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Elife. 2019 Aug 12;8. pii: e49253. doi: 10.7554/eLife.49253.

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

Author information

1
Department of Pharmacology, Columbia University, Vagelos College of Physicians and Surgeons, New York, United States.
2
Department of Physiology and Cellular Biophysics, Columbia University, Vagelos College of Physicians and Surgeons, New York, United States.

Abstract

Inhibiting high-voltage-activated calcium channels (HVACCs; CaV1/CaV2) 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 CaVβ subunits. Nb.F3 by itself is functionally inert, providing a convenient vehicle to target active moieties to CaVβ-associated channels. Nb.F3 fused to the catalytic HECT domain of Nedd4L (CaV-aβlator), an E3 ubiquitin ligase, ablated currents from diverse HVACCs reconstituted in HEK293 cells, and from endogenous CaV1/CaV2 channels in mammalian cardiomyocytes, dorsal root ganglion neurons, and pancreatic β cells. In cardiomyocytes, CaV-aβlator redistributed CaV1.2 channels from dyads to Rab-7-positive late endosomes. This work introduces CaV-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.

KEYWORDS:

Nedd4; biochemistry; calcium channel; calcium channel beta; cavia porcellus; cell biology; chemical biology; mouse; nanobody; ubiquitin

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