Properties of Clca Channels in Vascular Myocytes

申请者描述(申请人提供)：申请者研究计划的长期目标是解开在健康和疾病中涉及控制大管道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活跃地积聚在细胞质中。如此高的内部氯离子水平(~40 mM)导致预测的氯离子(ECL)平衡比血管平滑肌细胞的静息膜电位(RMP)(~-40~-60 mV)更正(-25 mV)。由于ECL和RMP之间的这种偏差，任何对氯离子通透性的增加都会导致被动的氯离子外流，膜去极化和血管张力增加。尽管我们知道平滑肌细胞对氯离子的高静息膜通透性，但这种基础阴离子电导的性质仍然不清楚。本研究旨在阐明钙调素依赖的激酶II(CaMKII)和丝氨酸/苏氨酸磷酸酶(CaMKII)等磷酸化机制对细胞内钙离子(CLCA)激活的氯通道的调节机制及其对肺循环电活动和血管活性的影响。本研究提出了4个具体目标：(1)确定一般磷酸化状态对兔肺动脉平滑肌细胞CLCA通道生物物理性质的影响；(2)确定CaMKII和丝氨酸/苏氨酸磷酸酶在调节兔肺动脉平滑肌细胞CLCA通道中的相对作用；(3)确定CLCA通道的磷酸酶调节对肺动脉平滑肌细胞和完整的肺动脉膜电位、钙瞬变和张力的生理影响；(4)克隆和表达3个编码兔肺动脉钙激活的氯通道的候选基因，并评价它们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，以及共焦成像技术和计算机模拟来实现上述目标。尽管离子机制缺陷可能在这种疾病中起作用，但人们对人类肺动脉高压(PH)的病因学仍知之甚少。由于CLCA通道作为肺动脉兴奋机制的潜在重要性，CLCA通道调节和/或表达受损可能参与了PH的发生。拟议的研究不仅将促进我们对它们的基本性质的了解，而且也应该为未来治疗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.