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By occluding the VGKC pore, CTX blocks passive K+ flow\u2014a defining function of these membrane proteins. Due to its mechanistic simplicity and high signal-to-noise ratio, the CTX\u2013VGKC interaction is an ideal system to investigate the molecular details of binding and unbinding. CTX bound to the Shaker VGKC exhibits thermal motion (wobbling) that permits access of external K+ to the channel pore. To test whether this wobbling is part of the reaction pathway during toxin\u2013channel interaction, the energetic role of external K+ was examined in the association and dissociation kinetics. A high-affinity Shaker K427E-VGKC variant was expressed in Xenopus oocytes, and its activity was monitored via two-electrode voltage clamp between \u223c10 and \u223c30\u00b0C. Nanomolar applications of CTX to open and closed channels, in the presence of high external Na+ or high K+ concentrations, were used to measure blockade kinetics at different voltages and temperatures. In high K+, both the dissociation and association rates showed higher activation enthalpies, by \u223c15 kJ\/mol and \u223c25 kJ\/mol, respectively, compared with high Na+ conditions. However, the association rates under high Na+ and K+ were equal at \u223c20\u00b0C, indicating a compensatory K+-induced activation entropy. We propose transient CTX-wobbling intermediates in both directions of the reaction pathway. Such a wobbling intermediate could enhance the diversity of productive collisions during association, increasing the efficacy of the scorpion venom.<\/jats:p>","DOI":"10.1085\/jgp.202413590","type":"journal-article","created":{"date-parts":[[2025,7,2]],"date-time":"2025-07-02T02:20:29Z","timestamp":1751422829000},"update-policy":"https:\/\/doi.org\/10.1085\/jgp.crossmarkpolicy","source":"Crossref","is-referenced-by-count":2,"title":["Charybdotoxin binding to Shaker K+ channels is temperature sensitive in high external K+ but not in high external Na+"],"prefix":"10.1085","volume":"157","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-6398-5929","authenticated-orcid":true,"given":"Nieves","family":"Navarro-Quezada","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00h9jrb69","id-type":"ROR","asserted-by":"publisher"}],"name":"Instituto de Neurociencia, Universidad de Valpara\u00edso 1 , Valpara\u00edso,","place":["Chile"]},{"name":"3Centro Interdisciplinario de Neurociencia de Valpara\u00edso, Valpara\u00edso, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-7749-2654","authenticated-orcid":true,"given":"Francisca","family":"Salas-Sepulveda","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01s4gpq44","id-type":"ROR","asserted-by":"publisher"}],"name":"Universidad de Talca 2 Centro de Bioinform\u00e1tica, Simulaci\u00f3n y Modelado (CBSM), Facultad de Ingenier\u00eda, , Talca,","place":["Chile"]},{"name":"4Doctorado en Ciencias menci\u00f3n Modelado de Sistemas Qu\u00edmicos y Biol\u00f3gicos, Universidad de Talca, Talca, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2650-3565","authenticated-orcid":true,"given":"Horacio","family":"Poblete","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01s4gpq44","id-type":"ROR","asserted-by":"publisher"}],"name":"Universidad de Talca 2 Centro de Bioinform\u00e1tica, Simulaci\u00f3n y Modelado (CBSM), Facultad de Ingenier\u00eda, , Talca,","place":["Chile"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3482-5126","authenticated-orcid":true,"given":"David","family":"Naranjo","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00h9jrb69","id-type":"ROR","asserted-by":"publisher"}],"name":"Instituto de Neurociencia, Universidad de Valpara\u00edso 1 , Valpara\u00edso,","place":["Chile"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"291","published-online":{"date-parts":[[2025,8,7]]},"reference":[{"key":"2025080710564899300_bib1","doi-asserted-by":"publisher","first-page":"1169","DOI":"10.1016\/s0896-6273(00)80143-9","article-title":"Contribution of the S4 segment to gating charge in the Shaker K+ channel","volume":"16","author":"Aggarwal","year":"1996","journal-title":"Neuron"},{"key":"2025080710564899300_bib2","doi-asserted-by":"publisher","first-page":"317","DOI":"10.1085\/jgp.91.3.317","article-title":"Charybdotoxin block of single Ca2+-activated K+ channels. 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