Properties of Clca Channels in Vascular Myocytes

血管肌细胞 Clca 通道的特性

• 批准号: 7839247
• 负责人: Normand Leblanc
• 金额: $ 13.54万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7839247
• 关键词: Anions; Architecture; Arteries; Behavior; Biochemical; Biochemistry; Blood Vessels; Calcineurin; Calmodulin; Candidate Disease Gene; Cell Line; Cell Membrane Permeability; Cell Volumes; Cell membrane; Chloride Ion; Computer Simulation; Coronary; Cytoplasm; Data; Dependence; Development; Disease; Equilibrium; Etiology; Follow-Up Studies; Future; Gene Family; Goals; Grant; Health; Human; Image; Imaging technology; Journals; Kinetics; Knowledge; Lung; Maintenance; Mammalian Cell; Measures; Mechanics; Membrane; Membrane Potentials; Messenger RNA; Modeling; Molecular; Molecular Biology Techniques; Muscle Cells; Nature; Oryctolagus cuniculus; Permeability; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Play; Property; Protein Dephosphorylation; Protein Isoforms; Protein Phosphatase 2A Regulatory Subunit PR53; Protein Serine/Threonine Phosphatase; Pulmonary Circulation; Pulmonary Hypertension; Pulmonary artery structure; Pump; Recommendation; Regulation; Relative (related person); Reporting; Research; Research Personnel; Resistance; Rest; Role; Side; Smooth Muscle; Smooth Muscle Myocytes; Specificity; Swelling; System; Testing; Transcript; Vascular Smooth Muscle; Vasoconstrictor Agents; Veins; calcineurin phosphatase; calmodulin-dependent protein kinase II; cell type; member; novel; patch clamp; programs; research study; symporter; vascular bed; voltage

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参与控制健康和疾病中大型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-) 并不是被动分布在细胞膜上。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。如此高的内部 Cl- 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，对 Cl- 通透性的任何增加都会导致 Cl- 被动外流、膜去极化和血管张力增加。尽管我们了解平滑肌细胞中对 Cl- 的静息膜通透性较高，但这种基础阴离子电导的性质仍然不明确。该提案的重点是阐明磷酸化机制调节所涉及的机制。 涉及肺动脉平滑肌细胞中由细胞内 Ca2+ (ClCa ) 激活的 Cl-通道的钙调蛋白依赖性激酶 II (CaMKII) 和丝氨酸/苏氨酸磷酸酶（钙调神经磷酸酶和 PP2A），以及这如何影响肺循环的电和血管活性特性。本次赠款提出了 4 个具体目标： (1) 确定总体效果 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/苏氨酸磷酸酶在调节兔肺动脉平滑肌细胞ClCa通道中的相对作用； (3)确定磷酸酶调节ClCa通道对肺膜电位、Ca2+瞬变和张力的生理影响 动脉平滑肌细胞和完整的肺动脉； (4)在兔肺动脉中克隆并表达编码Ca2+激活的Cl-通道的3个候选基因，并评估它们与天然通道的生理相关性。强大的合作研究团队将使用广泛的电生理学、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，但对人类肺动脉高压 (PH) 的病因仍知之甚少。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。拟议的研究不仅将增进我们对其基本特性的了解，而且还应为 开发未来治疗 PH 的疗法的方法。