Kyle John W, Minogue Peter J, Thomas Bettina C, Domowicz Denise A Lopez, Berthoud Viviana M, Hanck Dorothy A, Beyer Eric C
Department of Medicine, Section of Cardiology, University of Chicago, Chicago, IL 60637, USA.
J Cell Sci. 2008 Aug 15;121(Pt 16):2744-50. doi: 10.1242/jcs.032482. Epub 2008 Jul 29.
The cytoplasmic N-termini of connexins have been implicated in protein trafficking, oligomerization and channel gating. To elucidate the role of the N-terminus in connexin37 (CX37), we studied mutant constructs containing partial deletions of its 23 N-terminal amino acids and a construct with a complete N-terminus in which residues 2-8 were replaced with alanines. All mutants containing nine or more N-terminal amino acids form gap junction plaques in transiently transfected HeLa cells, whereas most of the longer deletions do not. Although wild-type CX37 allowed intercellular transfer of microinjected neurobiotin in HeLa cells and formed conducting hemichannels in Xenopus oocytes, none of the mutant constructs tested show evidence of channel function. However, in coexpression experiments, N-terminal mutants that formed gap junction plaques potently inhibit hemichannel conductance of wild-type CX37 suggesting their co-oligomerization. We conclude that as much as half the length of the connexin N-terminus can be deleted without affecting formation of gap junction plaques, but an intact N-terminus is required for hemichannel gating and intercellular communication.
连接蛋白的胞质N端与蛋白质运输、寡聚化及通道门控有关。为阐明N端在连接蛋白37（CX37）中的作用，我们研究了含有其23个N端氨基酸部分缺失的突变体构建体以及一个完整N端中2至8位残基被丙氨酸取代的构建体。所有含有九个或更多N端氨基酸的突变体在瞬时转染的HeLa细胞中形成间隙连接斑，而大多数更长缺失的突变体则不能。虽然野生型CX37在HeLa细胞中允许微注射的神经生物素进行细胞间转移，并在非洲爪蟾卵母细胞中形成有传导功能的半通道，但所测试的突变体构建体均未显示出通道功能的证据。然而，在共表达实验中，形成间隙连接斑的N端突变体强烈抑制野生型CX37的半通道电导，提示它们可共同寡聚化。我们得出结论，连接蛋白N端长度的一半被删除仍不影响间隙连接斑的形成，但半通道门控和细胞间通讯需要完整的N端。
