Gap junction channels formed by coexpressed connexin40 and connexin43
Gap junction channels formed by coexpressed connexin40 and connexin43
复制标题
DOI：
10.1152/ajpheart.2001.281.4.h1675
复制
发表时间：
2001-10-01
影响因子：
4.8
通讯作者：
Beyer, EC
中科院分区：
文献类型：
--
作者：
Valiunas, V;Gemel, J;Beyer, EC
许多心血管细胞共表达多种连接蛋白（Cx），导致潜在的混合（异聚）间隙连接半通道的形成，其生物物理特性可能不同于只含有一种连接蛋白类型的同源通道。我们研究了连续稳定转染这两种连接蛋白的HeLa细胞中连接蛋白Cx43和Cx40的潜在相互作用。免疫印迹证实了生产的可比量的两个连接蛋白，交联显示，两个连接蛋白形成的寡聚体，和免疫荧光显示广泛的共定位。此外，通过去污剂溶解的连接子的亲和层析，Cx40与（His）（6）-标记的Cx43共纯化，证明在一些半通道中存在两种连接蛋白。采用双全细胞膜片钳技术，比较了HeLa细胞Cx43/Cx40细胞缝隙连接的门控特性，其中Cx40-Cx40和Cx43-Cx43为同型缝隙连接，Cx40-Cx43为异型缝隙连接。许多观察到的单通道电导类似于同型或异型通道。共表达细胞对的稳态连接电导（g（j，ss））显示与同型间隙连接和/或异型间隙连接的不对称Vj依赖性相比，对细胞间电压（V-j）的敏感性降低。这些门控特性可以使用同型和异型通道特性的组合来拟合。因此，虽然我们的生化证据表明，Cx40和Cx43形成异聚体连接，我们的结论是，他们是功能无关紧要的电压依赖性门控。
Many cardiovascular cells coexpress multiple connexins (Cx), leading to the potential formation of mixed (heteromeric) gap junction hemichannels whose biophysical properties may differ from homomeric channels containing only one connexin type. We examined the potential interaction of connexin Cx43 and Cx40 in HeLa cells sequentially stably transfected with these two connexins. Immunoblots verified the production of comparable amounts of both connexins, cross-linking showed that both connexins formed oligomers, and immunofluorescence showed extensive colocalization. Moreover, Cx40 copurified with (His)(6)-tagged Cx43 by affinity chromatography of detergent-solubilized connexons, demonstrating the presence of both connexins in some hemichannels. The dual whole cell patch-clamp method was used to compare the gating properties of gap junctions in HeLa Cx43/Cx40 cells with homotypic (Cx40-Cx40 and Cx43-Cx43) and heterotypic (Cx40-Cx43) gap junctions. Many of the observed single channel conductances resembled those of homotypic or heterotypic channels. The steady-state junctional conductance (g(j,ss)) in coexpressing cell pairs showed a reduced sensitivity to the voltage between cells (V-j) compared with homotypic gap junctions and/or an asymmetrical Vj dependence reminiscent of heterotypic gap junctions. These gating properties could be fit using a combination of homotypic and heterotypic channel properties. Thus, whereas our biochemical evidence suggests that Cx40 and Cx43 form heteromeric connexons, we conclude that they are functionally insignificant with regard to voltage-dependent gating.