Gap junctions in Vascular Smooth Muscle: growth control

描述(申请人提供)：缝隙连接蛋白(连接蛋白Cx)的表达缺陷引起的人类疾病，包括：周围神经疾病、心脏发育畸形、耳聋、白内障和皮肤病，对人类健康的重要性日益明显。Cx基因的去除和替换在小鼠中产生的不同的表型进一步支持了缝隙连接对心血管系统的发育和功能的重要性，特别是对动物的重要性。缝隙连接的主要功能是调节细胞间信号分子的交换，从而在健康和疾病中产生协调的组织功能。这种交换途径的选择性显然是Cx特异性的，并且受到强烈的调控。目前这项提议的长期目标是确定Cx40和Cx43顺序和同时表达的功能后果。特别是，将研究由Cx43、Cx40和这两个连接蛋白形成的连接的选择性，以及通过磷酸化依赖的机制调节连接选择性的机制和结构基础。在目标1中，研究了磷酸化/去磷酸化对由通道和连接组成的Cx43或Cx40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。通过电喷雾电离(ES)和高效液相色谱(LC)相结合的强大组合，可以快速和灵敏地分析蛋白质和多肽，从而在线分离多肽混合物，然后进行串联质谱分析(MS/MS)，从而识别每个CX中特定激酶所针对的氨基酸残基。将使用这些位点的定点突变来探索每个CX中已识别的磷酸化位点的功能意义。在目标3中，研究了混合通道形成对连接选择性的影响，通过磷酸化依赖的机制对其进行调节，以及观察到的调节的结构基础。拟议的研究结果应该为差异Cx表达的功能后果提供新的见解，这是理解缝隙连接通道选择性及其调控的结构基础的基本信息，也是发展关于Cx相互作用规则及其在体内的潜在后果的可检验假说的必要数据。

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.