Vasoregulation by the Cav1.2 channel C-terminus

描述（由申请人提供）：电阻大小的脑动脉调节脑区域血压和血流。动脉平滑肌细胞电压依赖性钙（CaV1.2）通道是血管收缩性的关键调节因子。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（[Ca2+]i）浓度增加和血管收缩。CaV1.2通道可以被酶裂解，产生截断的、短形式的CaV1.2通道和c端蛋白片段（CCT）。目前尚不清楚CCT是否存在于动脉平滑肌细胞中并调节血管收缩性。这一应用源于新的初步数据，表明CCT表现出核定位，并减弱脑动脉平滑肌细胞中功能性CaV1.2的表达。初步数据还表明，血管收缩剂通过降低CCT蛋白，提高CaV1.2通道表达，诱导血管收缩。本研究的核心假设是CCT控制脑动脉平滑肌细胞CaV1.2通道的表达和质膜电流。Aim 1将研究截断的CaV1.2通道c端抑制动脉平滑肌细胞中CaV1.2转录、表面CaV1.2通道表达和CaV1.2电流的假设。目的2将检验血管收缩剂通过调节动脉平滑肌细胞中CCT的总量和核定位来控制CaV1.2通道表达和活性的假设。目的3将验证CCT调节动脉平滑肌细胞[Ca2+]i，从而调节收缩性的假设。使用的技术包括实时PCR、Western blotting、重组CCT表达、RNA干扰、免疫荧光和免疫荧光共振能量转移（immunofluorescence resonance energy transfer, immunoFRET）、膜片钳电生理、钙成像和加压动脉肌图。本研究将揭示CaV1.2通道c端调控血管收缩力的新机制。

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.