Properties of Clca Channels in Vascular Myocytes

描述（由申请人提供）：申请人研究计划的长期目标是阐明健康和疾病中大导管和阻力大小血管平滑肌细胞电和机械活动控制所涉及的离子机制。在平滑肌细胞中，氯离子（Cl-）并不是被动地分布在细胞膜上。据认为，这种阴离子通过各种阴离子转运蛋白在细胞质中积极积累。如此高的Cl-内部水平（~ 40 mM）导致Cl-（ECl）的预测平衡比血管平滑肌细胞的静息膜电位（RMP）（~ -40至-60 mV）更正（~ 25 mV）。由于ECl和RMP之间的这种偏差，Cl-渗透性的任何增加都将导致Cl-被动流出、膜去极化和血管张力增加。尽管我们的知识高静息膜渗透性的Cl-在平滑肌细胞中，这种基础阴离子电导的性质仍然不确定。该提案的重点是阐明磷酸化机制参与调节的机制，磷酸化机制涉及钙调蛋白依赖性激酶II（CaMKII）和丝氨酸/苏氨酸磷酸酶（钙调磷酸酶和PP 2A）的Cl-通道激活细胞内Ca 2+（ClCa）在肺动脉平滑肌细胞，以及这可能如何影响肺循环的电气和血管活性特性。4.本研究的具体目的：（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CaMK Ⅱ和丝氨酸/苏氨酸磷酸酶在兔肺动脉平滑肌细胞ClCa通道调节中的相对作用;（3）确定ClCa通道的磷酸酶调节对肺动脉平滑肌细胞和完整肺动脉中的膜电位、Ca 2+瞬变和张力的生理影响;（4）克隆和表达3个兔肺动脉钙激活氯离子通道的候选基因，并评价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，生物化学和分子生物学技术，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）的病因学仍然知之甚少，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发挥作用。由于它们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，而且还应为未来PH治疗方法的开发铺平道路。

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.