago, IL 60637, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francisco","family":"Bezanilla","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA"},{"name":"Centro Interdisciplinario de Neurociencias de Valparaiso and Facultad de Ciencias del Mar y Recursos Naturales, Universidad de Valpara\u00edso, Valpara\u00edso, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026091914350796000_2026.09.12.751153v1.1","unstructured":"Hille, B. 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