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: 8278680
• 负责人: MICHAEL X ZHU
• 金额: $ 29.41万
• 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 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8278680
• 关键词: Abbreviations; Acetylcholine; Acute; Binding; Binding Sites; Biochemistry; C-terminal; Calcium; Calmodulin; Cations; Cavia; Cell Surface Receptors; Cells; Characteristics; Codependence; Complex; Coupled; Coupling; Cytoskeleton; Dependence; Dissection; Electrophysiology (science); Enteral; Environment; Family; Functional disorder; G-Protein Signaling Pathway; GTP-Binding Proteins; Gastrointestinal tract structure; Genetic; Goals; Health; Heterotrimeric GTP-Binding Proteins; Inflammatory Bowel Diseases; Interstitial Cell of Cajal; Intestines; Irritable Bowel Syndrome; Knock-out; Knowledge; Learning; Light; Mediating; Membrane; Minor; Molecular; Molecular Biology; Molecular Models; Motor Neurons; Mus; Muscarinic Acetylcholine Receptor; Muscarinic Agonists; Muscarinics; Muscle Cells; Muscle Contraction; Nerve; Neurites; Neurotransmitters; Pathogenesis; Pertussis Toxin; Phosphatidylinositol 4,5-Diphosphate; Phospholipase C; Physiological; Physiology; Play; Property; Protein Isoforms; Proteins; Regulation; Relaxation; Research; Role; Signal Pathway; Signal Transduction; Site; Site-Directed Mutagenesis; Smooth Muscle; Smooth Muscle Myocytes; Structure of thyroid parafollicular cell; Synaptic Transmission; System; TRP channel; TRPC4 ion channel; Transgenic Mice; Urge Incontinence; Visceral; Work; base; cell motility; cholinergic; desensitization; gastrointestinal; gastrointestinal system; human disease; interdisciplinary approach; interest; member; molecular modeling; mutant; neurodevelopment; neurotransmitter release; overexpression; protein complex; protein protein interaction; receptor; receptor coupling; receptor operated channel; transmission process; vasoconstriction

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发射器。它在控制胃肠道运动性中起着核心作用。 G蛋白偶联的毒蕈碱乙酰胆碱受体（MACHRS）在后骨后细胞 - 平滑肌细胞和Cajal的间质细胞中接受了兴奋性输入。大多数内脏平滑肌共表达2型和3型MACHR（M2R和M3R）。 M2R和M3R之间的相互作用是了解胃肠道肌肉的胆碱能传播和收缩的核心。在平滑肌细胞中，毒蕈碱激动剂对M2R和M3R的共刺激会激活阳离子电流Micat。最近的研究表明，MICAT主要是由规范瞬态受体电位4（TRPC4）介导的。我们已经表明，与Micat一样，TRPC4通道的激活取决于GQ/11和GI/O偶联受体的共刺激。 TRPC4电流与MICAT具有许多生物物理特性和调节特征。拟议项目的目的是将TRPC4用作分子模型，以检查有关MICAT激活机制及其在胃肠道平滑肌生理的影响的两个独特而出色的特征。第一个是通道激活的GQ/11和GI/O信号通路的相互依赖性，第二个是细胞内Ca2+的双重调控。该目标与我们在阐明TRP通道的调节机制和生理功能方面的长期目标是一致的。我们假设两个G蛋白信号通路在TRPC4通道上协同工作以激活MICAT。 GI/O蛋白通过G1I/O或G23亚基的物理相互作用或TRPC4蛋白的物理相互作用作用，而Ca2+通过在通道分子的不同位点上的钙调蛋白结合发挥多种调节作用。该项目具有两个具体的目的：1）确定GI/O介导的MICAT激活的生理意义和分子机制； 2）通过细胞内Ca2+剖析MICAT调节（TRPC4）的分子机制。一种结合了分子生物学（异源表达和定位诱变），生物化学（蛋白质 - 蛋白质相互作用），电生理学（全细胞和单个通道记录）以及遗传方法（转基因小鼠（将表达TRPC同工型和突变通道的转基因小鼠）可以使用提出建议的研究）的多学科方法。这项研究将增强我们对平滑肌肉的激发反应耦合以及其他收缩功能的理解，并阐明了由平滑肌功能障碍引起的广泛的人类疾病的发病机理和新治疗，例如炎症性肠病，可激发的肠肠综合征和急诊无关。要生成的TRPC4调节的分子细节也将显着影响我们在其他生理系统中的知识，在其他生理系统中，TRPC4和相关的TRPC5通道已知涉及血管收缩/松弛，突触传播，神经突传播，神经突出/神经发展/神经发展和学习等功能。公共卫生相关性：该项目着重于毒蕈碱乙酰胆碱受体诱发的阳离子的激活和调节的分子机制，这些阳离子在肠平滑肌细胞中发现。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，从而导致胃肠道系统中的平滑肌收缩。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理和新治疗，例如炎症性肠病，肠易激综合症和敦促尿失禁。

项目成果

Acute Treatment with a Novel TRPC4/C5 Channel Inhibitor Produces Antidepressant and Anxiolytic-Like Effects in Mice.

• DOI: 10.1371/journal.pone.0136255
• 发表时间: 2015
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Yang LP;Jiang FJ;Wu GS;Deng K;Wen M;Zhou X;Hong X;Zhu MX;Luo HR
• 通讯作者: Luo HR

Selective potentiation of 2-APB-induced activation of TRPV1-3 channels by acid.

酸选择性增强 2-APB 诱导的 TRPV1-3 通道激活。

• DOI: 10.1038/srep20791
• 发表时间: 2016-02-15
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Gao L;Yang P;Qin P;Lu Y;Li X;Tian Q;Li Y;Xie C;Tian JB;Zhang C;Tian C;Zhu MX;Yao J
• 通讯作者: Yao J

Molecular mechanism of constitutive endocytosis of Acid-sensing ion channel 1a and its protective function in acidosis-induced neuronal death.

酸敏感离子通道1a组成型内吞的分子机制及其对酸中毒所致神经元死亡的保护作用

• DOI: 10.1523/jneurosci.5206-12.2013
• 发表时间: 2013-04-17
• 期刊: The Journal of neuroscience : the official journal of the Society for Neuroscience
• 作者: Zeng WZ;Liu DS;Duan B;Song XL;Wang X;Wei D;Jiang W;Zhu MX;Li Y;Xu TL
• 通讯作者: Xu TL

Bimodal voltage dependence of TRPA1: mutations of a key pore helix residue reveal strong intrinsic voltage-dependent inactivation.

TRPA1 的双峰电压依赖性：关键孔螺旋残基的突变揭示了强烈的内在电压依赖性失活

• DOI: 10.1007/s00424-013-1345-6
• 发表时间: 2014-07
• 期刊: PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY
• 影响因子: 4.5
• 作者: Wan, Xia;Lu, Yungang;Chen, Xueqin;Xiong, Jian;Zhou, Yuanda;Li, Ping;Xia, Bingqing;Li, Min;Zhu, Michael X.;Gao, Zhaobing
• 通讯作者: Gao, Zhaobing

The acid test: the discovery of two-pore channels (TPCs) as NAADP-gated endolysosomal Ca(2+) release channels.

• DOI: 10.1007/s00424-009-0682-y
• 发表时间: 2009-09
• 期刊: PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY
• 影响因子: 4.5
• 作者: Galione, Antony;Evans, A. Mark;Ma, Jianjie;Parrington, John;Arredouani, Abdelilah;Cheng, Xiaotong;Zhu, Michael X.
• 通讯作者: Zhu, Michael X.