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局部血流。L型电压依赖性钙通道（Ca 2+，CaV1.2）是阻力型动脉的主要钙通道，调节收缩性和基因表达等生理功能。CaV1.2通道由多个亚基形成，包括调节通道性质的成孔11和辅助124和2。尽管血管CaV1.2通道的重要性，很少有人知道关于肌细胞剪接变异体和辅助亚基的功能意义。在高血压中，动脉肌细胞Cav1.2电流增加，导致血管收缩和血压升高，但介导这种病理变化的机制尚不清楚。类似地，很少有选择性靶向Cav1.2通道以降低血管收缩性的方法。这一建议源于初步的数据表明，肌细胞的阻力大小脑动脉表达一种新的CaV1.2 11亚基剪接变异体，是唯一的辅助124亚基调制。数据还表明，在高血压中，通过124改变的肌细胞Cav1.2通道调节导致Cav1.2电流升高和血管收缩。本申请的总体目标是扩大我们对阻力大小脑动脉肌细胞CaV1.2通道分子生理学的了解，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将研究动脉肌细胞CaV1.2 11亚基剪接变异体在正常血压和高血压和测试的假设，即分子靶向的肌细胞特异性N-末端变异体引起血管舒张。目的2将研究的假设，124调制心肌细胞CaV1.2电流和高血压是与改变调节，导致Cav1.2电流升高和血管收缩。目的3将探讨的假设，在动脉肌细胞中，124是必要的质膜插入的CaV 1.2 11亚基和高血压的上调导致血管收缩。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，蛋白质印迹，RNA干扰，细胞内Ca 2+测量，和加压动脉直径肌电图。这些研究将提高知识的分子身份，亚基调节，生理学和病理生理学的CaV1.2通道，在肌细胞的阻力大小的动脉。公共卫生相关性：CaV1.2家族的电压依赖性钙（Ca 2+）通道是动脉平滑肌细胞中主要的Ca 2+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。CaV1.2通道亚基的分子特性和相关功能在动脉平滑肌细胞中表达，调节血压和正常血压和高血压的流量，目前还知之甚少。我们的建议将调查的假设，分子不同的CaV1.2通道在动脉平滑肌细胞中表达，这些通道的分子组成是改变高血压，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.