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.

项目摘要 在电力器官（例如胃肠道（GI）园）中，需要产生离子通道 驱动收缩的电活动。反过来，机械力会影响离子通道功能，因此 电活动，称为机械电源反馈。因此，离子通道机械效率为 对于正常功能和异常可能导致疾病。在上一个赠款周期中，我们有 表明由SCN5A编码的机械敏感电压门控钠通道NAV1.5 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）相关。 NAV1.5电流密度和 对正常和异常机械电反馈的机械敏感性尚不清楚。中心假设 该建议的是NAV1.5机械敏感和电流密度对于控制人GI至关重要 平滑肌肉兴奋性，并且都受到调节和目标。我们将在3中检验中心假设 具体目的（SAS）。我们将在以下确定：SA1中IBS NAV1.5突变如何影响机械敏感性以及如何 机械敏度的变化会影响机械电气反馈； SA2 NAV1.5孔确定 某些药物的机械敏感性和机械敏感性阻滞； SA3 NAV1.5如何受miRNA的调节 平滑肌细胞以及miRNA和药物对GI平滑肌细胞的调节的影响 功能。 SAS得到了广泛的初步数据的支持。 1）30％与IBS相关的SCN5A突变 导致机械敏感性异常降低机械电气反馈。 2）NAV1.5机械敏感性 取决于离子通道孔，而NAV1.5诸如ranolazine等药物的NAV1.5机械敏感性是 机械上与峰值电流块分开。 3）在慢速过境便秘的GI平滑肌中 患者NAV1.5被下调，一小部分miRNA被上调，而miRNA let-7f与 NAV1.5表达，下调NAV1.5电流并改变电慢波活性。 4）患者 雷诺嗪延迟了结肠运输，而大鼠gi transit被雷诺嗪延迟。调查中央 假设我们使用多种尖端技术，包括全电池和单通道电压 - 电流夹电生理学和光遗传学与超快速压力递送结合 CRISPR-CAS将患者突变引入细胞，具有设计器功能域的细菌NAV通道， Western印迹，IHC，慢病毒的miRNA模拟物，大鼠器官培养物和前瞻性临床 审判。成功完成拟议的研究具有基本意义和临床影响。因此 在以前的赠款周期中所做的工作以及本提案中提供的初步数据，我们将 显着提高了我们对NAV1.5通道机械敏感性分子机制的理解， NAV1.5机械敏感性对GI中NAV1.5的机械反馈和调节的贡献 平滑肌以了解如何靶向NAV1.5来调节异常的gi功能。