Mechanotransduction in Intestinal Smooth Muscle Cells

描述（由申请人提供）：机械敏感性是有效胃肠功能所必需的。本提案的总体目标是了解平滑肌和间质细胞Cajal （ICC）机械敏感性的离子机制，确定离子通道病变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。本研究的中心假设是，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+通道由Na+通道相互作用蛋白（SCIPs）控制，l型Ca2+通道由脂质双分子层控制。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。Specific Aim 1将验证人体肠平滑肌细胞和ICC中的机械敏感Na+电流由构成功能单元的SCIPs调节，以及构成功能单元的蛋白质突变可导致疾病的假设。特异性目标2将测试假设，即l型Ca2+通道的机械敏感性是通道和脂质双分子层之间相互作用的结果。中心假设得到了初步数据的支持，这些数据表明，SCIPs，包括telethonin，与机械感觉Na+通道协同作用，创造一个功能单元，编码Nav1.5的基因（SCN5A）突变导致胃肠道症状，在本建议中新发现的SCIPs突变，在运动障碍患者中被发现，脂质双分子层的张力机械地关闭l型Ca2+通道并改变开放概率。PI将通过结合电生理、分子和基于人口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、Western blots、RT-PCR、SCPCR、qRT-PCR、酵母双杂交、GST-pulldowns、变性高效液相色谱和基于问卷的技术。本研究的成功完成既有基础意义又有临床意义。我们现在准备显著推进我们对功能机械敏感的Nav1.5通道的组成部分和l型Ca2+通道机械敏感的机制的理解。这项工作还将提供机制信息，不仅与胃肠道有关，而且与心脏等器官有关，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蛋白。了解离子通道突变和离子通道相互作用蛋白在运动障碍中的作用不仅有助于确定这些疾病的原因，而且有助于确定未来治疗胃肠道运动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.