Dept. of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, NY 11794-8661, USA.
Am J Physiol Cell Physiol. 2011 Mar;300(3):C600-9. doi: 10.1152/ajpcell.00316.2010. Epub 2010 Dec 9.
Gap junction channels formed by different connexins exhibit specific permeability to a variety of larger solutes including second messengers, polypeptides, and small interfering RNAs. Here, we report the permeability of homotypic connexin26 (Cx26), Cx40, Cx43, and Cx45 gap junction channels stably expressed in HeLa cells to solutes with different size and net charge. Channel permeability was determined using simultaneous measurements of junctional conductance and the cell-cell flux of a fluorescent probe. All four connexins allowed passage of both cationic and anionic probes, but the transfer rates were connexin dependent. The negatively charged probes [Lucifer yellow (LY; median axial diameter 9.9 Å, charge -2), carboxyfluorescein (CF; 8.2 Å; -2), and Alexa Fluor350 (AF350, 5.4 Å; -1)] exhibited the following permeability order: Cx43 > Cx45 > Cx26 > Cx40. In contrast, for the positively charged species permeability, the orders were as follows: Cx26 ≈ Cx43 ≈ Cx40 ≈ Cx45 for N,N,N-trimethyl-2-[methyl-(7-nitro-2,1,3-benzoxadiol-4-yl) amino] ethanaminium (NBD-m-TMA; 5.5 Å, +1) and Cx26 ≥ Cx43 ≈ Cx40 > Cx45 for ethidium bromide (10.3 Å, +1). Comparison of probe permeability relative to K(+) revealed that Cx43 and Cx45 exhibited similar permeability for NBD-m-TMA and AF350, indicating weak charge selectivity. However, lesser transfer of CF and LY through Cx45 relative to Cx43 channels suggests stronger size-dependent discrimination of solute. The permeability of NBD-m-TMA for Cx40 and Cx26 channels was approximately three times higher than to anionic AF350 despite the fact that both have similar minor diameters, suggesting charge selectivity. In conclusion, these results confirm that channels formed from individual connexins can discriminate for solutes based on size and charge, suggesting that channel selectivity may be a key factor in cell signaling.
不同连接蛋白形成的缝隙连接通道对包括第二信使、多肽和小干扰 RNA 在内的多种较大溶质具有特定的通透性。在这里，我们报告了稳定表达于 HeLa 细胞中的同种连接蛋白 26（Cx26）、Cx40、Cx43 和 Cx45 缝隙连接通道对不同大小和净电荷的溶质的通透性。通过同时测量连接电导和荧光探针的细胞-细胞通量来确定通道通透性。所有四种连接蛋白都允许阳离子和阴离子探针通过，但传输速率与连接蛋白有关。带负电荷的探针[荧光素黄（LY；中轴直径 9.9 Å，电荷-2）、羧基荧光素（CF；8.2 Å；-2）和 Alexa Fluor350（AF350，5.4 Å；-1）]的通透性顺序如下：Cx43>Cx45>Cx26>Cx40。相比之下，对于带正电荷的物质，通透性顺序如下：Cx26≈Cx43≈Cx40≈Cx45（对于 N,N,N-三甲基-2-[甲基-（7-硝基-2,1,3-苯并恶二唑-4-基）氨基]乙胺（NBD-m-TMA；5.5 Å，+1））和 Cx26≥Cx43≈Cx40>Cx45（对于溴化乙锭（10.3 Å，+1））。与 K+相比，探针通透性的比较表明 Cx43 和 Cx45 对 NBD-m-TMA 和 AF350 具有相似的通透性，表明其电荷选择性较弱。然而，与 Cx43 通道相比，CF 和 LY 通过 Cx45 通道的传递较少，表明溶质对大小的依赖性更强。尽管 NBD-m-TMA 的直径与阴离子 AF350 相似，但 Cx40 和 Cx26 通道对其的通透性约为阴离子 AF350 的三倍，这表明存在电荷选择性。总之，这些结果证实，由单个连接蛋白形成的通道可以根据大小和电荷对溶质进行区分，这表明通道选择性可能是细胞信号传递的关键因素。
