Cao F, Eckert R, Elfgang C, Nitsche J M, Snyder S A, H-ulser D F, Willecke K, Nicholson B J
Department of Biological Sciences, State University of New York at Buffalo, Buffalo, NY 14260, USA.
J Cell Sci. 1998 Jan;111 ( Pt 1):31-43. doi: 10.1242/jcs.111.1.31.
Gap junctions provide direct intercellular communication by linking adjacent cells with aqueous pores permeable to molecules up to 1 kDa in molecular mass and 8-14 A in diameter. The identification of over a dozen connexins in the mammalian gap junction family has stimulated interest in the functional significance of this diversity, including the possibility of selectivity for permeants as seen in other channel classes. Here we present a quantitative comparison of channel permeabilities of different connexins expressed in both HeLa transfectants (rat Cx26, rat Cx32 and mouse Cx45) and Xenopus oocytes (rat Cx26 and rat Cx32). In HeLa cells, we examined permeability to two fluorescent molecules: Lucifer Yellow (LY: anionic, MW 457) and 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI, cationic, MW 350). A comparison of the kinetics of fluorescent dye transfer showed Cx32, Cx26 and Cx45 to have progressively decreasing permeabilities to LY, but increasing permeabilities to DAPI. This pattern was inconsistent with selection based on physical size of the probe, nor could it be accounted for by the differences between clones in the electrical conductance of the monolayers. In Xenopus oocytes, where electrical and dye coupling could be assessed in the same cells, Cx32 coupled oocytes showed an estimated 6-fold greater permeability to LY than those coupled by Cx26, a comparable result to that seen in HeLa cells, where an approximately 9-fold difference was seen. The oocyte system also allowed an examination of Cx32/Cx26 heterotypic gap junction that proved to have a permeability intermediate between the two homotypic forms. Thus, independent of the expression system, it appears that connexins show differential permeabilities that cannot be predicted based on size considerations, but must depend on other features of the probe, such as charge.
间隙连接通过将相邻细胞与水通道相连，提供直接的细胞间通讯。这些水通道对分子量高达1 kDa、直径为8 - 14 Å的分子具有通透性。在哺乳动物间隙连接家族中已鉴定出十几种连接蛋白，这激发了人们对这种多样性功能意义的兴趣，包括像在其他通道类型中所见的对通透分子的选择性。在此，我们对在HeLa转染细胞（大鼠Cx26、大鼠Cx32和小鼠Cx45）和非洲爪蟾卵母细胞（大鼠Cx26和大鼠Cx32）中表达的不同连接蛋白的通道通透性进行了定量比较。在HeLa细胞中，我们检测了对两种荧光分子的通透性：路西法黄（LY：阴离子型，分子量457）和4',6-二脒基-2-苯基吲哚二盐酸盐（DAPI，阳离子型，分子量350）。荧光染料转移动力学的比较表明，Cx32、Cx26和Cx45对LY的通透性逐渐降低，但对DAPI的通透性逐渐增加。这种模式与基于探针物理大小的选择不一致，也不能用单层膜电导的克隆差异来解释。在非洲爪蟾卵母细胞中，可以在同一细胞中评估电偶联和染料偶联，表达Cx32的偶联卵母细胞对LY的通透性估计比表达Cx26的偶联卵母细胞高6倍，这与在HeLa细胞中观察到的结果相当，在HeLa细胞中两者相差约9倍。卵母细胞系统还允许对Cx32/Cx26异型间隙连接进行检测，结果证明其通透性介于两种同型形式之间。因此，无论表达系统如何，连接蛋白似乎都表现出不同的通透性，这不能基于大小考虑来预测，而必须取决于探针的其他特征，如电荷。
