CX43 AND CX37 GAP JUNCTION CHANNEL ACTIVITY

这项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限制下。目标是： 1)确定未转染组的门控特性 使用双全细胞批式钳制的Cx43衍生缝隙连接通道 (DWCP)。人血管平滑肌-细胞对(HVSM)来源于 已知含有连接蛋白43的海绵体将被用作 以及含有大鼠连接蛋白43的NRK细胞对。缝隙结通道 门控将通过测量平均打开时间(S)、平均 闭合时间(S)、开路概率和电压灵敏度。此外，我们 确定是否存在协作门控(非独立门控 在缝隙结通道之间。2a)在N2A细胞中使用DWCP RCx43或Human Cx37，并确定异型Cx43/Cx37缺口的行为 连接通道。Cx37的同型行为在文献中有记载，我们 给出了rCx43转基因细胞的初步结果中的数据。我们 将继续研究同型形式并确定它们的门控 与未转染的rCx43比较的特性。同型 指连接成缝隙连接通道的两个相同的半通道 (Cx43/Cx43)，异型指的是两个不相同的半通道 链接在一起(Cx43/Cx37)，其中每个半通道仅由一个 连接蛋白类型。 2B)将Cx43和Cx37共转染入N2A细胞(一种潜在的 内皮细胞细胞间通讯)，并监测多通道 行为。在N2a单元中形成的低数量的通道 与我们的分析相结合，将会揭示出 同型和异型缝隙结通道以及缝隙结 由异构体(异构体=两种不同形式的半通道，Cx43)制成的 和Cx37在同一半通道中发现)。我们的假设是 异构体和异型体是可以相互区分的，并且 关于参数的同型形式，例如开放概率、平均值 打开时间、平均关闭时间、合作门控、电压依赖和 么正电导。 3.)用直接膜片钳技术对HVSM细胞和转染的N2A细胞进行膜片钳 识别新分离细胞表面的缝隙连接通道。 4)阐明单间隙的门到门的相互作用(或称门控) 使用DWCP数据的交汇点通道，其中应用了较大的增量Vj步长 以及对宏观电流和单一通道活动进行建模。我们 也将使用渗透负荷(Zimmerberg和Parsegian，1986)来 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.