Site-directed mutations in the transmembrane domain M3 of human connexin37 alter channel conductance and gating
Site-directed mutations in the transmembrane domain M3 of human connexin37 alter channel conductance and gating
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DOI：
10.1006/bbrc.2000.4121
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发表时间：
2001-01-19
影响因子：
3.1
通讯作者：
Brink, PR
中科院分区：
文献类型：
--
作者：
Kumari, SS;Varadaraj, K;Brink, PR
连接蛋白37（Cx 37）主要在内皮细胞中表达。我们已经在多态性人Cx 37（Cx 37-S319）的推定孔衬里段M3中引入了个体点突变（Cx 37-V156 D或Cx 37-K162 E），并在N2 A和RIN细胞中表达它们。用RT-PCR和免疫荧光显微镜证实蛋白的表达。对稳定转染的细胞进行电生理学研究。使用双全细胞膜片钳方法在细胞对上进行实验。单通道记录显示，两种突变体均显示出多种导电状态（Cx 37-V156 D，47-250 pS; Cx 37-K162 E，58-342 pS），而Cx 37-S319报道的典型高电导为340-375 pS和亚导电状态为60-80 pS。Cx 37-K162 E的宏观数据的分析揭示了变宽的Vo，表明突变对电压门控的影响。我们的数据表明，取代保守的残基与带电残基可能会导致的主要状态和/或孔的大小的变化。M3结构域中的这些特定残基可能与Cx 37蛋白中的几个其他结构域静电相互作用。(C)北京：科学出版社.
Connexin37 (Cx37) is expressed principally in endothelial cells. We have introduced individual point mutations (Cx37-V156D or Cx37-K162E) in the putative pore lining segment M3 of a polymorphic human Cx37 (Cx37-S319) and expressed them in N2A and RIN cells. RT-PCR and immunofluorescence microscopy were used to confirm the expression of the proteins. Stably transfected cells were subjected to electrophysiological studies. Experiments were performed on cell pairs using the dual whole cell patch-clamp method. Single channel records showed that both mutants display a variety of conductive states (Cx37-V156D, 47-250 pS; Cx37-K162E, 58-342 pS) in contrast to the typical high conductance of 340-375 pS and subconductive state of 60-80 pS reported for Cx37-S319. Analysis of the macroscopic data for Cx37-K162E revealed a broadened Vo indicating the influence of the mutation on voltage gating. Our data indicate that substitution of a conserved residue with a charged residue could cause changes in the main state and/or in the size of the pore. It is possible that these particular residues in the M3 domain interact electrostatistically with several of the other domains in the Cx37 protein. (C) 2001 Academic Press.