Regulation of Calcium-activated Chloride Conductance in Smooth Muscle Vasculature

描述（由申请人提供）：钙激活氯离子通道（Clca）在许多细胞中广泛表达，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的肺动脉高压（PH）的病因学。本课题组最近的工作为钙调蛋白依赖性蛋白激酶II（CaMKII）和钙离子依赖性钙调神经磷酸酶（CaN; PP 2B）分别通过磷酸化和去磷酸化调节动脉SMC中的钙激活氯电流（Icuca）提供了证据。由于丝氨酸/苏氨酸磷酸酶CaN参与Clca电导的上调，可能也有其他磷酸酶参与其活性的上调。在兔肺动脉平滑肌细胞中，本研究的主要目的是：1）确定在静态和动态细胞内钙水平下CaMK II和CaN在调节CICa通道中的相对作用; 2）确定赋予CaNAa相对于CaNAa特异性的结构决定因素（3）在中国国际商会的规管方面;和3）检查其他丝氨酸/苏氨酸磷酸酶的可能参与（PP 1/PP 2A）在ICi（ca）的调制中的作用。为了完成该任务，将实施各种技术，包括使用药理学试剂来抑制蛋白磷酸酶，细胞膜片钳条件下，在兔PASMC，以确定在CICa电导的PP 1和PP 2A的相对贡献。这项技术也将被证明是有用的，以确定是否CaN和其他（S）Ser/Thr磷酸酶靶向共同或不同的磷酸化位点，以调节心血管疾病（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，CVD是美国所有死亡人数中超过37%的潜在死亡原因。由于离子通道在调节血管张力中起着重要作用，因此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将导致新的血管靶点，用于开发新药，以改善患有心血管疾病（如PH）、中风、心肌心绞痛和梗死的患者的状况。

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.