Vasoregulation by the Cav1.2 channel C-terminus

描述(申请人提供)：阻力大小的大脑动脉调节大脑区域的血压和流量。动脉平滑肌细胞电压依赖性钙通道(CaV1.2)是血管收缩的关键调节因子。血管CaV1.2通道被膜去极化和血管收缩激动剂激活，导致细胞内钙离子浓度升高和血管收缩。CaV1.2通道可以被酶切割，产生截短的短形CaV1.2通道和一个C-末端蛋白片段(CCT)。CCT是否存在于动脉平滑肌细胞中并调节血管收缩性能尚不清楚。这一应用源于新的初步数据表明，CCT显示出核定位，并减弱了大脑动脉平滑肌细胞中功能性CaV1.2的表达。初步数据还表明，血管收缩药减少CCT蛋白，上调CaV1.2通道的表达，诱导血管收缩。这一建议的中心假设是CCT控制大脑动脉平滑肌细胞CaV1.2通道的功能表达和质膜电流。目的1探讨CaV1.2通道C末端被截断后抑制CaV1.2转录、表面CaV1.2通道表达和抑制动脉平滑肌细胞CaV1.2电流的假说。目的2验证血管紧张剂通过调节CCT在动脉平滑肌细胞中的总量和核定位来控制CaV1.2通道表达和活性的假说。目的3将验证CCT调节动脉平滑肌细胞[Ca~(2+)]i，从而调节收缩能力的假设。可使用的技术包括实时聚合酶链式反应、Western blotting、重组CCT的表达、RNA干扰、免疫荧光和免疫荧光共振能量转移、膜片钳电生理、钙成像和加压动脉肌成像。本研究将揭示CaV1.2通道C末端调节血管收缩的新机制。 公共卫生相关性：动脉平滑肌细胞电压依赖性钙通道在调节全身和局部血流方面发挥着重要作用。影响动脉平滑肌细胞电压依赖性钙通道功能的机制尚不清楚。这项建议将研究电压依赖性钙通道切割对表达、电流和动脉收缩性能的影响。

DESCRIPTION (provided by applicant): Resistance-size cerebral arteries modulate brain regional blood pressure and flow. Arterial smooth muscle cell voltage-dependent calcium (CaV1.2) channels are key regulators of vascular contractility. Vascular CaV1.2 channels are activated by membrane depolarization and vasoconstrictor agonists, leading to an increase in intracellular calcium ([Ca2+]i) concentration and vasoconstriction. CaV1.2 channels can undergo enzymatic cleavage, yielding truncated, short-form CaV1.2 channels and a C-terminal protein fragment (CCT). Whether CCT exists in arterial smooth muscle cells and regulates vascular contractility is unclear. This application stems from novel preliminary data indicating that CCT exhibits nuclear localization and attenuates functional CaV1.2 expression in cerebral artery smooth muscle cells. Preliminary data also indicate that vasoconstrictors reduce CCT protein to elevate CaV1.2 channel expression and induce vasoconstriction. The central hypothesis of this proposal is that the CCT controls functional CaV1.2 channel expression and plasma membrane currents in cerebral artery smooth muscle cells. Aim 1 will investigate the hypothesis that the truncated CaV1.2 channel C-terminus inhibits CaV1.2transcription, surface CaV1.2 channel expression, and CaV1.2currents in arterial smooth muscle cells. Aim 2 will examine the hypothesis that vasoconstrictors control CaV1.2 channel expression and activity by modulating the total amount and nuclear localization of CCT in arterial smooth muscle cells. Aim 3 will test the hypothesis that the CCT regulates arterial smooth muscle cell [Ca2+]i, thereby modulating contractility. Techniques to be used include real-time PCR, Western blotting, expression of recombinant CCT, RNA interference, immunofluorescence and immunofluorescence resonance energy transfer (immunoFRET), patch-clamp electrophysiology, calcium imaging, and pressurized artery myography. This research will reveal novel mechanisms of vascular contractility regulation by the CaV1.2 channel C-terminus. PUBLIC HEALTH RELEVANCE: Arterial smooth muscle cell voltage-dependent Ca2+ channels play a significant role in the regulation of systemic and regional blood flow. Mechanisms that influence voltage-dependent Ca2+ channel function in arterial smooth muscle cells are unclear. This proposal will study the effects of voltage-dependent Ca2+ channel cleavage on expression, currents, and arterial contractility.