Properties of Clca Channels in Vascular Myocytes

血管肌细胞 Clca 通道的特性

• 批准号: 6983085
• 负责人: Normand Leblanc
• 金额: $ 35.13万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6983085
• 关键词: biophysics; calcineurin; calcium; calmodulin dependent protein kinase; chloride channels; computer simulation; confocal scanning microscopy; enzyme activity; enzyme mechanism; enzyme model; gene expression; laboratory rabbit; membrane potentials; molecular cloning; muscle cells; phosphatase inhibitor; phosphorylation; pulmonary artery; pulmonary circulation; pulmonary hypertension; tissue /cell culture; vascular endothelium; vascular smooth muscle; vasomotion; voltage /patch clamp

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.

描述（由申请人提供）：申请人研究计划的长期目标是揭示与健康和疾病中大型导管和阻力尺寸血管平滑肌细胞的电气和机械活性有关的离子机制。在平滑肌细胞中，氯离子（Cl-）并非被动地分布在细胞膜上。人们认为，该阴离子由各种阴离子转运蛋白积极积累。如此高的内部Cl-（〜40 mm）水平导致Cl-（ECL）的预测平衡比血管平滑肌细胞的静息膜电位（RMP）更正（-25 mV）（〜-40至-60 mV）。由于ECL和RMP之间的这种偏差，对Cl-的渗透性的任何增加都会导致Cl'Cl“外排”，膜去极化和血管张力的增加。尽管我们对高静止膜通透性的了解，但在平滑肌细胞中对Cl- cl- cl-在平滑肌细胞中的了解仍然不确定，但这种机制仍然涉及这种机制。该机构仍然涉及该机制的范围。涉及钙调蛋白依赖性激酶II（CAMKII）和丝氨酸/苏氨酸磷酸酶（钙调蛋白和PP2A）在肺动脉平滑肌细胞中被细胞内Ca2+（CLCA）激活的CL通道，以及这可能会影响电气和血管的孔子循环效果。兔肺动脉平滑肌细胞中CLCA通道的生物物理特性上的磷酸化状态； （3）确定CLCA通道调节磷酸酶对肺动脉平滑肌细胞和完整肺动脉的磷酸酶调节的生理影响； （4）在兔肺动脉中编码Ca2+激活的CL-通道的克隆和表达3个候选基因，并评估其与天然通道的生理相关性。一个强大的合作调查人员团队将使用各种电生理，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）的病因仍然尚不清楚，尽管越来越清楚的是，有缺陷的离子机制可能在该疾病中起作用。由于它们作为肺动脉中的兴奋性机制的潜在重要性，因此CLCA通道的调节和/或表达受损可能会参与pH。拟议的研究不仅将提高我们对其基本特性的了解，还应为开发未来治疗pH的疗法铺平道路。