Mechanotransduction in Intestinal Smooth Muscle Cells

描述(由申请人提供)：有效的胃肠道功能需要机械敏感性。这项建议的总体目标是了解Cajal平滑肌和间质细胞(ICC)机械敏感性的离子机制，确定离子通道病变在运动性疾病中的作用，并确定离子通道相互作用的蛋白质和脂质环境在健康和疾病中调节机械感觉离子通道的新角色。这一建议的中心假设是，在人类肠道平滑肌细胞和ICC中表达的机械感觉离子通道受到特定机制的调节。Na+通道受钠离子通道相互作用蛋白(SCIPs)调控，L类钙通道受脂质双层调控。通道亚单位本身和/或通道相互作用蛋白的突变可能导致运动障碍。核心假设将在两个具体目标上得到检验。具体目标1将检验这样一种假设，即人类肠道平滑肌细胞和ICC中的机械敏感钠电流受组成功能单位的SCIP调节，组成功能单位的蛋白质突变可导致疾病。具体目标2将验证L类钙离子通道的机械敏感性是该通道与脂质双分子层相互作用的结果的假设。中心假说得到了初步数据的支持，这些数据表明，SCIP，包括端乙醇胺，与机械感觉Na+通道共同作用，创建一个功能单位；编码NaV1.5的基因(SCN5A)的突变会导致胃肠道症状；本研究中新发现的SCIP突变在动力障碍患者中被发现；以及脂质双层中的张力机械地关闭L类型的钙通道并改变开放概率。PI将通过电生理、分子和基于群体的技术的组合来检验中心假设。这些技术包括膜片钳技术、免疫组织化学技术、免疫印迹技术、RT-PCR技术、SCPCR技术、qRT-PCR技术、酵母双杂交技术、GST-Pull-Down技术、变性高效液相色谱技术和基于问卷的技术。建议研究的顺利完成具有基础意义和临床影响。我们现在准备显著提高我们对功能性机械敏感性NaV1.5通道的组成以及L类型钙离子通道机械敏感性的机制的理解。这项工作还将提供机制信息，这些信息不仅与胃肠道有关，还与心脏等器官有关，这些器官也表达机械感觉离子通道和/或离子通道相互作用的蛋白质。了解离子通道和离子通道相互作用蛋白突变在动力障碍中的作用，不仅有助于确定这些障碍的原因，还将有助于确定未来治疗胃肠道动力障碍的靶点和策略。

DESCRIPTION (provided by applicant): Mechanosensitivity is required for effective gastrointestinal function. The overall objective of this proposal is to understand the ionic mechanisms that underlie smooth muscle and interstitial cell of Cajal (ICC) mechanosensitivity, to determine the role of ion channelopathies in motility disorders and to establish new roles for ion channel interacting proteins and the lipid environment in the regulation of mechanosensory ion channels in health and in disease. The central hypothesis of this proposal is that mechanosensory ion channels expressed in human intestinal smooth muscle cells and ICC are regulated by specific mechanisms. The Na+ channel is gated by Na+ channel interacting proteins (SCIPs) and the L-type Ca2+ channel by the lipid bilayer. Mutations in the channel subunits themselves and/or the channel interacting proteins can contribute to motility disorders. The central hypothesis will be tested in two specific aims. Specific Aim 1 will test the hypothesis that the mechanosensitive Na+ current in human intestinal smooth muscle cells and ICC is regulated by SCIPs that make up a functional unit and that mutations in proteins that make up the functional unit can result in disease. Specific Aim 2 will test the hypothesis that mechanosensitivity of L-type Ca2+ channels is a result of an interaction between the channel and the lipid bilayer. The central hypothesis is supported by preliminary data that show that SCIPs, including telethonin, act in concert with the mechanosensory Na+ channel to create a functional unit, that mutations in the gene (SCN5A) that encodes for Nav1.5 result in gastrointestinal symptoms, that mutations in SCIPs, newly identified in this proposal, are found in patients with motility disorders, and that tension in the lipid bilayer mechanically gates the L-type Ca2+ channel and alters open probability. The PI will test the central hypothesis by the combination of electrophysiological, molecular, and population based techniques. These include patch clamp, immunohistochemistry, Western blots, RT-PCR, SCPCR, qRT-PCR, yeast two hybrid, GST-pulldowns, denaturing high performance liquid chromatography and questionnaire based techniques. Successful completion of the proposed studies has both basic significance and clinical impact. We are now poised to significantly advance our understanding of the components of a functional mechanosensitive Nav1.5 channel and on the mechanism of L-type Ca2+ channel mechanosensitivity. The work will also provide mechanistic information that is relevant to not only the gastrointestinal tract but also to organs such as the heart that also express mechanosensory ion channels and/or ion channel interacting proteins. Understanding the role mutations in ion channels and in ion channel interacting proteins play in motility disorders will not only help determine the cause of these disorders but will also serve to identify future therapeutic targets and strategies to treat motility disorders of the gastrointestinal tract.