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Dynein regulates Kv7.4 channel trafficking from the cell membrane

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  • Jennifer van der Horst, University of Copenhagen
  • ,
  • Salomé Rognant, University of Copenhagen
  • ,
  • Geoffrey W. Abbott, University of California at Irvine
  • ,
  • Lijo Cherian Ozhathil, University of Copenhagen
  • ,
  • Per Hägglund, University of Copenhagen
  • ,
  • Vincenzo Barrese, St. George's University of London, University of Naples Federico II
  • ,
  • Christine Y. Chuang, University of Copenhagen
  • ,
  • Thomas Jespersen, University of Copenhagen
  • ,
  • Michael J. Davies, University of Copenhagen
  • ,
  • Iain A. Greenwood, St. George's University of London
  • ,
  • Pontus Gourdon, University of Copenhagen, Lund University
  • ,
  • Christian Aalkjær
  • Thomas A. Jepps, University of Copenhagen

The dynein motor protein transports proteins away from the cell membrane along the microtubule network. 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-Kv7.5 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-mediated 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.

Original languageEnglish
Article numbere202012760
JournalJournal of General Physiology
Number of pages19
Publication statusPublished - 2021

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© 2021 van der Horst et al. This article is distributed under the terms of an Attribution-Noncommercial-Share Alike-No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution-Noncommercial-Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

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