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。本提案的总体目标是了解平滑肌和Cajal间质细胞（ICC）机械敏感性的离子机制，以确定离子通道病在运动障碍中的作用，并建立离子通道相互作用蛋白和脂质环境在健康和疾病中机械感觉离子通道调节中的新作用。该建议的中心假设是，在人肠平滑肌细胞和ICC中表达的机械感觉离子通道由特定机制调节。Na+通道由Na+通道相互作用蛋白（SCIPs）门控，L-型Ca 2+通道由脂质双层门控。通道亚基本身和/或通道相互作用蛋白的突变可导致运动障碍。中心假设将在两个具体目标中得到检验。具体目标1将检验以下假设：人肠平滑肌细胞和ICC中的机械敏感性Na+电流由构成功能单元的SCIP调节，并且构成功能单元的蛋白质突变可能导致疾病。具体目标2将检验L型Ca 2+通道的机械敏感性是通道与脂质双层之间相互作用的结果这一假设。初步数据支持了中心假设，这些数据表明，SCIP（包括telethonin）与机械感觉Na+通道协同作用以产生一个功能单位，编码Nav1.5的基因（SCN 5A）突变导致胃肠道症状，该提案中新发现的SCIP突变发现于运动障碍患者中，并且脂质双层中的张力机械地门控L型Ca 2+通道并改变开放概率。PI将通过结合电生理学、分子和基于群体的技术来检验中心假设。这些技术包括膜片钳、免疫组织化学、蛋白质印迹、RT-PCR、SCPCR、qRT-PCR、酵母双杂交、GST-下拉、变性高效液相色谱和基于问卷的技术。成功完成拟定研究具有基础意义和临床影响。我们现在准备显着推进我们的理解的一个功能性机械敏感Nav1.5通道的组成部分和L型Ca 2+通道机械敏感性的机制。这项工作还将提供不仅与胃肠道相关，而且与心脏等也表达机械感觉离子通道和/或离子通道相互作用蛋白质的器官相关的机械信息。了解离子通道和离子通道相互作用蛋白在动力障碍中的作用突变不仅有助于确定这些疾病的原因，而且还有助于确定未来治疗胃肠道动力障碍的治疗靶点和策略。

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.