Molecular mechanism of regulation of mI(CAT) in intestinal smooth muscle cells

肠平滑肌细胞mI(CAT)调控的分子机制

• 批准号: 8207618
• 负责人: MICHAEL X ZHU
• 金额: $ 20.15万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8207618
• 关键词: Molecular; mechanism; regulation; mI; CAT

DESCRIPTION (provided by applicant): Acetylcholine is the primary transmitter released by enteric excitatory motor neurons. It plays a central role in the control of motility of the gastrointestinal tract. The excitatory input is received by G protein-coupled muscarinic acetylcholine receptors (mAChRs) expressed in postjunctional cells - smooth muscle cells and interstitial cells of Cajal. Most visceral smooth muscles co-express both type 2 and type 3 mAChRs (M2R and M3R). Interactions between M2R and M3R are central to understand cholinergic transmission and contraction of gastrointestinal smooth muscles. In the smooth muscle cells, co-stimulation of M2R and M3R by muscarinic agonists activates a cation current, mICAT. Recent studies demonstrate that mICAT is mainly mediated by canonical transient receptor potential 4 (TRPC4). We have shown that like mICAT, the activation of TRPC4 channel is dependent on the co-stimulation of both Gq/11 and Gi/o-coupled receptors. The TRPC4 currents share many biophysical properties and regulatory features with mICAT. The goal of the proposed project is to use TRPC4 as the molecular model to examine two unique and outstanding features concerning the activation mechanisms of mICAT and their implications in gastrointestinal smooth muscle physiology. The first is the codependence on Gq/11 and Gi/o signaling pathways for channel activation and the second is the dual regulation by intracellular Ca2+. This goal is consistent with our long-term objective in elucidating the regulatory mechanisms and physiological functions of TRP channels. We hypothesize that the two G protein signaling pathways work synergistically on TRPC4 channel to activate mICAT. Gi/o proteins act via physical interaction of the G1i/o or G23 subunits, or both, with the TRPC4 protein, and Ca2+ exerts multiple regulatory actions through calmodulin binding at distinct sites of the channel molecule. The project has two specific aims: 1) to determine the physiological significance and molecular mechanism of Gi/o-mediated mICAT activation; 2) to dissect the molecular mechanisms of regulation of mICAT (TRPC4) by intracellular Ca2+. A multidisciplinary approach that combines molecular biology (heterologous expression and site-directed mutagenesis), biochemistry (protein-protein interactions), electrophysiology (whole-cell and single channel recordings), and genetic approaches (transgenic mice that express defined TRPC isoforms and mutant channels) will be used to accomplish the proposed research. The study will enhance our understanding on excitation-contraction coupling and other contractile functions of smooth muscles and shed light on the pathogenesis and new treatment of a wide range of human diseases caused by smooth muscle dysfunctions, such as inflammatory bowel disease, irritable bowel syndrome, and urge incontinence. The molecular details of TRPC4 regulation to be generated will also significantly impact our knowledge in other physiological systems, where TRPC4 and related TRPC5 channels are known to involve in functions such as vasoconstriction/relaxation, synaptic transmission, neurite outgrowth/neural development, and learning. PUBLIC HEALTH RELEVANCE: This project focuses on the molecular mechanism of activation and regulation of muscarinic acetylcholine receptor-evoked cation current found in intestinal smooth muscle cells. The study is aimed to provide a better understanding on how neurotransmitters trigger membrane depolarization and the subsequent intracellular calcium increase to cause smooth muscle contraction in the gastrointestinal system. This will shed light on the pathogenesis and new treatment of a wide range of human diseases caused by smooth muscle dysfunctions, such as inflammatory bowel disease, irritable bowel syndrome, and urge incontinence.

描述（由申请人提供）：乙酰胆碱是肠兴奋性运动神经元释放的主要递质。它在胃肠道运动的控制中起着核心作用。兴奋性输入由连接后细胞（平滑肌细胞和卡哈尔间质细胞）中表达的 G 蛋白偶联毒蕈碱乙酰胆碱受体 (mAChR) 接收。大多数内脏平滑肌同时表达 2 型和 3 型 mAChR（M2R 和 M3R）。 M2R 和 M3R 之间的相互作用对于了解胆碱能传递和胃肠平滑肌收缩至关重要。在平滑肌细胞中，毒蕈碱激动剂对 M2R 和 M3R 的共同刺激会激活阳离子电流 mICAT。最近的研究表明，mICAT 主要由经典瞬时受体电位 4 (TRPC4) 介导。我们已经证明，与 mICAT 一样，TRPC4 通道的激活依赖于 Gq/11 和 Gi/o 偶联受体的共同刺激。 TRPC4 电流与 mICAT 具有许多相同的生物物理特性和调控特征。该项目的目标是使用 TRPC4 作为分子模型来研究有关 mICAT 激活机制的两个独特且突出的特征及其对胃肠道平滑肌生理学的影响。第一个是通道激活对 Gq/11 和 Gi/o 信号通路的相互依赖性，第二个是细胞内 Ca2+ 的双重调节。这一目标与我们阐明 TRP 通道的调节机制和生理功能的长期目标是一致的。我们假设这两条 G 蛋白信号通路在 TRPC4 通道上协同作用以激活 mICAT。 Gi/o 蛋白通过 G1i/o 或 G23 亚基或两者与 TRPC4 蛋白的物理相互作用发挥作用，Ca2+ 通过在通道分子的不同位点结合钙调蛋白发挥多种调节作用。该项目有两个具体目标：1）确定Gi/o介导的mICAT激活的生理意义和分子机制； 2) 剖析细胞内Ca2+调节mICAT (TRPC4)的分子机制。一种多学科方法，结合了分子生物学（异源表达和定点突变）、生物化学（蛋白质-蛋白质相互作用）、电生理学（全细胞和单通道记录）和遗传方法（表达特定 TRPC 亚型和突变通道的转基因小鼠） ）将用于完成拟议的研究。该研究将增进我们对平滑肌兴奋-收缩耦合和其他收缩功能的理解，并揭示由平滑肌功能障碍引起的多种人类疾病的发病机制和新的治疗方法，例如炎症性肠病、肠易激综合征、并急迫失禁。 TRPC4 调控的分子细节也将显着影响我们对其他生理系统的了解，其中 TRPC4 和相关的 TRPC5 通道已知参与血管收缩/松弛、突触传递、神经突生长/神经发育和学习等功能。公共健康相关性：该项目重点研究肠道平滑肌细胞中毒蕈碱乙酰胆碱受体诱发的阳离子电流的激活和调节的分子机制。该研究旨在更好地了解神经递质如何触发膜去极化以及随后的细胞内钙增加以引起胃肠系统平滑肌收缩。这将揭示由平滑肌功能障碍引起的多种人类疾病的发病机制和新的治疗方法，例如炎症性肠病、肠易激综合征和急迫性尿失禁。