The permeability of gap junction channels to probes of different size is dependent on connexin composition and permeant-pore affinities.
The permeability of gap junction channels to probes of different size is dependent on connexin composition and permeant-pore affinities.
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DOI：
10.1529/biophysj.103.036350
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发表时间：
2004-08
影响因子：
3.4
通讯作者：
Paul A Weber;H. Chang;Kris E. Spaeth;J. Nitsche;B. Nicholson
中科院分区：
文献类型：
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作者：
Paul A Weber;H. Chang;Kris E. Spaeth;J. Nitsche;B. Nicholson
间隙连接传统上被表征为细胞之间的非特异性孔，其传递分子量高达1 kDa的分子。然而，越来越明显的是，连接蛋白（Cx）家族的不同成员介导非常不同的生理过程，并且通常是不可互换的。与这一观察结果一致，不同的连接蛋白对天然代谢物的渗透性差异已被报道，尽管选择性的物理基础尚未建立。对连接蛋白家族不同成员的比较研究为离子电荷选择性提供了证据，但令人惊讶的是，关于连接蛋白组合物如何影响孔的大小知之甚少。我们采用了一系列的Alexa染料，具有相似的结构特征，但在分子量350至760的大小范围内，探测不同的连接蛋白通道在非洲爪蟾卵母细胞中表达的渗透性和大小限制。相关的染料转移和电气测量每个细胞对，结合在卵母细胞系统中的染料扩散的三维数学模型，使我们能够获得所有三种染料在六个同型和四个异型通道的单通道渗透性。Cx43和Cx 32通道以相似的效率通过所有三种染料，而Cx 26、Cx40和Cx45通道显示出最大染料的渗透性显著下降。Cx 37通道仅对较小的两种染料显示出显著的渗透性，但比测试的其他连接蛋白低2 - 6倍。在异型的情况下研究（Cx 26/Cx 32和Cx43/Cx 37），渗透性特征被发现类似于更严格的父母同型通道。该研究的最令人惊讶的发现是，除了一个例外，在该研究中计算的所有间隙连接通道的绝对渗透率比纯粹基于受阻孔扩散预测的至少大2个数量级。因此，探针和孔之间的亲和力产生了能量上有利的孔内环境，这将提高孔内的渗透物浓度并因此提高通量，这是强烈暗示的。
Gap junctions have traditionally been characterized as nonspecific pores between cells passing molecules up to 1 kDa in molecular mass. Nonetheless, it has become increasingly evident that different members of the connexin (Cx) family mediate quite distinct physiological processes and are often not interchangeable. Consistent with this observation, differences in permeability to natural metabolites have been reported for different connexins, although the physical basis for selectivity has not been established. Comparative studies of different members of the connexin family have provided evidence for ionic charge selectivity, but surprisingly little is known about how connexin composition affects the size of the pore. We have employed a series of Alexa dyes, which share similar structural characteristics but range in size from molecular weight 350 to 760, to probe the permeabilities and size limits of different connexin channels expressed in Xenopus oocytes. Correlated dye transfer and electrical measurements on each cell pair, in conjunction with a three-dimensional mathematical model of dye diffusion in the oocyte system, allowed us to obtain single channel permeabilities for all three dyes in six homotypic and four heterotypic channels. Cx43 and Cx32 channels passed all three dyes with similar efficiency, whereas Cx26, Cx40, and Cx45 channels showed a significant drop-off in permeability with the largest dye. Cx37 channels only showed significant permeability for the smaller two dyes, but at two- to sixfold lower levels than other connexins tested. In the heterotypic cases studied (Cx26/Cx32 and Cx43/Cx37), permeability characteristics were found to resemble the more restrictive parental homotypic channel. The most surprising finding of the study was that the absolute permeabilities calculated for all gap junctional channels in this study are, with one exception, at least 2 orders of magnitude greater than predicted purely on the basis of hindered pore diffusion. Consequently, affinity between the probes and the pore creating an energetically favorable in-pore environment, which would elevate permeant concentration within the pore and hence the flux, is strongly implicated.