From the Vascular Research Centre, Institute of Molecular and Clinical Sciences, St George's, University of London, United Kingdom (V.B., J.B.S., H.B.F., I.A.G.)
From the Vascular Research Centre, Institute of Molecular and Clinical Sciences, St George's, University of London, United Kingdom (V.B., J.B.S., H.B.F., I.A.G.).
Hypertension. 2018 Jun;71(6):1091-1100. doi: 10.1161/HYPERTENSIONAHA.118.11116. Epub 2018 Apr 23.
Voltage-gated K7.4 channels have been implicated in vascular smooth muscle cells' activity because they modulate basal arterial contractility, mediate responses to endogenous vasorelaxants, and are downregulated in several arterial beds in different models of hypertension. Angiotensin II (Ang II) is a key player in hypertension that affects the expression of several classes of ion channels. In this study, we evaluated the effects of Ang II on the expression and function of vascular K7.4. Western blot and quantitative polymerase chain reaction revealed that in whole rat mesenteric artery, Ang II incubation for 1 to 7 hours decreased K7.4 protein expression without reducing transcript levels. Moreover, Ang II decreased XE991 (K7)-sensitive currents and attenuated membrane potential hyperpolarization and relaxation induced by the K7 activator ML213. Ang II also reduced K7.4 staining at the plasma membrane of vascular smooth muscle cells. Proteasome inhibition with MG132 prevented Ang II-induced decrease of K7.4 levels and counteracted the functional impairment of ML213-induced relaxation in myography experiments. Proximity ligation assays showed that Ang II impaired the interaction of K7.4 with the molecular chaperone HSP90 (heat shock protein 90), enhanced the interaction of K7.4 with the E3 ubiquitin ligase CHIP (C terminus of Hsp70-interacting protein), and increased K7.4 ubiquitination. Similar alterations were found in mesenteric vascular smooth muscle cells isolated from Ang II-infused mice. The effect of Ang II was emulated by 17-AAG (17-demethoxy-17-(2-propenylamino) geldanamycin) that inhibits HSP90 interactions with client proteins. These results show that Ang II downregulates K7.4 by altering protein stability through a decrease of its interaction with HSP90. This leads to the recruitment of CHIP and K7.4 ubiquitination and degradation via the proteasome.
电压门控 K7.4 通道与血管平滑肌细胞的活性有关，因为它们调节基础动脉收缩性、介导对内源性血管舒张剂的反应，并在高血压的几种动脉床中下调。血管紧张素 II（Ang II）是高血压的关键参与者，它影响几类离子通道的表达。在这项研究中，我们评估了 Ang II 对血管 K7.4 表达和功能的影响。Western blot 和定量聚合酶链反应显示，在整个大鼠肠系膜动脉中，Ang II 孵育 1 至 7 小时会降低 K7.4 蛋白表达，而不会降低转录水平。此外，Ang II 降低了 XE991（K7）敏感电流，并减弱了 K7 激活剂 ML213 引起的膜电位超极化和松弛。Ang II 还减少了血管平滑肌细胞质膜上的 K7.4 染色。蛋白酶体抑制剂 MG132 可防止 Ang II 诱导的 K7.4 水平降低，并在肌动图实验中抵消了 ML213 诱导的松弛功能障碍。邻近连接测定显示，Ang II 损害了 K7.4 与分子伴侣 HSP90（热休克蛋白 90）的相互作用，增强了 K7.4 与 E3 泛素连接酶 CHIP（Hsp70 相互作用蛋白 C 端）的相互作用，并增加了 K7.4 的泛素化。在从 Ang II 输注小鼠分离的肠系膜血管平滑肌细胞中也发现了类似的改变。17-AAG（17-去甲氧基-17-（2-丙烯基氨基）格尔德霉素）可模拟 Ang II 的作用，该物质抑制 HSP90 与客户蛋白的相互作用。这些结果表明，Ang II 通过降低其与 HSP90 的相互作用来改变蛋白质稳定性，从而下调 K7.4。这导致 CHIP 的募集和 K7.4 的泛素化和通过蛋白酶体降解。
