Vasoregulation by the Cav1.2 channel C-terminus

Cav1.2 通道 C 端的血管调节

• 批准号: 8531708
• 负责人: Kirk W Evanson
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8531708
• 关键词: Agonist; Arteries; Attenuated; Binding; Biological Assay; Biotinylation; Blood Pressure; Blood Vessels; Blood flow; Brain; C-terminal; Calcium; Caliber; Calmodulin; Cardiac Myocytes; Cell membrane; Cerebrum; Cleaved cell; Coupled; Data; Detection; Electrophysiology (science); Endothelin; Endothelin-1; Energy Transfer; Ensure; Exhibits; Fluorescence Microscopy; Fura-2; Genes; Genetic Transcription; Glass; Goals; Image; Immunofluorescence Immunologic; Ion Channel; Knowledge; L-type calcium channel alpha(1C); Length; Luc Gene; Measurement; Measures; Membrane; Membrane Potentials; Membrane Proteins; Messenger RNA; Methods; Microelectrodes; Muscle Cells; Myography; Myosin Light Chain Kinase; Neurons; Nuclear; Phospholipase C; Physiological; Play; Protein Fragment; Protein Kinase; Proteins; Purinoceptor; RNA Interference; Recombinants; Regional Blood Flow; Regulation; Reporter; Research; Resistance; Role; Signal Pathway; Smooth Muscle Myocytes; Stimulus; Surface; Techniques; Testing; Time; Transcript; Uridine; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents; Western Blotting; Work; capillary bed; cerebral artery; channel blockers; constriction; mutant; novel; overexpression; patch clamp; pressure; ratiometric; research study; stem; tripolyphosphate; vasoconstriction; voltage

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.

描述（由申请人提供）：阻力大小的脑动脉调节脑局部血压和血流。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剂激动剂激活，导致细胞内钙（[Ca2+]i）浓度增加和血管收缩。CaV1.2通道可以进行酶切，产生截短的短形式CaV1.2通道和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尚不清楚。这项应用源于新的初步数据表明，CCT表现出核定位和减弱功能性CaV1.2在脑动脉平滑肌细胞的表达。初步数据还表明，血管收缩剂减少CCT蛋白，以提高CaV1.2通道的表达，并诱导血管收缩。该提议的中心假设是CCT控制脑动脉平滑肌细胞中功能性CaV1.2通道表达和质膜电流。目的1：研究截短的CaV1.2通道C端对动脉平滑肌细胞CaV1.2转录、表面CaV1.2通道表达和CaV1.2电流的抑制作用。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将检验CCT调节动脉平滑肌细胞[Ca2+]i从而调节收缩性的假设。待使用的技术包括实时PCR、蛋白质印迹、重组CCT的表达、RNA干扰、免疫荧光和免疫荧光共振能量转移（immunofluorescence resonance energy transfer，immunFRET）、膜片钳电生理学、钙成像和加压动脉肌造影。本研究将揭示CaV1.2通道C端调控血管收缩力的新机制。