Bai Donglin, del Corsso Cristiane, Srinivas Miduturu, Spray David C
Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada.
J Pharmacol Exp Ther. 2006 Dec;319(3):1452-8. doi: 10.1124/jpet.106.112045. Epub 2006 Sep 19.
2-Aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-triphosphate receptor modulator, inhibits capacitive current transients measured in normal rat kidney and human embryonic kidney 293 cells, an indication of blocking gap junction channels between these cells. Here, we used the dual whole-cell patch-clamp method to study the actions of 2-APB on gap junction channels formed by selected connexins expressed in a communication-deficient neuroblastoma cell line (N2A). 2-APB dose-dependently and reversibly blocked junctional currents of connexin (Cx) 50 gap junction channels. The concentration-inhibition curve of 2-APB on the junctional current indicated an IC(50) of 3.7 microM, lower than that of most gap junction inhibitors. At a concentration of 20 microM, 2-APB also significantly blocked junctional conductance in cell pairs coupled by Cx26, Cx30, Cx36, Cx40, and Cx45 but did not appreciably affect coupling in cell pairs expressing Cx32, Cx43, and Cx46. Although concentration inhibition curves of 2-APB on Cx36 channels were similar to Cx50 (Cx36; IC(50), 3.0 microM), IC(50) values were higher for Cx43 (51.6 microM), Cx45 (18.1 microM), and Cx46 (29.4 microM). The blocking action of 2-APB did not substantially alter transjunctional voltage-dependent gating of Cx50 gap junction channels, and recordings from poorly coupled pairs of Cx50-transfected N2A cells indicated that 2-APB reduced gap junction channel open probability without changing the main state single-channel conductance. The differential efficacy of block by 2-APB of gap junction channels formed by different connexins may provide a useful tool that could be exploited in gap junction research to selectively block certain gap junction channel subtypes.
2-氨基乙氧基二苯硼酸盐（2-APB）是一种肌醇1,4,5-三磷酸受体调节剂，可抑制在正常大鼠肾脏和人胚肾293细胞中测得的电容性电流瞬变，这表明其可阻断这些细胞之间的缝隙连接通道。在此，我们使用双全细胞膜片钳方法研究2-APB对由在缺乏通讯的神经母细胞瘤细胞系（N2A）中表达的选定连接蛋白形成的缝隙连接通道的作用。2-APB剂量依赖性且可逆地阻断连接蛋白（Cx）50缝隙连接通道的连接电流。2-APB对连接电流的浓度抑制曲线表明其半数抑制浓度（IC50）为3.7微摩尔，低于大多数缝隙连接抑制剂。在20微摩尔的浓度下，2-APB还显著阻断了由Cx26、Cx30、Cx36、Cx40和Cx45耦合的细胞对中的连接电导，但对表达Cx32、Cx43和Cx46的细胞对中的耦合没有明显影响。尽管2-APB对Cx36通道的浓度抑制曲线与Cx50相似（Cx36；IC50，3.0微摩尔），但Cx43（51.6微摩尔）、Cx45（18.1微摩尔）和Cx46（29.4微摩尔）的IC50值更高。2-APB的阻断作用并未实质性改变Cx50缝隙连接通道的跨连接电压依赖性门控，并且对转染Cx50的N2A细胞的弱耦合对的记录表明，2-APB降低了缝隙连接通道的开放概率，而没有改变主要状态下单通道电导。2-APB对由不同连接蛋白形成的缝隙连接通道的不同阻断效力可能提供一种有用的工具，可用于缝隙连接研究中选择性地阻断某些缝隙连接通道亚型。
