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.
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February 03 2021
Dynein regulates Kv7.4 channel trafficking from the cell membrane
Jennifer van der Horst
,
Jennifer van der Horst
1
Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
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Salomé Rognant
,
Salomé Rognant
1
Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
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Geoffrey W. Abbott
,
Geoffrey W. Abbott
2
Bioelectricity Laboratory, Department of Physiology and Biophysics, School of Medicine, University of California, Irvine, CA
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Lijo Cherian Ozhathil
,
Lijo Cherian Ozhathil
1
Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
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Per Hägglund
,
Per Hägglund
1
Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
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Vincenzo Barrese
,
Vincenzo Barrese
3
St. George’s, University of London, London, UK
4
Department of Neuroscience, Reproductive Science and Dentistry, University of Naples “Federico II,” Naples, Italy
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Christine Y. Chuang
,
Christine Y. Chuang
1
Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
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Thomas Jespersen
,
Thomas Jespersen
1
Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
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Michael J. Davies
,
Michael J. Davies
1
Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
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Iain A. Greenwood
,
Iain A. Greenwood
3
St. George’s, University of London, London, UK
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Pontus Gourdon
,
Pontus Gourdon
1
Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
5
Department of Medical Sciences, Lund University, Lund, Sweden
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Christian Aalkjær
,
Christian Aalkjær
1
Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
6
Department of Biomedicine, Aarhus University, Aarhus, Denmark
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Thomas A. Jepps
1
Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
Correspondence to Thomas A. Jepps: tjepps@sund.ku.dk
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Jennifer van der Horst
1
Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
Salomé Rognant
1
Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
Geoffrey W. Abbott
2
Bioelectricity Laboratory, Department of Physiology and Biophysics, School of Medicine, University of California, Irvine, CA
Lijo Cherian Ozhathil
1
Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
Per Hägglund
1
Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
Vincenzo Barrese
3
St. George’s, University of London, London, UK
4
Department of Neuroscience, Reproductive Science and Dentistry, University of Naples “Federico II,” Naples, Italy
Christine Y. Chuang
1
Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
Thomas Jespersen
1
Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
Michael J. Davies
1
Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
Iain A. Greenwood
3
St. George’s, University of London, London, UK
Pontus Gourdon
1
Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
5
Department of Medical Sciences, Lund University, Lund, Sweden
Christian Aalkjær
1
Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
6
Department of Biomedicine, Aarhus University, Aarhus, Denmark
Thomas A. Jepps
1
Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
Correspondence to Thomas A. Jepps: tjepps@sund.ku.dk
This work is part of a special collection on myofilament function and disease.
Received:
September 20 2020
Revision Received:
December 21 2020
Accepted:
January 08 2021
Online Issn: 1540-7748
Print Issn: 0022-1295
Funding:
Carlsbergfondet
(CF16-0136)
Danmarks Frie Forskningsfond
(9039-00409B)
H2020 Marie Skłodowska-Curie Actions
(801199)
Horizon 2020
(NO AWARD)
Lundbeckfonden
(R323-2018-3674)
National Institute of General Medical Sciences
(GM130377)
National Institute of Neurological Disorders and Stroke
(NS107671)
Novo Nordisk Fonden
(NNF13OC0004294, NNF18OC0031634)
© 2021 van der Horst et al.
2021
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/).
J Gen Physiol (2021) 153 (3): e202012760.
Article history
Received:
September 20 2020
Revision Received:
December 21 2020
Accepted:
January 08 2021
Citation
Jennifer van der Horst, Salomé Rognant, Geoffrey W. Abbott, Lijo Cherian Ozhathil, Per Hägglund, Vincenzo Barrese, Christine Y. Chuang, Thomas Jespersen, Michael J. Davies, Iain A. Greenwood, Pontus Gourdon, Christian Aalkjær, Thomas A. Jepps; Dynein regulates Kv7.4 channel trafficking from the cell membrane. J Gen Physiol 1 March 2021; 153 (3): e202012760. doi: https://doi.org/10.1085/jgp.202012760
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