Properties of Clca Channels in Vascular Myocytes

描述（由申请人提供）：申请人研究项目的长期目标是阐明在健康和疾病中参与控制大导管和阻力大小血管的平滑肌细胞的电和机械活动的离子机制。在平滑肌细胞中，氯离子（Cl-）不是被动地分布在细胞膜上。这种阴离子被认为是通过各种阴离子转运体在细胞质中积极积累的。如此高的内部Cl-水平（~ 40 mM）导致Cl- (ECl)的预测平衡（-25 mV）比血管平滑肌细胞的静息膜电位（RMP）（~ -40至-60 mV）更正。由于ECl和RMP之间的这种偏差，任何对Cl-通透性的增加都会导致被动Cl"外排，膜去极化和血管张力增加。尽管我们知道平滑肌细胞对Cl-的高静息膜通透性，但这种基础阴离子传导的性质仍然不明确。该建议的重点是阐明磷酸化机制所涉及的调节机制，包括钙调素依赖性激酶II （CaMKII）和丝氨酸/苏氨酸磷酸酶（Calcineurin和PP2A）在肺动脉平滑肌细胞内由细胞内Ca2+ (ClCa)激活的Cl-通道，以及这可能如何影响肺循环的电和血管活性特性。本研究的具体目的是：(1)确定一般磷酸化状态对兔肺动脉平滑肌细胞Clca通道生物物理特性的影响；(2)确定CaMKII和丝氨酸/苏氨酸磷酸酶在兔肺动脉平滑肌细胞ClCa通道调控中的相对作用；(3)确定磷酸酶调控ClCa通道对肺动脉平滑肌细胞和完整肺动脉的膜电位、Ca2+瞬态和张力的生理影响；(4)克隆并表达3个家兔肺动脉Ca2+激活Cl-通道候选基因，并评估其与天然通道的生理相关性。一个强大的合作研究团队将使用广泛的电生理、生化和分子生物学技术，以及共聚焦成像技术和计算机模拟来实现上述目标。人类肺动脉高压（PH）的病因尚不清楚，尽管越来越清楚的是，有缺陷的离子机制可能在这种疾病中起作用。由于其作为肺动脉兴奋机制的潜在重要性，Clca通道的调节和/或表达受损可能参与PH。拟议的研究不仅将推进我们对其基本特性的了解，而且还将为未来治疗PH的疗法的发展铺平道路。

DESCRIPTION (provided by applicant): The long term objective of the applicant's research program is to unravel the ionic mechanisms that are involved in the control of electrical and mechanical activities of the smooth muscle cells of large conduit and resistance-sized vessels in health and disease. In smooth muscle cells, chloride ions (Cl-) are not passively distributed across the cell membrane. It is thought that this anion is actively accumulated in the cytoplasm by various anion transporters. Such a high internal level of Cl- (~ 40 mM) results in a predicted equilibrium for Cl- (ECl) that is more positive (-25 mV) than the resting membrane potential (RMP) of vascular smooth muscle cells (~ -40 to -60 mV). Because of this deviation between ECl and RMP, any increase in permeability to Cl- would result in passive Cl" efflux, membrane depolarization and increased vascular tone. In spite of our knowledge about high resting membrane permeability to Cl- in smooth muscle cells, the nature of this basal anion conductance still remains undefined. This proposal is focused on elucidating the mechanisms involved in the regulation by phosphorylation mechanisms involving calmodulin-dependent kinase II (CaMKII) and serine/threonine phosphatases (Calcineurin and PP2A) of a Cl- channel activated by intracellular Ca2+ (ClCa ) in pulmonary arterial smooth muscle cells, and how this might impact on the electrical and vasoactive properties of the pulmonary circulation. 4 Specific Aims are proposed in this grant: (1) determine the effect of general phosphorylation status on the biophysical properties of Clca channels in rabbit pulmonary artery smooth muscle cells; (2) determine the relative role of CaMKII and serine/threonine phosphatases in the regulation of ClCa channels in rabbit pulmonary artery smooth muscle cells; (3) determine the physiological impact of phosphatase regulation of ClCa channels on membrane potential, Ca2+ transients and tone in pulmonary arterial smooth muscle cells and intact pulmonary arteries; and (4) clone and express 3 candidate genes encoding Ca2+-activated Cl- channels in rabbit pulmonary artery and evaluate their physiological relevance to the native channels. A strong team of collaborating investigators will use a wide array of electrophysiological, biochemical and molecular biology techniques, as well as confocal imaging technology and computer simulations to accomplish the above goals. The etiology of pulmonary hypertension (PH) in humans is still poorly understood although it is becoming increasingly clear that defective ionic mechanisms may play a role in this disease. Because of their potential importance as an excitatory mechanism in pulmonary arteries, impaired regulation and/or expression of Clca channels could potentially participate in PH. The proposed studies will not only advance our knowledge about their basic properties, but should also pave the way for the development of future therapies to treat PH.