Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark.
Bioelectricity Laboratory, Department of Physiology and Biophysics, School of Medicine, University of California, Irvine, CA.
J Gen Physiol. 2021 Mar 1;153(3). doi: 10.1085/jgp.202012760.
The dynein motor protein transports proteins away from the cell membrane along the microtubule network. Recently, we found the microtubule network was important for regulating the membrane abundance of voltage-gated Kv7.4 potassium channels in vascular smooth muscle. Here, we aimed to investigate the influence of dynein on the microtubule-dependent internalization of the Kv7.4 channel. Patch-clamp recordings from HEK293B cells showed Kv7.4 currents were increased after inhibiting dynein function with ciliobrevin D or by coexpressing p50/dynamitin, which specifically interferes with dynein motor function. Mutation of a dynein-binding site in the Kv7.4 C terminus increased the Kv7.4 current and prevented p50 interference. Structured illumination microscopy, proximity ligation assays, and coimmunoprecipitation showed colocalization of Kv7.4 and dynein in mesenteric artery myocytes. Ciliobrevin D enhanced mesenteric artery relaxation to activators of Kv7.2-Kv7.5 channels and increased membrane abundance of Kv7.4 protein in isolated smooth muscle cells and HEK293B cells. Ciliobrevin D failed to enhance the negligible S-1-mediated relaxations after morpholino-mediated knockdown of Kv7.4. Mass spectrometry revealed an interaction of dynein with caveolin-1, confirmed using proximity ligation and coimmunoprecipitation assays, which also provided evidence for interaction of caveolin-1 with Kv7.4, confirming that Kv7.4 channels are localized to caveolae in mesenteric artery myocytes. Lastly, cholesterol depletion reduced the interaction of Kv7.4 with caveolin-1 and dynein while increasing the overall membrane expression of Kv7.4, although it attenuated the Kv7.4 current in oocytes and interfered with the action of ciliobrevin D and channel activators in arterial segments. Overall, this study shows that dynein can traffic Kv7.4 channels in vascular smooth muscle in a mechanism dependent on cholesterol-rich caveolae.
动力蛋白沿微管网络将蛋白质从细胞膜运走。最近，我们发现微管网络对于调节血管平滑肌中电压门控 Kv7.4 钾通道的膜丰度很重要。在这里，我们旨在研究动力蛋白对 Kv7.4 通道的微管依赖性内化的影响。HEK293B 细胞的膜片钳记录显示，抑制动力蛋白功能后，Kv7.4 电流增加，例如用睫状神经麻痹素 D 或共表达 p50/dynamitin 进行抑制，后者特异性干扰动力蛋白的运动功能。在 Kv7.4 C 端的动力蛋白结合位点发生突变后，增加了 Kv7.4 电流并防止了 p50 的干扰。结构照明显微镜、临近连接分析和共免疫沉淀显示，Kv7.4 和动力蛋白在肠系膜动脉肌细胞中存在共定位。睫状神经麻痹素 D 增强了对 Kv7.2-Kv7.5 通道激活剂的肠系膜动脉松弛，并增加了分离的平滑肌细胞和 HEK293B 细胞中 Kv7.4 蛋白的膜丰度。睫状神经麻痹素 D 未能增强 Kv7.4 经 morpholino 介导的敲低后微不足道的 S-1 介导的松弛。质谱揭示了动力蛋白与 caveolin-1 的相互作用，通过临近连接和共免疫沉淀分析得到证实，这也为 caveolin-1 与 Kv7.4 的相互作用提供了证据，证实 Kv7.4 通道位于肠系膜动脉肌细胞中的 caveolae 中。最后，胆固醇耗竭减少了 Kv7.4 与 caveolin-1 和动力蛋白的相互作用，同时增加了 Kv7.4 的整体膜表达，尽管它在卵母细胞中减弱了 Kv7.4 电流，并干扰了睫状神经麻痹素 D 和通道激活剂在动脉段的作用。总体而言，这项研究表明，动力蛋白可以在依赖富含胆固醇的 caveolae 的机制中在血管平滑肌中运输 Kv7.4 通道。
