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Recently, we found the microtubule network was important for regulating the membrane abundance of voltage-gated Kv7.4 potassium channels in vascular smooth muscle. Here, we aimed to investigate the influence of dynein on the microtubule-dependent internalization of the Kv7.4 channel. Patch-clamp recordings from HEK293B cells showed Kv7.4 currents were increased after inhibiting dynein function with ciliobrevin D or by coexpressing p50\/dynamitin, which specifically interferes with dynein motor function. Mutation of a dynein-binding site in the Kv7.4 C terminus increased the Kv7.4 current and prevented p50 interference. Structured illumination microscopy, proximity ligation assays, and coimmunoprecipitation showed colocalization of Kv7.4 and dynein in mesenteric artery myocytes. Ciliobrevin D enhanced mesenteric artery relaxation to activators of Kv7.2\u2013Kv7.5 channels and increased membrane abundance of Kv7.4 protein in isolated smooth muscle cells and HEK293B cells. Ciliobrevin D failed to enhance the negligible S-1\u2013mediated relaxations after morpholino-mediated knockdown of Kv7.4. Mass spectrometry revealed an interaction of dynein with caveolin-1, confirmed using proximity ligation and coimmunoprecipitation assays, which also provided evidence for interaction of caveolin-1 with Kv7.4, confirming that Kv7.4 channels are localized to caveolae in mesenteric artery myocytes. Lastly, cholesterol depletion reduced the interaction of Kv7.4 with caveolin-1 and dynein while increasing the overall membrane expression of Kv7.4, although it attenuated the Kv7.4 current in oocytes and interfered with the action of ciliobrevin D and channel activators in arterial segments. Overall, this study shows that dynein can traffic Kv7.4 channels in vascular smooth muscle in a mechanism dependent on cholesterol-rich caveolae.<\/jats:p>","DOI":"10.1085\/jgp.202012760","type":"journal-article","created":{"date-parts":[[2021,2,6]],"date-time":"2021-02-06T05:16:52Z","timestamp":1612588612000},"update-policy":"https:\/\/doi.org\/10.1085\/jgp.crossmarkpolicy","source":"Crossref","is-referenced-by-count":20,"title":["Dynein regulates Kv7.4 channel trafficking from the cell membrane"],"prefix":"10.1085","volume":"153","author":[{"given":"Jennifer","family":"van der Horst","sequence":"first","affiliation":[{"name":"Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark 1"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3276-5887","authenticated-orcid":true,"given":"Salom\u00e9","family":"Rognant","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark 1"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4552-496X","authenticated-orcid":true,"given":"Geoffrey W.","family":"Abbott","sequence":"additional","affiliation":[{"name":"Bioelectricity Laboratory, Department of Physiology and Biophysics, School of Medicine, University of California, Irvine, CA 2"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4620-8507","authenticated-orcid":true,"given":"Lijo Cherian","family":"Ozhathil","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark 1"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6627-7518","authenticated-orcid":true,"given":"Per","family":"H\u00e4gglund","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark 1"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vincenzo","family":"Barrese","sequence":"additional","affiliation":[{"name":"St. George\u2019s, University of London, London, UK 3"},{"name":"Department of Neuroscience, Reproductive Science and Dentistry, University of Naples \u201cFederico II,\u201d Naples, Italy 4"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christine Y.","family":"Chuang","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark 1"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas","family":"Jespersen","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark 1"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5196-6919","authenticated-orcid":true,"given":"Michael J.","family":"Davies","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark 1"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Iain A.","family":"Greenwood","sequence":"additional","affiliation":[{"name":"St. George\u2019s, University of London, London, UK 3"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8631-3539","authenticated-orcid":true,"given":"Pontus","family":"Gourdon","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark 1"},{"name":"Department of Medical Sciences, Lund University, Lund, Sweden 5"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2618-4015","authenticated-orcid":true,"given":"Christian","family":"Aalkj\u00e6r","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark 1"},{"name":"Department of Biomedicine, Aarhus University, Aarhus, Denmark 6"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9903-714X","authenticated-orcid":true,"given":"Thomas A.","family":"Jepps","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark 1"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"291","published-online":{"date-parts":[[2021,2,3]]},"reference":[{"key":"2023072708385428100_bib1","doi-asserted-by":"publisher","first-page":"R1222","DOI":"10.1152\/ajpregu.00092.2007","article-title":"Arterial remodeling and plasma volume expansion in caveolin-1-deficient mice","volume":"293","author":"Albinsson","year":"2007","journal-title":"Am. 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