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.
Elife. 2019 Aug 12;8:e49253. doi: 10.7554/eLife.49253.
Inhibiting high-voltage-activated calcium channels (HVACCs; Ca1/Ca2) 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 Caβ subunits. Nb.F3 by itself is functionally inert, providing a convenient vehicle to target active moieties to Caβ-associated channels. Nb.F3 fused to the catalytic HECT domain of Nedd4L (Ca-aβlator), an E3 ubiquitin ligase, ablated currents from diverse HVACCs reconstituted in HEK293 cells, and from endogenous Ca1/Ca2 channels in mammalian cardiomyocytes, dorsal root ganglion neurons, and pancreatic β cells. In cardiomyocytes, Ca-aβlator redistributed Ca1.2 channels from dyads to Rab-7-positive late endosomes. This work introduces Ca-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.
抑制高电压激活钙通道（HVACCs；Ca1/Ca2）对多种心血管和神经疾病具有治疗作用。对于特定的应用，与小分子相比，基因编码的 HVACC 抑制剂可以实现更高的组织特异性和多功能性的通道抑制。在这里，我们通过首先分离一种免疫的羊驼纳米抗体（nb.F3）来构建一种基因编码的 HVACC 抑制剂，该纳米抗体与辅助 HVACC Caβ 亚基结合。nb.F3 本身没有功能活性，为将活性部分靶向到与 Caβ 相关的通道提供了便利的载体。nb.F3 与 Nedd4L（Ca-aβlator）的催化 HECT 结构域融合，后者是一种 E3 泛素连接酶，可消除在 HEK293 细胞中重组的各种 HVACCs 和哺乳动物心肌细胞、背根神经节神经元和胰岛β细胞中内源性 Ca1/Ca2 通道的电流。在心肌细胞中，Ca-aβlator 将 Ca1.2 通道从二联体重新分布到 Rab-7 阳性晚期内体。这项工作将 Ca-aβlator 作为一种有效的基因编码 HVACC 抑制剂引入，并描述了一种通用方法，可广泛用于生成用于大分子膜蛋白复合物的多功能调节剂。
