A potent voltage-gated calcium channel inhibitor engineered from a nanobody targeted to auxiliary CaVβ subunits

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Abstract

Inhibiting high-voltage-activated calcium channels (HVACCs; Ca_V1/Ca_V2) is therapeutic for myriad cardiovascular and neurological diseases. For particular applications, genetically-encoded HVACC blockers may enable channel inhibition with greater tissue-specificity and versatility than is achievable with small molecules. Here, we engineered a genetically-encoded HVACC inhibitor by first isolating an immunized llama nanobody (nb.F3) that binds auxiliary HVACC Ca_Vβ subunits. Nb.F3 by itself is functionally inert, providing a convenient vehicle to target active moieties to Ca_Vβ-associated channels. Nb.F3 fused to the catalytic HECT domain of Nedd4L (Ca_V-aβlator), an E3 ubiquitin ligase, ablated currents from diverse HVACCs reconstituted in HEK293 cells, and from endogenous Ca_V1/Ca_V2 channels in mammalian cardiomyocytes, dorsal root ganglion neurons, and pancreatic β cells. In cardiomyocytes, Ca_V-aβlator redistributed Ca_V1.2 channels from dyads to Rab-7-positive late endosomes. This work introduces Ca_V-aβlator as a potent genetically-encoded HVACC inhibitor, and describes a general approach that can be broadly adapted to generate versatile modulators for macro-molecular membrane protein complexes.

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All data generated or analysed during this study are included in the manuscript and supporting files.

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Travis Morgenstern

Department of Pharmacology, Columbia University, New York, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0003-2634-8470
Jinseo Park

Department of Pharmacology, Columbia University, New York, United States

Competing interests
The authors declare that no competing interests exist.
Qing R Fan

Department of Pharmacology, Columbia University, New York, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-9330-0963
Henry M Colecraft

Department of Pharmacology, Columbia University, New York, United States

For correspondence
hc2405@cumc.columbia.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-2340-8899

Funding

National Institutes of Health (R01 HL142111)

Henry M Colecraft

National Institutes of Health (R01 GM107585)

Henry M Colecraft

National Institutes of Health (F31 DK118866)

Travis Morgenstern

National Institutes of Health (S10RR027050)

Travis Morgenstern
Jinseo Park
Qing R Fan
Henry M Colecraft

National Institutes of Health (P30 CA013696)

Travis Morgenstern
Jinseo Park
Qing R Fan
Henry M Colecraft

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#A3007-01) of Columbia University. Primary cultures of adult guinea pig heart ventricular cells were prepared in accordance with the guidelines of Columbia University Animal Care and Use Committee protocols (AC-AAAS5410). All surgery was performed under isoflurane anesthesia, and every effort was made to minimize suffering.

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This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.