Dynein regulates Kv7.4 channel trafficking from the cell membrane

Author:

van der Horst Jennifer1,Rognant Salomé1ORCID,Abbott Geoffrey W.2ORCID,Ozhathil Lijo Cherian1ORCID,Hägglund Per1ORCID,Barrese Vincenzo34,Chuang Christine Y.1,Jespersen Thomas1,Davies Michael J.1ORCID,Greenwood Iain A.3,Gourdon Pontus15ORCID,Aalkjær Christian16ORCID,Jepps Thomas A.1ORCID

Affiliation:

1. Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark

2. Bioelectricity Laboratory, Department of Physiology and Biophysics, School of Medicine, University of California, Irvine, CA

3. St. George’s, University of London, London, UK

4. Department of Neuroscience, Reproductive Science and Dentistry, University of Naples “Federico II,” Naples, Italy

5. Department of Medical Sciences, Lund University, Lund, Sweden

6. Department of Biomedicine, Aarhus University, Aarhus, Denmark

Abstract

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.

Funder

Carlsberg Foundation

Lundbeck Foundation

Danmarks Frie Forskningsfond

Horizon 2020

Marie Skłodowska-Curie

Novo Nordisk Foundation

National Institute of General Medical Sciences

National Institute of Neurological Disorders and Stroke

Publisher

Rockefeller University Press

Subject

Physiology

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