CX43 AND CX37 GAP JUNCTION CHANNEL ACTIVITY

该建议的重点是阐明连接蛋白的门控机制 43和37在定义的离子和表达式约束下。其目标是： 1)确定未转染的细胞群的门控特征， 连接蛋白43衍生的间隙连接通道，使用双全细胞分批钳 （DWCP）。人血管平滑肌细胞对（HVSM）来源于 已知含有连接蛋白43的阴茎海绵体将被用作 以及含有大鼠连接蛋白43的NRK细胞对。间隙连接通道 将通过测量平均开放时间、平均 闭合时间、断开概率和电压灵敏度。我们还 确定是否存在协作选通（非独立选通 间隙连接通道之间。2a）在用以下转染的N2 A细胞中使用DWCP： rCx 43或humanCx 37，并确定异型Cx43/Cx 37缺口的行为 连接通道。Cx 37的同型行为在文献中，我们 在rCx 43转染细胞的初步结果中提供数据。我们 将继续研究同型形式并确定它们的门控 与未转染的rCx 43进行比较。同型 是指连接形成间隙连接通道的两个相同的半通道 (Cx43/Cx43）和异型是指两个不相同的半通道 连接在一起（Cx43/Cx 37），其中每个半通道仅由一个 连接蛋白型。 2b）将Cx43和Cx 37共转染到N2 A细胞中（一种潜在的模型， 内皮细胞间通讯）和监测多通道 行为N2 A细胞中形成的通道数量较少， 结合我们的分析将揭示任何门控差异之间 同型和异型间隙连接通道以及间隙连接 由异聚体组成（异聚体=两种不同形式的半通道，Cx43 和Cx 37发现于相同的半通道中）。我们的假设是 异聚体和异型形式彼此可区分， 同型形式的参数，如开放概率，平均值 开放时间、平均闭合时间、合作门控、电压依赖性和 幺正电导 3.）第三章在HVSM细胞和转染的N2 A细胞上使用直接膜片钳， 鉴定新鲜分离的细胞表面上的间隙连接通道。 4)阐明单缺口的门-门相互作用（偶然门控） 使用DWCP数据的交汇通道，其中应用了较大的deltaVj步长 以及对宏观电流和单一通道活动两者进行建模。 我们 也将使用渗透负荷（Zimmerberg和Parsegian，1986）， 区分间隙的不同电导状态之间的体积变化 连接通道。

This proposal is focused on elucidating the gating mechanisms of connexins 43 and 37 under defined ionic and expression constraints. The aims are: 1) Determine the gating characteristics of a non-transfected population of connexin43 derived gap junction channels using dual whole cell batch clamp (DWCP). Human vascular smooth muscle-cell pairs (HVSM) derived from corpora cavernosa, which are known to contain connexin43, will be used as well as NRK cell pairs which contain rat connexin43. Gap junction channel gating will be monitored by measurement of the mean open time(s), mean closed time(s), open probabilities and voltage sensitivity. Further, we ascertain whether there is cooperative gating (non-independent gating between gap junction channels. 2a) Use DWCP in N2A cells transfected with rCx43 or humanCx37 and determine the behavior of heterotypic Cx43/Cx37 gap junction channels. Homotypic behavior for Cx37 is in the literature and we present data in the preliminary results for rCx43 transfected cells. We will continue to study the homotypic forms and determine their gating characteristics for comparison with non-transfected rCx43. Homotypic refers to two identical hemichannels linked to form a gap junction channel (Cx43/Cx43) and a heterotypic refers to two non-identical hemichannels linked together (Cx43/Cx37) where each hemichannel is composed of only one connexin type. 2b) Co-transfect Cx43 and Cx37 into N2A cells (a potential model for endothelial cell intercellular communication) and monitor the multichannel behavior. The low number of channels formed in the N2A cells in combination with our analysis will reveal any gating differences between homotypic and heterotypic gap junction channels as well as gap junctions made of heteromers (heteromers=hemichannels of two different forms,Cx43 and Cx37 found in the same hemichannel). Our assumption is that heteromeric and heterotypic forms are distinguishable from each other and homotypic forms in terms of parameters such as open probabilities, mean open times, mean closed times, cooperative gating, voltage dependence and unitary conductance. 3.) Use direct patch clamp on HVSM cells and transfected N2A cells to identify gap junction channels on the surface of freshly isolated cells. 4) Elucidate gate-to-gate interactions (contingent gating) for single gap junction channels using DWCP data where large deltaVj steps are applied and modeling both macroscopic currents and unitary channel activity. We will also use osmotic loading (Zimmerberg and Parsegian, 1986) to distinguish volume changes between different conductance states of gap junction channels.