Calcium channels in arterial smooth muscle cells

描述（由申请人提供）：阻力大小，肌源性动脉调节全身血压和区域血流。l型电压依赖性钙（Ca2+, CaV1.2）通道是阻力大小动脉肌细胞中的主要Ca2+进入途径，并调节包括收缩性和基因表达在内的生理功能。CaV1.2通道由多个亚单元形成，包括孔隙形成11和辅助调节通道性质的124和2。尽管血管CaV1.2通道很重要，但关于肌细胞剪接变异体和辅助亚基的功能意义知之甚少。高血压患者动脉肌细胞Cav1.2电流增加，导致血管收缩和血压升高，但介导这种病理改变的机制尚不清楚。同样，选择性靶向Cav1.2通道以降低血管收缩性的方法也很少。这一建议源于初步数据，这些数据表明，抵抗大小的脑动脉肌细胞表达一种新的CaV1.2 11亚基剪接变体，该变体由辅助124亚基唯一调节。数据还表明，在高血压中，心肌细胞Cav1.2通道调节改变124导致Cav1.2电流升高和血管收缩。本应用的总体目标是扩大我们对脑动脉阻力大小肌细胞中CaV1.2通道的分子生理学知识，并研究与高血压相关的功能改变。将研究三个具体目标。目的1将检查血压正常和高血压患者的动脉肌细胞CaV1.2 11亚基剪接变异，并验证肌细胞特异性n端变异的分子靶向导致血管舒张的假设。目的2将研究124调节心肌细胞CaV1.2电流的假设，高血压与调节改变有关，导致CaV1.2电流升高和血管收缩。Aim 3将探讨在动脉肌细胞中，124对于CaV1.2 11亚基的质膜插入是必要的，并且高血压的上调会导致血管收缩。为了研究这些目标，我们将使用各种各样的技术，包括定量聚合酶链反应，膜片钳电生理学，激光扫描共聚焦显微镜，Western blotting， RNA干扰，细胞内Ca2+测量和加压动脉直径肌图。这些研究将提高对在阻力大小动脉肌细胞中表达的CaV1.2通道的分子特性、亚基调控、生理学和病理生理学的认识。CaV1.2家族的电压依赖性钙（Ca2+）通道是动脉平滑肌细胞的主要Ca2+内流途径，调节包括收缩性在内的多种生理功能，并在高血压中上调，导致血管收缩和血压升高。在正常血压和高血压中调节血压和血流的动脉平滑肌细胞中表达的CaV1.2通道亚基的分子特性和相关功能尚不清楚。我们的建议将研究一种假设，即在动脉平滑肌细胞中表达分子上不同的CaV1.2通道，并且这些通道的分子组成在高血压中发生改变，导致血管收缩。

DESCRIPTION (provided by applicant): Resistance-size, myogenic arteries regulate both systemic blood pressure and regional flow. L-type voltage- dependent calcium (Ca2+, CaV1.2) channels are the primary Ca2+ entry pathway in myocytes of resistance-size arteries and regulate physiological functions including contractility and gene expression. CaV1.2 channels are formed from multiple subunits, including a pore forming 11 and an auxiliary 124 and 2 which modulate channel properties. Despite the importance of vascular CaV1.2 channels, little is known regarding the functional significance of myocyte splice variants and auxiliary subunits. In hypertension there is an increase in arterial myocyte Cav1.2 currents, leading to an elevation in vascular contraction and blood pressure, but mechanisms mediating this pathological alteration are unclear. Similarly, there are few approaches to selectively target Cav1.2 channels to reduce vascular contractility. This proposal stems from preliminary data which suggest that myocytes of resistance-size cerebral arteries express a novel CaV1.2 11 subunit splice variant that is uniquely modulated by the auxiliary 124 subunit. Data also indicate that in hypertension, altered myocyte Cav1.2 channel regulation by 124 leads to an elevation in Cav1.2 currents and vasoconstriction. The overall goal of this application is to expand our knowledge of the molecular physiology of CaV1.2 channels in myocytes of resistance-size cerebral arteries and to study functional alterations that are associated with hypertension. Three specific aims will be investigated. Aim 1 will examine arterial myocyte CaV1.2 11 subunit splice variants in normotension and hypertension and test the hypothesis that molecular targeting of a myocyte-specific N-terminal variant causes vasodilation. Aim 2 will investigate the hypothesis that 124 modulates myocyte CaV1.2 currents and that hypertension is associated with altered regulation, leading to a Cav1.2 current elevation and vasoconstriction. Aim 3 will explore the hypothesis that in arterial myocytes, 124 is necessary for plasma membrane insertion of CaV1.2 11 subunits and that upregulation in hypertension leads to vasoconstriction. To investigate these aims, we will use a wide variety of techniques, including quantitative polymerase chain reaction, patch-clamp electrophysiology, laser-scanning confocal microscopy, Western blotting, RNA interference, intracellular Ca2+ measurements, and pressurized arterial diameter myography. These studies will improve knowledge of the molecular identity, subunit regulation, physiology, and pathophysiology of CaV1.2 channels that are expressed in myocytes of resistance-size arteries. PUBLIC HEALTH RELEVANCE: Project Narrative Voltage-dependent calcium (Ca2+) channels of the CaV1.2 family are the principal Ca2+ influx pathway in arterial smooth muscle cells, regulate a variety of physiological functions including contractility, and are upregulated in hypertension leading to vasoconstriction and elevated blood pressure. The molecular identity and associated functions of CaV1.2 channel subunits that are expressed in smooth muscle cells of arteries that regulate blood pressure and flow in normotension and hypertension is poorly understood. Our proposal will investigate the hypothesis that molecularly distinct CaV1.2 channels are expressed in arterial smooth muscle cells, and that the molecular composition of these channels is altered in hypertension, leading to vasoconstriction.