Mechanotransduction in Intestinal Smooth Muscle Cells

肠平滑肌细胞的力转导

• 批准号: 9905495
• 负责人: Arthur Beyder
• 金额: $ 35.78万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9905495
• 关键词: Address; Affect; Animals; Binding Sites; Biophysics; Cell physiology; Cells; Clinical; Clinical Trials; Closure by clamp; Clustered Regularly Interspaced Short Palindromic Repeats; Colon; Constipation; Data; Development; Disease; Electrophysiology (science); Feedback; Functional disorder; Gastrointestinal Diseases; Gastrointestinal Motility; Gastrointestinal Transit; Gastrointestinal tract structure; Generations; Grant; Habits; Health; Heart; Human; In Vitro; Interstitial Cell of Cajal; Intestines; Ion Channel; Ion Channel Gating; Irritable Bowel Syndrome; Lead; Mechanics; Mediating; Methods; MicroRNAs; Molecular; Motor; Mutation; Organ; Patients; Pharmaceutical Preparations; Phenotype; Population; Process; Property; Rattus; Regulation; Small Intestines; Smooth Muscle; Smooth Muscle Myocytes; Sodium Channel; Stretching; Subfamily lentivirinae; Symptoms; Techniques; Testing; Time; Tissues; Transducers; Translating; Validation; Western Blotting; Work; base; cohort; density; effective therapy; experimental study; gastrointestinal; gastrointestinal function; in vivo; loss of function; mechanical force; mechanotransduction; motility disorder; novel; optogenetics; pressure; prospective; ranolazine; single molecule; voltage; voltage gated channel

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.

项目概要 在机电器官中，例如胃肠道 (GI)，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，离子通道机械敏感性为 对于正常功能很重要，异常可能导致疾病。在之前的资助周期中，我们有 研究表明，由 SCN5A 编码的机械敏感电压门控钠通道 NaV1.5 存在于 人类小肠和结肠的胃肠道平滑肌细胞。此外，SCN5A突变是 与肠易激综合症（IBS）有关。 NaV1.5电流密度和的贡献 对正常和异常机电反馈的机械敏感性尚不清楚。中心假设 该提案的要点是 NaV1.5 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，两者都是可调节且可靶向的。我们将在3中检验中心假设 具体目标（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影响机械敏感性 机械敏感性的变化影响机械电反馈； SA2 NaV1.5孔如何决定 机械敏感性和某些药物的机械敏感性阻断； SA3 NaV1.5如何受miRNA调控 平滑肌细胞及miRNA和药物调控NaV1.5对胃肠道平滑肌细胞的影响 功能。 SA 得到了广泛的初步数据的支持。 1) 30% 的 IBS 相关 SCN5A 突变 导致机械敏感性异常，减少机械电反馈。 2) NaV1.5机械敏感性 取决于离子通道孔，雷诺嗪等药物对 NaV1.5 的机械敏感性阻断是 机械上与峰值电流块分开。 3) 慢传输型便秘引起的胃肠道平滑肌 患者 NaV1.5 下调，而一小部分 miRNA 上调，并且 miRNA let-7f 与 NaV1.5 表达下调 NaV1.5 电流并改变电慢波活动。 4) 患者 雷诺嗪会延迟结肠转运，并且雷诺嗪会延迟大鼠胃肠道转运。为调查中央 假设我们使用多种尖端技术，包括全细胞和单通道电压- 电流钳电生理学和光遗传学与超快速压力传输相结合， CRISPR-Cas 将患者突变引入细胞、具有设计功能域的细菌 NaV 通道、 蛋白质印迹、IHC、慢病毒递送 miRNA 模拟物、大鼠器官型培养物和前瞻性临床 审判。拟议研究的成功完成既具有基础意义，又具有临床影响。因此 根据之前拨款周期中完成的工作以及本提案中提供的初步数据，我们将 显着促进我们对 NaV1.5 通道机械敏感性分子机制的理解， NaV1.5 机械敏感性对胃肠道中 NaV1.5 机械电反馈和调节的贡献 平滑肌，以了解如何靶向 NaV1.5 来调节异常的胃肠道功能。