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

• DOI: 10.7554/elife.49253
• Publication Date: 2019-08-12T12:00:19Z
• Authors: Travis J Morgenstern; Jinseo Park; Qing R Fan; Henry M Colecraft
• Keywords: 0301 basic medicine; 570; QH301-705.5; Science; 610; cavia porcellus; 03 medical and health sciences; Biochemistry and Chemical Biology; ubiquitin; calcium channel beta; Animals; Humans; Biology (General); Pharmacology; Biological Products; Q; R; Single-Domain Antibodies; Calcium Channel Blockers; 3. Good health; nanobody; Calcium channels; HEK293 Cells; Medicine; calcium channel; Calcium Channels; Nedd4; Calcium--Antagonists; Camelids, New World; Protein Binding

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.