CONNEXONS IN CARDIOVASCULAR CELL COMMUNICATION

描述（改编自申请人摘要）：心电图 传导依赖于离子通过间隙连接的细胞间通道 渠道缝隙连接分布和亚基蛋白的改变 表达的研究表明， 心律不齐间隙连接通道是由两个 六聚体半通道（连接子）由亚基蛋白质组成，称为 连接蛋白（Cx）。单个连接蛋白形成具有不同 生物物理特性许多心血管细胞含有多种 连接蛋白经常共同定位以识别间隙连接。主要 目前建议的目标是确定混合的参数， 心血管连接蛋白和混合的功能后果。 研究人员假设， 连接蛋白的混合导致混合六聚体的形成 （异聚连接子）。调查人员提出了一个由三部分组成的计划 其将特别关注主要心脏连接蛋白的混合 Cx43与Cx 37或Cx40之间的关系，因为Cx 37和Cx40在Cx43中大量表达， 不同的表达模式（主要是Cx43的子集） 分布），但这些蛋白质单独使通道， 与Cx43有很大不同。具体目标如下： 1）以生物化学的方式检查， 心血管连接蛋白形成混合连接。连接蛋白对（Cx 37 + Cx43，Cx40 + Cx43）将通过稳定转染 永生化细胞系。交联和免疫共沉淀将是 用于确定这些细胞内连接蛋白的混合程度， 2）在生理上定义，如何 细胞间通讯通过异聚体的形成而改变 渠道在co-cells中连接通道的生理特性 将转染的细胞与仅含有单个 连接蛋白利用双通道方法研究了单位电导和通道门控。 全细胞和双重透化贴片方法。信道 渗透性/选择性将通过细胞间转移来评估， 显微注射荧光示踪剂; 3）确定功能性 连接蛋白混合对心脏传导的影响。Cx40或Cx 37将 在心肌细胞中过表达（通过产生转基因小鼠或 腺病毒感染）。通道门控和动作电位延长 将在细胞对中确定。阵列中的传导 培养的新生肌细胞将通过光学标测进行检查。 心电图和心外膜标测将用于检查 心室传导。

DESCRIPTION (adapted from the applicant's abstract): Cardiac electrical conduction depends on intercellular passage of ions through gap-junction channels. Alterations in gap-junction distribution and subunit protein expression have been implicated in the pathogenesis of re-entrant arrhythmias. Gap-junction channels are formed by the joining of two hexameric hemichannels (connexons) composed of subunit proteins called connexins (Cx). Individual connexins form channels with different biophysical properties. Many cardiovascular cells contain multiple connexins which frequently co-localize to identify gap-junction. The major goals of the current proposal is to define the parameters of mixing of the cardiovascular connexins and the functional consequence of that mixing. The investigators hypothesize that the most physiological consequential mixing of connexins results in the formation of mixed hexamers (heteromeric connexons). The investigators propose a three-part program which will focus particularly on the mixing of the major cardiac connexin (Cx43) with Cx37 or Cx40, since Cx37 and Cx40 are abundantly expressed in distinct expression patterns (which are largely subsets of the Cx43 distribution), but these proteins individually make channels with substantially different properties from Cx43. The specific aims are as follows 1) To examine, biochemically, the extent to which combinations of the cardiovascular connexins form mixed connexons. Connexin pairs (Cx37 plus Cx43, Cx40 plus Cx43) will be expressed by stable transfection of immortalized cell lines. Cross-linking and co-immunoprecipitation will be used to determine the extend of mixing of connexins within these cells and within individual channels; 2) To define physiologically, how intercellular communication is modified by the formation of heteromeric channels. Physiologic properties of the juncitonal channels in co- transfected cells will be compared to ones containing only a single connexin. Unitary conductance and channel gating will be studied by double whole-cell and dual permeabilized patch methods. Channel permeability/selectivity will be assessed by intercellular transfer of micro-injected fluorescent tracers; 3) To determine the functional consequences of connexin mixing on cardiac conduction. Cx40 or Cx37 will be over-expressed in cardiac myocytes (by production of transgenic mice or adenoviral infection). Channel gating and action-potential prolongation will be determined in cell pairs. Conduction in patterned arrays in cultured neonatal myocytes will be examined by optical mapping. Electrocardiograms and epicardial mapping will be used to examine ventricular conduction.