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

描述(申请人提供)：乙酰胆碱是肠道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。兴奋性信息由G蛋白偶联的M受体(MAChRs)接受，mAChRs表达于Cajal的连接后细胞--平滑肌细胞和间质细胞。大多数内脏平滑肌同时表达2型和3型mAChRs(M2R和M3R)。M2R和M3R之间的相互作用是理解胃肠道平滑肌胆碱能传递和收缩的核心。在平滑肌细胞中，M受体激动剂对M2R和M3R的共同刺激激活了阳离子电流mICAT。最近的研究表明，mICAT主要是由典型的瞬时受体电位4(TRPC4)介导的。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具有许多生物物理特性和调节特性。该项目的目标是使用TRPC4作为分子模型来研究mICAT的两个独特而突出的激活机制及其在胃肠道平滑肌生理学中的意义。其一是相互依赖Gq/11和Gi/o信号通路激活通道，其二是细胞内钙离子的双重调节。这一目标与我们阐明色氨酸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，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，而Ca~(2+)通过与通道分子不同位置的钙调蛋白结合而发挥多种调节作用。该项目有两个具体目的：1)确定Gi/o介导的mICAT激活的生理意义和分子机制；2)剖析细胞内钙离子调节mICAT(TRPC4)的分子机制。将使用结合分子生物学(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)的多学科方法来完成拟议的研究。这项研究将加深我们对平滑肌兴奋-收缩偶联和其他收缩功能的理解，并有助于揭示由平滑肌功能障碍引起的一系列人类疾病的发病机制和新的治疗方法，如炎症性肠病、肠易激综合征和急迫性失禁。TRPC4调控的分子细节也将显著影响我们在其他生理系统中的知识，在这些系统中，TRPC4和相关的TRPC5通道被认为参与了血管收缩/松弛、突触传递、轴突生长/神经发育和学习等功能。与公共健康相关：本项目重点研究在肠道平滑肌细胞中发现的M胆碱受体诱发的阳离子电流的激活和调节的分子机制。这项研究的目的是为了更好地了解神经递质如何触发膜去极化和随后的细胞内钙增加，从而导致胃肠道系统的平滑肌收缩。这将有助于阐明由平滑肌功能障碍引起的一系列人类疾病的发病机制和新的治疗方法，如炎症性肠病、肠易激综合征和尿失禁。

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.