Connexons in Cardiovascular Cell Communication

描述（由申请人提供）：心脏电传导依赖于离子通过间隙连接通道的细胞间通道。间隙连接分布和亚单位蛋白（连接蛋白，Cx）表达的改变与再入性心律失常的发病机制有关。连接蛋白对心脏的正常发育和非兴奋性组织的功能也至关重要，这意味着它在较大的溶质（如信号分子）的通过中起着重要作用。单个连接蛋白形成具有不同性质（包括电导、磁导率和门控）的通道。工作心肌中的主要连接蛋白是Cx43，但在各种心血管细胞中也发现了Cx37、Cx40和Cx45。多个连接蛋白经常共定位于相同的间隙连接斑块，它们可以形成异寡聚通道。Cx43与其他连接蛋白的共表达和异质通道的形成可能是调节和调节心血管细胞间通讯的主要机制。1. 生化研究将检查亚基连接蛋白之间相互作用的分子决定因素。我们假设特定的序列（可能是跨膜结构域）允许连接蛋白亚基之间的相互作用形成半通道。我们将使用免疫荧光、Ni2+NTA树脂亲和层析和速度沉降来检测不同嵌合突变体的寡聚化，以确定涉及亚基相互作用的序列。细菌表达系统（TOXCAT）将用于确定相互作用跨膜域的关键残基。2. 生理研究将检查在稳定转染细胞系中共表达的功能后果，在这些细胞系中，我们可以用Cx43控制Cx40或Cx45的相对表达。我们假设这些连接蛋白的不同相对量的共表达将影响电导率、渗透性/选择性和通过蛋白激酶激活的门控。我们将使用双全细胞膜片钳技术和荧光显微镜同时定量电导和染料通量。3. 细胞和生理学研究将检查Cx43羧基尾部近端区域的截短和点突变，以检查其在间隙连接的组装和功能中的作用。

DESCRIPTION (provided by applicant): Cardiac electrical conduction depends on intercellular passage of ions through gap junction channels. Altered gap junction distribution and subunit protein (connexin, Cx) expression have been implicated in the pathogenesis of reentrant arrhythmias. Connexins are also crucial for normal development of the heart and for the function of non-excitable tissues, implying a role in the passage of larger solutes (like signaling molecules). Individual connexins form channels with different properties (including conductance, permeability, and gating). The major connexin in working myocardium is Cx43, but Cx37, Cx40, and Cx45 are also found in various cardiovascular cells. Multiple connexins frequently co-localize to identical gap junction plaques, and they can form hetero-oligomeric channels. Co-expression and formation of heteromeric channels between Cx43 and other connexins may be a major mechanism for modulating and regulating cardiovascular intercellular communication. 1. Biochemical studies will examine the molecular determinants of interactions between subunit connexin proteins. We hypothesize that specific sequences (likely the transmembrane domains) allow interactions between connexin subunits to form hemi-channels. We will use immunofluorescence, affinity chromatography with Ni2+NTA resins, and velocity sedimentation to examine oligomerization of different chimeric mutants to determine sequences involved in subunit interactions. A bacterial expression system (TOXCAT) will be used to define critical residues in the interacting transmembrane domains. 2. Physiological studies will examine the functional consequences of co-expression in stably transfected cell lines in which we can control the relative expression of Cx40 or Cx45 with Cx43. We hypothesize that co-expression of different relative amounts of these connexins will affect electrical conductance, permeability/selectivity, and gating by protein kinase activation. We will use the double whole-cell patch-clamp technique and fluorescence microscopy to quantitate conductance and dye flux simultaneously. 3. Cellular and physiologic studies will examine truncations and point mutants of the proximal region of the carboxyl tail of Cx43 to examine its role in the assembly and function of gap junctions.