Gap junctions in Vascular Smooth Muscle: growth control

描述（由申请人提供）：从缺陷间隙连接蛋白（connexins Cx）表达引起的人类疾病来看，间隙连接介导的通讯对人类健康的重要性越来越明显，包括：周围神经病变、心脏发育畸形、耳聋、白内障和皮肤病。Cx基因消融和替代在小鼠中引起的不同表型进一步支持间隙连接对心血管系统发育和功能的重要性，特别是对动物的重要性。间隙连接的主要功能是介导细胞间信号分子的交换，从而在健康和疾病中协调组织功能。这种交换途径的选择性显然是Cx特异性的，并且受到严格调控。当前提案的长期目标是确定顺序和同时表达Cx40和Cx43的功能后果。特别地，Cx43、Cx40和两种连接蛋白形成的连接的选择性，以及磷酸化依赖机制调节连接选择性的机制和结构基础将被研究。在目的1中，探讨了磷酸化/去磷酸化对Cx43或Cx40组成通道和连接的选择性的影响。选择性将使用最先进的电生理学，荧光显微镜和数学建模技术进行量化。在目标2中，确定了目标1中描述的反应的结构基础。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，这允许快速和敏感地分析蛋白质和肽，再加上高效液相色谱（LC），允许在线分离肽混合物，然后是串联质谱（MS/MS）。每个Cx中鉴定的磷酸化位点的功能意义将通过这些位点的位点导向突变来探索。在Aim 3中，混合通道形成对连接选择性的影响，磷酸化依赖机制对其的调节，以及观察到的调节的结构基础进行了检查。这些研究结果将为Cx差异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控的结构基础的重要信息，也是关于Cx相互作用规则及其在体内环境中潜在后果的可测试假设发展的必要数据。

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.