Vasoregulation by the Cav1.2 channel C-terminus

Cav1.2 通道 C 端的血管调节

基本信息

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
项目类别：
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

项目摘要

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大脑区域的血压和流量。动脉平滑肌细胞电压依赖性钙 (CaV1.2) 通道是血管收缩力的关键调节因子。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 通道可以经历酶裂解，产生截短的短形式 CaV1.2 通道和 C 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力尚不清楚。该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的功能性 CaV1.2 表达。初步数据还表明，血管收缩剂可减少 CCT 蛋白，从而提高 CaV1.2 通道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的功能性 CaV1.2 通道表达和质膜电流。目标 1 将研究截短的 CaV1.2 通道 C 端抑制动脉平滑肌细胞中的 CaV1.2 转录、表面 CaV1.2 通道表达和 CaV1.2 电流的假设。目标 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。目标 3 将检验 CCT 调节动脉平滑肌细胞 [Ca2+]i 从而调节收缩力的假设。使用的技术包括实时 PCR、蛋白质印迹、重组 CCT 表达、RNA 干扰、免疫荧光和免疫荧光共振能量转移 (immunoFRET)、膜片钳电生理学、钙成像和加压动脉肌动描记术。这项研究将揭示 CaV1.2 通道 C 末端调节血管收缩力的新机制。