Regulation of Calcium-activated Chloride Conductance in Smooth Muscle Vasculature

描述（由申请人提供）：钙活化的氯离子通道（Clca）广泛表达于许多细胞中，包括上皮细胞、神经元细胞、心脏细胞和平滑肌细胞（SMC）。CICa参与了许多重要的细胞功能，如上皮分泌、神经元和心肌的膜兴奋性以及平滑肌血管张力的调节。由于CICa在肺动脉平滑肌细胞（PASMC）中的表达，这些通道可能参与各种形式肺动脉高压（PH）的病因学。我们小组最近的工作提供了钙激活氯电流（Icuca）在动脉SMC中分别通过钙调蛋白依赖性蛋白激酶II （CaMKII）和Ca2+依赖性钙调神经磷酸酶（CaN; PP2B）的磷酸化和去磷酸化来调节的证据。由于丝氨酸/苏氨酸磷酸酶CaN参与了Clca电导的上调，因此可能还有其他磷酸酶也参与了其活性的上调。在兔肺动脉SMCs中，本研究的主要目的是：1)确定静态和动态细胞内钙水平下CaMKII和CaN在CICa通道调控中的相对作用；2)确定在CICa调控中CaN Aa特异性高于CaN A(3)的结构决定因素；3)检查其他丝氨酸/苏氨酸磷酸酶（PP1/PP2A）在lCi（ca）调节中的可能参与-为了完成这项任务，将实施多种技术，包括在兔PASMC全细胞膜片钳条件下使用药物抑制蛋白磷酸酶，以确定PP1和PP2A在CICa电导中的相对贡献。这项技术也将被证明在确定CaN和其他丝氨酸/苏氨酸磷酸酶是否靶向一个共同的或不同的磷酸化位点来调节心血管疾病（CVD）方面是有用的，心血管疾病（CVD）仍然是美国最大的杀手之一。根据美国心脏协会的数据，2003年心血管疾病是美国37%以上死亡的潜在原因。由于离子通道在调节血管张力中发挥着重要作用，我们希望更好地了解离子通道（如Clca）在心血管系统中的潜在机制，从而为开发新的血管靶点提供新药，以改善心血管疾病（如PH、卒中、心绞痛和梗死）患者的病情。

DESCRIPTION (provided by applicant): Calcium-activated chloride channels (Clca) are widely expressed in numerous cells including epithelial, neuronal, cardiac and smooth muscle cells (SMC). CICa have been implicated in many important cellular functions such as epithelial secretion, membrane excitability in neurons and cardiac muscle as well as regulation of smooth muscle vascular tone. As a result of CICa expression in pulmonary artery smooth muscle cells (PASMC), there is a possibility that these channels may participate in the etiology of various forms of pulmonary hypertension (PH). Recent work by our group has provided evidence for the modulation of calcium-activated chloride currents (Icuca)) through phoshorylation and dephosphorylation by calmodulin- dependent protein kinase II (CaMKII) and Ca2+dependent Calcineurin (CaN; PP2B) respectively in arterial SMC. As a result of the serine/threonine phosphatase CaN involvement in up-regulation of Clca conductance, it is possible that there may be other phophatases contributing in up-regulation their activity as well. In rabbit pulmonary artery SMCs, the major goals of this research project are to: 1) determine the relative role of CaMKII and CaN in the regulation of CICa channels under static and dynamic intracellular calcium levels; 2) determine the structural determinants conferring specificity of CaN Aa over CaN A(3 in the regulation of CICa; and 3) examine the possible involvement of other serine/threonine phosphatases (PP1/PP2A) in the modulation of lCi(ca)- To accomplish this task, a variety of techniques will be implemented, including the use of pharmacological agents to inhibit protein phosphatases under whole-cell patch clamp conditions in rabbit PASMC to determine the relative contribution of PP1 and PP2A in CICa conductance. This technique will also prove to be useful in determining whether CaN and the other(s) ser/thr phosphatases target a common or distinct phosphorylation site to modulate Cardiovascular disease (CVD) remains to be one of the largest killers in the United States. According to the American Heart Association, in 2003 CVD was the underlying cause of death for more than 37% of all deaths in the United States. Since ion channels play an important role in the regulation of vascular tone, it is our hope that a better understanding of the underlying mechanisms of ion channels (like Clca) in the cardiovascular system will lead to novel vascular targets for the development of new drugs to improve the conditions of patients suffering from CVDs such as PH, stroke, myocardial angina and infarctions.