Mechanotransduction in Intestinal Smooth Muscle Cells

描述(申请人提供)：离子通道需要产生电活动，以驱动胃肠道和心脏等器官的收缩。在以前的授予周期中，我们已经证明了人小肠平滑肌细胞(SMC)表达电压敏感的Na+通道NaV1.5，其a亚基由SCN5A编码，并且NaV1.5是机械敏感的。NAV1.5的机械调节是高度相关的，因为它对电压的敏感度很高，通道动力学的微小变化明显影响生理。NaV1.5是选择性表达的。它在人类、狗和大鼠的肠道中产生Na+电流，但在其他几个物种如豚鼠和小鼠中不产生。NaV1.5的突变会导致疾病。这一建议的中心假设是，NAV1.5的机械敏感性是由于电压传感器的物理变化(S)，并且与生理相关的机械刺激显著改变了NAV1.5的功能。我们还假设，在肠易激综合征(IBS)的一组患者中，SCN5A的特定突变导致NaV1.5的电生理和机械敏感性改变，NaV1.5调节人SMC的膜电位和钙动力学。我们将在三个具体目标上检验中心假说。在SA 1中，我们将确定离子通道机械敏感性的基本机制。在SA 2中，我们将确定IBS中发现的NaV1.5突变的生理学相关性。在SA 3中，我们将确定NaV1.5的生理作用。初步数据表明，约3%的IBS患者(超过135万人)存在SCN5A突变，IBS SCN5A突变可改变NaV1.5的电生理，突变体和毒素可调节机械敏感性，机械敏感性也可被FDA批准的药物调控，NaV1.5的敲除和药理阻断可使人肠道环状SMC膜电位超极化，改变慢波频率，NaV1.5聚集在细胞膜上，通过NaV1.5进入细胞内可设置局部细胞内Na+，并通过Na+/Ca2+交换器(NCX)调节钙离子。我们将使用膜片钳技术、超快压力传递、高分辨率斑片成像、免疫组织化学、Western blots、单细胞聚合酶链式反应、定量聚合酶链式反应、慢病毒RNA击倒技术、器官和单细胞培养、蛋白质全内反射荧光(TIRF)成像、钙离子和钠离子以及微电极记录来研究这一中心假说。建议研究的顺利完成具有基础意义和临床影响。由于在之前的资助周期中所做的工作以及本提案中提出的初步数据，我们现在准备在亚分子水平上显著推进我们对机械敏感性的基本机制的理解，对NaV1.5在正常和异常人类肠道生理中所起的作用的理解，并在IBS患者的子集中建立离子通道病的作用。

DESCRIPTION (provided by applicant): Ion channels are required to generate electrical activity that drives contractility in organs such as the gastrointestinal tract and the heart. In previous grant cycles we have shown that human small intestinal smooth muscle cells (SMC) express a voltage-sensitive Na+ channel, Nav1.5, the a subunit of which is encoded by SCN5A and that Nav1.5 is mechanosensitive. Mechano-regulation of Nav1.5 is highly relevant because of the steep voltage-sensitivity, with small changes in channel kinetics markedly affecting physiology. Nav1.5 is selectively expressed. It generates a Na+ current in the intestinal tract of humans, dogs and rats but not in several other species such as guinea pig and mouse. Mutations in Nav1.5 cause disease. The central hypothesis of this proposal is that mechanosensitivity of the Nav1.5 is due to physical changes in the voltage sensor(s), and that physiologically relevant mechanical stimuli markedly alter Nav1.5 function. We also hypothesize that in a subset of patients with irritable bowel syndrome (IBS), specific mutations in SCN5A result in altered electrophysiology and mechanosensitivity of Nav1.5 and that Nav1.5 regulates membrane potential and Ca2+ dynamics of human SMC. We will test the central hypothesis in 3 specific aims. In SA 1 we will determine the basic mechanisms that underlie ion channel mechanosensitivity. In SA 2 we will determine the physiological relevance of Nav1.5 mutations found in IBS. In SA 3 we will determine the physiological role of Nav1.5. The specific aims are supported by preliminary data which show that SCN5A mutations are found in approximately 3% of patients with IBS (over 1.35 million), that IBS SCN5A mutations change the electrophysiology of Nav1.5, that mutants and toxins modulate mechanosensitivity, that mechanosensitivity can also be modulated by FDA approved drugs, that knockdown and pharmacological block of Nav1.5 hyperpolarize human intestinal circular SMC membrane potential and change slow wave frequency, that Nav1.5 is clustered on the cell membrane and that Na+ entry through Nav1.5 sets local intracellular Na+ and regulates Ca2+ through Na+/Ca2+ exchanger (NCX). We will use patch clamp techniques, ultrafast pressure delivery, high resolution patch imaging, immunohistochemistry, Western blots, single cell PCR, quantitative PCR, lentivirus RNA knock down techniques, organotypic and single cell cultures, total internal reflection fluorescence (TIRF) imaging of proteins, Ca2+ and Na+ as well as microelectrode recordings to investigate the central hypothesis. 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 are now poised to significantly advance our understanding, at a sub- molecular level, of the fundamental mechanisms that underlie mechanosensitivity, of the role Nav1.5 plays in normal and abnormal human intestinal physiology and to establish a role of ion channelopathies in a subset of patients with IBS.