L-type CaV1.2 current (ICa,L) links electrical excitation to contraction in cardiac myocytes. ICa,L is tightly regulated to control cardiac output. Rad is a Ras-related, monomeric protein that binds to L-type calcium channel β subunits (CaVβ) to promote inhibition of ICa,L. In addition to CaVβ interaction conferred by the Rad core motif, the highly conserved Rad C-terminus can direct membrane association in vitro and inhibition of ICa,L in immortalized cell lines. In this work, we test the hypothesis that in cardiomyocytes the polybasic C-terminus of Rad confers t-tubular localization, and that membrane targeting is required for Rad-dependent ICa,L regulation. We introduced a 3xFlag epitope to the N-terminus of the endogenous mouse Rrad gene to facilitate analysis of subcellular localization. Full-length 3xFlag-Rad (Flag-Rad) mice were compared with a second transgenic mouse model, in which the extended polybasic C-termini of 3xFlag-Rad was truncated at alanine 277 (Flag-RadΔCT). Ventricular cardiomyocytes were isolated for anti-Flag-Rad immunocytochemistry and ex vivo electrophysiology. Full-length Flag-Rad showed a repeating t-tubular pattern whereas Flag-RadΔCT failed to display membrane association. ICa,L in Flag-RadΔCT cardiomyocytes showed a hyperpolarized activation midpoint and an increase in maximal conductance. Additionally, current decay was faster in Flag-RadΔCT cells. Myocardial ICa,L in a Rad C-terminal deletion model phenocopies ICa,L modulated in response to β-AR stimulation. Mechanistically, the polybasic Rad C-terminus confers CaV1.2 regulation via membrane association. Interfering with Rad membrane association constitutes a specific target for boosting heart function as a treatment for heart failure with reduced ejection fraction.
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2 September 2024
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July 11 2024
The C-terminus of Rad is required for membrane localization and L-type calcium channel regulation
Garrett Elmore
,
Garrett Elmore
*
(Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing - original draft, Writing - review & editing)
1Department of Physiology,
University of Kentucky
, Lexington, KY, USA
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Brooke M. Ahern
,
Brooke M. Ahern
*
(Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Visualization, Writing - review & editing)
1Department of Physiology,
University of Kentucky
, Lexington, KY, USA
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Nicholas M. McVay
,
Nicholas M. McVay
(Formal analysis, Investigation, Writing - review & editing)
1Department of Physiology,
University of Kentucky
, Lexington, KY, USA
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Kyle W. Barker
,
Kyle W. Barker
(Formal analysis, Investigation, Methodology, Validation, Visualization, Writing - original draft)
1Department of Physiology,
University of Kentucky
, Lexington, KY, USA
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Sarisha S. Lohano
,
Sarisha S. Lohano
(Data curation, Formal analysis, Investigation, Visualization)
1Department of Physiology,
University of Kentucky
, Lexington, KY, USA
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Nemat Ali
,
Nemat Ali
(Data curation, Formal analysis, Investigation, Methodology, Writing - review & editing)
2Department of Molecular and Cellular Biochemistry,
University of Kentucky
, Lexington, KY, USA
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Andrea Sebastian
,
Andrea Sebastian
(Investigation, Methodology, Resources, Writing - review & editing)
1Department of Physiology,
University of Kentucky
, Lexington, KY, USA
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Douglas A. Andres
,
Douglas A. Andres
(Conceptualization, Funding acquisition, Investigation, Methodology, Supervision, Validation, Writing - review & editing)
2Department of Molecular and Cellular Biochemistry,
University of Kentucky
, Lexington, KY, USA
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Jonathan Satin
,
(Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing)
1Department of Physiology,
University of Kentucky
, Lexington, KY, USA
Correspondence to Jonathan Satin: [email protected]
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Bryana M. Levitan
Bryana M. Levitan
(Data curation, Formal analysis, Investigation)
1Department of Physiology,
University of Kentucky
, Lexington, KY, USA
3
Gill Heart and Vascular Institute
, Lexington, KY, USA
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Garrett Elmore
Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing - original draft, Writing - review & editing
*
1Department of Physiology,
University of Kentucky
, Lexington, KY, USA
Brooke M. Ahern
Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Visualization, Writing - review & editing
*
1Department of Physiology,
University of Kentucky
, Lexington, KY, USA
Nicholas M. McVay
Formal analysis, Investigation, Writing - review & editing
1Department of Physiology,
University of Kentucky
, Lexington, KY, USA
Kyle W. Barker
Formal analysis, Investigation, Methodology, Validation, Visualization, Writing - original draft
1Department of Physiology,
University of Kentucky
, Lexington, KY, USA
Sarisha S. Lohano
Data curation, Formal analysis, Investigation, Visualization
1Department of Physiology,
University of Kentucky
, Lexington, KY, USA
Nemat Ali
Data curation, Formal analysis, Investigation, Methodology, Writing - review & editing
2Department of Molecular and Cellular Biochemistry,
University of Kentucky
, Lexington, KY, USA
Andrea Sebastian
Investigation, Methodology, Resources, Writing - review & editing
1Department of Physiology,
University of Kentucky
, Lexington, KY, USA
Douglas A. Andres
Conceptualization, Funding acquisition, Investigation, Methodology, Supervision, Validation, Writing - review & editing
2Department of Molecular and Cellular Biochemistry,
University of Kentucky
, Lexington, KY, USA
Jonathan Satin
Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing
1Department of Physiology,
University of Kentucky
, Lexington, KY, USA
Bryana M. Levitan
Data curation, Formal analysis, Investigation
1Department of Physiology,
University of Kentucky
, Lexington, KY, USA
3
Gill Heart and Vascular Institute
, Lexington, KY, USA
Correspondence to Jonathan Satin: [email protected]
*
G. Elmore and B.M. Ahern shared co-first authors.
Disclosures: The authors declare no competing interests exist.
Received:
December 05 2023
Revision Received:
May 17 2024
Accepted:
June 26 2024
Online ISSN: 1540-7748
Print ISSN: 0022-1295
Funder(s):
American Heart Association
- Award Id(s): 19PRE34380909
Funder(s):
National Heart, Lung and Blood Institute
- Award Id(s): HL166280
Funder(s):
Department of Defense
- Award Id(s): W81WXH-20-1-0418
© 2024 Elmore et al.
2024
Elmore 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/).
J Gen Physiol (2024) 156 (9): e202313518.
Article history
Received:
December 05 2023
Revision Received:
May 17 2024
Accepted:
June 26 2024
Citation
Garrett Elmore, Brooke M. Ahern, Nicholas M. McVay, Kyle W. Barker, Sarisha S. Lohano, Nemat Ali, Andrea Sebastian, Douglas A. Andres, Jonathan Satin, Bryana M. Levitan; The C-terminus of Rad is required for membrane localization and L-type calcium channel regulation. J Gen Physiol 2 September 2024; 156 (9): e202313518. doi: https://doi.org/10.1085/jgp.202313518
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