Hoang Quan V, Qian Haohua, Ripps Harris
Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL 60612, USA.
Mol Vis. 2010 Jul 15;16:1343-52.
The gap junctions (GJs) mediating direct cell-cell interaction are formed by clusters of membrane-spanning proteins known as connexins (Cxs). These channels play a key role in signal transmission, and their permeability, time-, and voltage-dependence are governed by the properties of the specific Cxs forming the gap junctions. Retinal pigment epithelium (RPE) cells express Cx43 and Cx46. Here, we employed a heterologous expression system to explore the functional properties of the hemichannels and GJs that could be formed by different combinations of these Cxs. Specifically, we examined the response kinetics of GJs formed by pairing cells expressing Cx43 or Cx46, or those expressing both, i.e., designated as Cx43*Cx46.
The Xenopus oocyte expression system and a two-electrode voltage clamp technique were used to study the properties of hemichannels and GJs formed in oocytes transfected with Cx43 and/or Cx46 mRNA.
Depolarizing voltages activated hemicurrents of similar amplitude from single oocytes transfected with Cx46 or Cx43Cx46, but not in oocytes expressing Cx43 alone. Incorporating Cx43 with Cx46 altered the gating charge, but not the voltage sensitivity of the hemichannels. In addition, Cx43Cx46 hemichannel currents exhibited faster activation kinetics than homomeric Cx46 hemichannels. Both homotypic GJs formed by Cx43 and Cx46, and heteromeric Cx43Cx46 GJs exhibited large junctional conductances with amplitudes of 6.5+/-3.0 microS (Cx43), 8.9+/-3.4 microS (Cx46), and 8.5+/-1.8 microS (Cx4346); a significantly lower conductance (1.8+/-0.7 microS) was observed for heterotypic GJs formed by Cx43 and Cx46. There were also differences in their gating kinetics. Whereas the kinetics of homotypic Cx46 could be described by a single exponential function (tau=0.91 s), double exponential functions were required for homotypic Cx43 (tau(1)=0.24, tau(2)=3.4 s), heterotypic Cx43/Cx46 (tau(1)=0.29, tau(2)=3.6 s), and heteromeric Cx43Cx46/Cx43Cx46 (tau(1)=1.2, tau(2)=8.1 s) junctions.
The failure of oocytes expressing Cx43 to exhibit hemichannel activity is an intrinsic membrane property of this Cx, and cannot be attributed to a lack of expression; western blot analysis showed clearly that Cx43 was expressed in oocytes in which it was injected. Our results provide further evidence that Cx43 and Cx46 form both heterotypic and heteromeric channels when co-expressed, an indication that various combinations of Cxs may participate in gap-junctional communication between RPE cells.
介导细胞间直接相互作用的间隙连接（GJ）由称为连接蛋白（Cx）的跨膜蛋白簇形成。这些通道在信号传递中起关键作用，其通透性、时间和电压依赖性由形成间隙连接的特定Cx的特性决定。视网膜色素上皮（RPE）细胞表达Cx43和Cx46。在此，我们采用异源表达系统来探索由这些Cx的不同组合形成的半通道和GJ的功能特性。具体而言，我们研究了表达Cx43或Cx46的细胞配对形成的GJ，或同时表达两者（即指定为Cx43*Cx46）的细胞配对形成的GJ的反应动力学。
采用非洲爪蟾卵母细胞表达系统和双电极电压钳技术研究用Cx43和/或Cx46 mRNA转染的卵母细胞中形成的半通道和GJ的特性。
去极化电压激活了用Cx46或Cx43Cx46转染的单个卵母细胞中幅度相似的半电流，但未激活仅表达Cx43的卵母细胞中的半电流。将Cx43与Cx46结合会改变门控电荷，但不会改变半通道的电压敏感性。此外，Cx43Cx46半通道电流表现出比同源Cx46半通道更快的激活动力学。由Cx43和Cx46形成的同型GJ以及异源Cx43Cx46 GJ均表现出较大的连接电导，幅度分别为6.5±3.0微西门子（Cx43）、8.9±3.4微西门子（Cx46）和8.5±1.8微西门子（Cx4346）；由Cx43和Cx46形成的异型GJ的电导明显较低（1.8±0.7微西门子）。它们的门控动力学也存在差异。虽然同型Cx46的动力学可用单指数函数描述（时间常数τ=0.91秒），但同型Cx43（τ1=0.24，τ2=3.4秒）、异型Cx43/Cx46（τ1=0.29，τ2=3.6秒）和异源Cx43Cx46/Cx43Cx46（τ1=1.2，τ2=8.1秒）连接需要双指数函数描述。
表达Cx43的卵母细胞未能表现出半通道活性是该Cx的固有膜特性，不能归因于表达不足；蛋白质免疫印迹分析清楚地表明Cx43在注射了它的卵母细胞中表达。我们的结果进一步证明，Cx43和Cx46共表达时形成异型和异源通道，这表明Cx的各种组合可能参与RPE细胞之间的间隙连接通讯。
