eLife | |
A potent voltage-gated calcium channel inhibitor engineered from a nanobody targeted to auxiliary CaVβ subunits | |
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[1] Department of Pharmacology, Columbia University, Vagelos College of Physicians and Surgeons, New York, United States;Department of Pharmacology, Columbia University, Vagelos College of Physicians and Surgeons, New York, United States;Department of Physiology and Cellular Biophysics, Columbia University, Vagelos College of Physicians and Surgeons, New York, United States; | |
关键词: calcium channel; nanobody; ubiquitin; Nedd4; calcium channel beta; cavia porcellus; Mouse; | |
DOI : 10.7554/eLife.49253 | |
来源: publisher | |
【 摘 要 】
10.7554/eLife.49253.001Inhibiting 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.
【 授权许可】
CC BY
【 预 览 】
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RO201911192538455ZK.pdf | 2629KB | download |