Mechanotransduction in Intestinal Smooth Muscle Cells

项目总结 在机电器官中，如胃肠道，需要离子通道来产生 驱动收缩的电活动。反过来，机械力影响离子通道功能，因此 电活动，称为机电反馈。因此，离子通道的机械敏感性是 对正常功能很重要，异常会导致疾病。在之前的赠款周期中，我们有 结果表明，由SCN5A编码的机械敏感的电压门控钠通道NaV1.5存在于 人小肠和结肠的胃肠平滑肌细胞。此外，SCN5A突变是 与肠易激综合征(IBS)相关。NaV1.5电流密度的贡献和 对正常和异常的机电反馈的机械敏感性尚不清楚。中心假说 这一建议的关键是NaV1.5的机械敏感性和电流密度对于控制人类GI至关重要 平滑肌兴奋性，两者都是可调节和靶向的。我们将在3个月测试中心假设 特定目标(SA)。我们将在：SA1中确定IBS NaV1.5突变如何影响机械敏感性以及如何 机械敏感度的变化影响机电反馈；SA2 NaV1.5气孔如何决定 机械敏感性和机械敏感性被某些药物阻断；SA3 NaV1.5是如何被miRNAs调控的 血管内皮细胞及miRNA和药物对NaV1.5的调节对胃肠平滑肌细胞的影响 功能。SA得到了大量初步数据的支持。1)30%的IBS相关SCN5A突变 结果异常的机械敏感性降低了机电反馈。2)NaV1.5机械敏感性 依赖于离子通道孔，而NaV1.5对雷诺嗪等药物的机械敏感性阻断是 从机械上与峰值电流阻挡隔离。3)慢传输型便秘引起的胃肠道平滑肌 患者NaV1.5下调，一小部分miRNAs上调，miRNA let-7f与 NaV1.5表达，下调NaV1.5电流，改变电慢波活动。4)患者在 雷诺嗪延缓了大鼠的结肠转运，延缓了大鼠的胃肠道转运。为了调查中央 假设我们使用了各种各样的尖端技术，包括全电池和单通道电压- 以及电流钳电生理学和光遗传学与超快压力输送相结合， CRISPR-CA将患者突变引入细胞，具有设计功能域的细菌NAV通道， 免疫印迹、免疫组化、慢病毒传递miRNA模拟物、大鼠器官培养和前瞻性临床研究 审判。建议研究的顺利完成具有基础意义和临床影响。结果 关于前几个赠款周期所做的工作和本提案中提出的初步数据，我们将 极大地提高了我们对NaV1.5通道机械敏感性的分子机制的理解， 胃肠道NaV1.5的机械敏感性在机电反馈和调节中的作用 为了了解如何靶向NAV1.5来调节异常的胃肠功能。

PROJECT SUMMARY In electro-mechanical organs, such as the gastrointestinal (GI) tract, ion channels are required to generate electrical activity that drives contractions. In turn, mechanical forces affect ion channel function and therefore electrical activity, which is termed mechano-electric feedback. Therefore, ion channel mechanosensitivity is important for normal function, and abnormalities can lead to disease. In the previous grant cycles we have shown that a mechano-sensitive voltage-gated sodium channel NaV1.5, encoded by SCN5A, is present in gastrointestinal smooth muscle cells of the human small bowel and colon. Further, SCN5A mutations are associated with irritable bowel syndrome (IBS). The contribution of NaV1.5 current density and mechanosensitivity to normal and abnormal mechano-electric feedback is not known. The central hypothesis of this proposal is that NaV1.5 mechanosensitivity and current density are critical for control of human GI smooth muscle excitability, and both are regulated and targetable. We will test the central hypothesis in 3 specific aims (SAs). We will determine in: SA1 how IBS NaV1.5 mutations affect mechanosensitivity and how changes in mechanosensitivity affect mechano-electric feedback; SA2 how NaV1.5 pore determines mechanosensitivity and mechanosensitivity block by certain drugs; SA3 how NaV1.5 is regulated by miRNAs in smooth muscle cells and the effects of NaV1.5 regulation by miRNA and drugs on GI smooth muscle cell function. The SAs are supported by extensive preliminary data. 1) 30% of IBS-associated SCN5A mutations result abnormal mechanosensitivity reducing mechano-electric feedback. 2) NaV1.5 mechanosensitivity depends on ion channel pore, and NaV1.5 mechanosensitivity blockade by drugs such as ranolazine is mechanistically separate from peak current block. 3) In GI smooth muscle from slow transit constipation patients NaV1.5 is down-regulated while a small set of miRNAs is upregulated and miRNA let-7f correlates with NaV1.5 expression, down-regulates NaV1.5 current and alters electrical slow wave activity. 4) Patients on ranolazine have delayed colon transit and rat GI transit is delayed by ranolazine. To investigate the central hypothesis we use a wide variety of cutting-edge techniques, including whole-cell and single-channel voltage- and current-clamp electrophysiology and optogenetics in combination with ultra-fast pressure delivery, CRISPR-Cas to introduce patient mutations into cells, bacterial NaV channels with designer functional domains, Western blots, IHC, delivery of miRNA mimics by lentivirus, rat organotypic cultures, and a prospective clinical trial. Successful completion of the proposed studies has both basic significance and clinical impact. As a result of the work done in the previous grant cycles and the preliminary data presented in this proposal, we will significantly advance our understanding of the molecular mechanisms of NaV1.5 channel mechanosensitivity, the contribution of NaV1.5 mechanosensitivity to mechano-electric feedback and regulation of NaV1.5 in GI smooth muscle in order to understand how to target NaV1.5 to modulate abnormal GI function.