CONNEXONS IN CARDIOVASCULAR CELL COMMUNICATION

描述(改编自申请人的摘要)：心脏电信号 传导依赖于离子通过缝隙连接的细胞间通道。 频道。缝隙连接分布和亚单位蛋白的变化 基因表达与折返性心脏病的发病机制有关 心律不齐。缝隙结通道是由两个通道连接而成 六聚体半连蛋白(连接蛋白)由亚基蛋白质组成，称为 连接蛋白(Cx)。单个连接蛋白以不同的方式形成通道 生物物理特性。许多心血管细胞含有多个 连接蛋白经常共定位以识别缝隙连接。少校 当前提案的目标是定义混合的参数 心血管连接蛋白及其混合的功能后果。 研究人员假设，最具生理学影响的 连接蛋白的混合导致混合六聚体的形成 (异构体连接外显子)。调查人员提出了一个由三部分组成的计划 它将特别关注主要的心脏连接蛋白的混合 (Cx43)与Cx37或Cx40结合，因为Cx37和Cx40在 不同的表达模式(主要是Cx43的子集 分布)，但这些蛋白质分别与 与Cx43的特性有很大的不同。具体目标如下 以下是1)从生物化学的角度来考察 心血管连接蛋白形成混合连接蛋白。连接蛋白对(Cx37 +Cx43、Cx40+Cx43)将通过稳定的转染获得表达 永生的细胞系。交联剂和免疫共沉淀法将 用来确定这些细胞内连接蛋白混合的程度和 在单独的渠道内；2)从生理上定义如何 异构体的形成改变了细胞间的通讯 频道。慢性阻塞性肺疾病交界区通道的生理特性 将转基因细胞与那些只含有单个 缝隙连接蛋白。一元电导和通道门控将通过 全细胞贴片法和双透膜贴片法。渠道 渗透性/选择性将通过细胞间转移来评估 微量注射荧光示踪剂；3)测定功能 缝隙连接蛋白混合对心脏传导的影响。Cx40或Cx37将 在心肌细胞中过度表达(通过生产转基因小鼠或 腺病毒感染)。通道门控与动作电位延长 将以细胞对的形式确定。In中图案化阵列的导电性 培养的新生儿心肌细胞将通过光学标测进行检查。 将使用心电图和心外膜标测检查 脑室传导。

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.