Dynein regulates Kv7.4 channel trafficking from the cell membrane.
Dynein regulates Kv7.4 channel trafficking from the cell membrane.
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动力蛋白调节Kv7.4通道从细胞膜的运输。
DOI：
10.1085/jgp.202012760
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发表时间：
2021-03-01
期刊：
影响因子：
--
通讯作者：
Jepps TA
中科院分区：
文献类型：
--
作者：
van der Horst J;Rognant S;Abbott GW;Ozhathil LC;Hägglund P;Barrese V;Chuang CY;Jespersen T;Davies MJ;Greenwood IA;Gourdon P;Aalkjær C;Jepps TA
van der Horst等人的研究表明，微管运动蛋白dynein在血管平滑肌细胞中将电压门控钾通道Kv7.4带离细胞膜，从而降低其功能影响。这些数据对我们对动脉收缩性的理解具有启示意义。动力蛋白运动蛋白沿着微管网络将蛋白质从细胞膜上运输出去。最近，我们发现微管网络在调节血管平滑肌电压门控Kv7.4钾通道的膜丰度中起重要作用。在这里，我们的目的是研究动力蛋白对Kv7.4通道微管依赖性内化的影响。来自HEK293B细胞的膜片钳记录显示，用纤毛球蛋白D抑制动力蛋白功能或通过共表达p50/动力蛋白（特异性干扰动力蛋白运动功能）抑制动力蛋白功能后，Kv7.4电流增加。Kv7.4 C端动力蛋白结合位点的突变增加了Kv7.4电流并阻止了p50干扰。结构照明显微镜、近端结扎实验和共免疫沉淀显示Kv7.4和动力蛋白在肠系膜动脉肌细胞中共定位。在离体平滑肌细胞和HEK293B细胞中，Ciliobrevin D增强了肠系膜动脉对Kv7.2-Kv7.5通道激活物的松弛，并增加了Kv7.4蛋白的膜丰度。在morpholino介导的Kv7.4下调后，纤毛球蛋白D未能增强可忽略的s -1介导的松弛。质谱分析显示动力蛋白与caveolin-1相互作用，通过近距离结扎和共免疫沉淀试验证实，这也为caveolin-1与Kv7.4相互作用提供了证据，证实了Kv7.4通道定位于肠系膜动脉肌细胞的小窝。最后，胆固醇降低降低了Kv7.4与caveolin-1和dynein的相互作用，同时增加了Kv7.4的整体膜表达，尽管它减弱了卵母细胞中的Kv7.4电流，并干扰了纤毛球蛋白D和动脉段通道激活剂的作用。总的来说，这项研究表明动力蛋白可以通过一种依赖于富含胆固醇的小泡的机制运输血管平滑肌中的Kv7.4通道。
van der Horst et al. show that dynein, a microtubule motor protein, carries the voltage-gated potassium channel Kv7.4 away from the cell membrane in vascular smooth muscle cells, thereby reducing its functional impact. These data have implications for our understanding of arterial contractility. 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.
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DOI：
10.1152/ajpregu.00092.2007
发表时间：
2007-09-01
影响因子：
2.8
作者：
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影响因子：
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DOI：
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发表时间：
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影响因子：
11.1
作者：
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影响因子：
13.8
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影响因子：
7.3
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