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

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。它在控制胃肠道运动中起着核心作用。兴奋性输入由G蛋白偶联的毒蕈碱乙酰胆碱受体（mAChR）接收，所述受体在连接后细胞-平滑肌细胞和卡哈尔间质细胞中表达。大多数内脏平滑肌共表达2型和3型mAChR（M2 R和M3 R）。M2 R和M3 R之间的相互作用是理解胆碱能传递和胃肠平滑肌收缩的核心。在平滑肌细胞中，毒蕈碱激动剂对M2 R和M3 R的共刺激激活阳离子电流miICAT。最近的研究表明，mICAT主要由典型瞬时受体电位4（TRPC 4）介导。我们已经表明，像mICAT一样，TRPC 4通道的激活依赖于Gq/11和Gi/o偶联受体的共刺激。TRPC 4电流与MICAT共享许多生物物理特性和调控特征。该项目的目标是使用TRPC 4作为分子模型，以研究两个独特的和突出的功能有关的激活机制的mICAT及其在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信号通路的共依赖性，第二个是细胞内Ca 2+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。我们推测这两条G蛋白信号通路协同作用于TRPC 4通道激活mICAT。Gi/o蛋白通过G1 i/o或G23亚基或两者与TRPC 4蛋白的物理相互作用发挥作用，并且Ca 2+通过钙调蛋白结合在通道分子的不同位点发挥多种调节作用。本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Ca 2+调节mICAT（TRPC 4）的分子机制。结合分子生物学（异源表达和定点诱变），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（表达定义的TRPC亚型和突变通道的转基因小鼠）的多学科方法将用于完成拟议的研究。这项研究将加强我们对平滑肌兴奋-收缩偶联和其他收缩功能的理解，并阐明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，如炎症性肠病，肠易激综合征和急迫性尿失禁。TRPC 4调节的分子细节也将显著影响我们在其他生理系统中的知识，其中已知TRPC 4和相关的TRPC 5通道参与诸如血管收缩/舒张、突触传递、神经突生长/神经发育和学习的功能。公共卫生相关性：本项目主要研究肠平滑肌细胞中毒蕈碱型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和随后的细胞内钙增加，从而引起胃肠系统平滑肌收缩。这将阐明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，如炎症性肠病、肠易激综合征和急迫性尿失禁。

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.