Gap junctions in Vascular Smooth Muscle: growth control

血管平滑肌的间隙连接：生长控制

• 批准号: 7039119
• 负责人: JANIS M BURT
• 金额: $ 33.15万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-15 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7039119
• 关键词: cell growth regulation; electrophysiology; electrospray ionization mass spectrometry; fluorescence microscopy; gap junctions; high performance liquid chromatography; mathematical model; membrane channels; membrane potentials; phosphorylation; protein structure function; site directed mutagenesis; tissue /cell culture; vascular smooth muscle

DESCRIPTION (provided by applicant): The significance of gap junction mediated communication to human health is increasingly evident from the human diseases caused by expression of defective gap junction proteins (connexins Cx), including: peripheral neuropathies, cardiac developmental malformations, deafness, cataracts and skin diseases. The diverse phenotypes in mice arising from Cx gene ablation and substitution lend further support to the importance of gap junctions to development and function of the cardiovascular system, in particular, and to the animal in general. The primary function of gap junctions is to mediate the intercellular exchange of the signaling molecules that result in coordinated tissue function in health and disease. The selectivity of this exchange pathway is apparently Cx specific and acutely regulated. The long-term goal of the current proposal is to identify the functional consequences of sequential and simultaneous expression of Cx40 and Cx43. In particular, the selectivity of the junctions formed by Cx43, Cx40 and both connexins, and the mechanisms and structural basis for regulation of junctional selectivity by phosphorylation-dependent mechanisms will be examined. In Aim 1 the consequences of phosphorylation/dephosphorylation on the selectivity of Cx43 or Cx40 comprised channels and junctions are explored. Selectivity will be quantified using state-of-the-art electrophysiology, fluorescence microscopy and mathematical modeling techniques. In Aim 2 the structural bases for the responses delineated in Aim 1 are determined. The amino acid residues targeted by specific kinases in each Cx are identified by the powerful combination of electrospray ionization (ES), which allows for rapid and sensitive analyses of proteins and peptides, coupled to an HPLC (LC), which allows for on-line separation of peptide mixtures, followed by tandem mass spectrometry (MS/MS). The functional significance of identified phosphorylation sites in each Cx will be explored using site-directed mutants of those sites. In Aim 3 the consequences of mixed channel formation on junctional selectivity, the regulation thereof by phosphorylation-dependent mechanisms, and the structural bases for observed regulation are examined. The results of the proposed studies should provide new insights on the functional consequences of differential Cx expression, which is essential information for understanding the structural basis for gap junction channel selectivity and regulation thereof, and necessary data for the development of testable hypotheses regarding the rules of Cx interaction and their potential consequences in the in vivo setting.

描述（由申请人提供）：间隙连接介导的通讯对人类健康的重要性越来越明显，因为间隙连接蛋白（连接蛋白Cx）表达缺陷引起的人类疾病包括：周围神经病、心脏发育畸形、耳聋、白内障和皮肤病。小鼠中 Cx 基因消融和替代产生的多样化表型进一步支持了间隙连接对于心血管系统（特别是整个动物）的发育和功能的重要性。间隙连接的主要功能是介导信号分子的细胞间交换，从而协调健康和疾病中的组织功能。这种交换途径的选择性显然是 Cx 特异性的并且受到严格调节。当前提案的长期目标是确定 Cx40 和 Cx43 顺序和同时表达的功能后果。特别是，将检查由 Cx43、Cx40 和两种连接蛋白形成的连接的选择性，以及通过磷酸化依赖性机制调节连接选择性的机制和结构基础。在目标 1 中，探讨了磷酸化/去磷酸化对 Cx43 或 Cx40 组成的通道和连接选择性的影响。将使用最先进的电生理学、荧光显微镜和数学建模技术来量化选择性。在目标 2 中，确定了目标 1 中描述的响应的结构基础。每个 Cx 中特定激酶靶向的氨基酸残基通过电喷雾电离 (ES) 的强大组合进行鉴定，电喷雾电离 (ES) 可以对蛋白质和肽进行快速、灵敏的分析，与 HPLC (LC) 相结合，可以在线分离肽混合物，然后进行串联质谱 (MS/MS)。将使用这些位点的定点突变体来探索每个 Cx 中已识别的磷酸化位点的功能意义。在目标 3 中，检查了混合通道形成对连接选择性的影响、磷酸化依赖性机制对其的调节以及观察到的调节的结构基础。所提出的研究结果应该为差异Cx表达的功能后果提供新的见解，这是理解间隙连接通道选择性及其调节的结构基础的重要信息，也是发展关于Cx相互作用规则及其在体内环境中潜在后果的可检验假设的必要数据。