Functional characterization of a GJA1 frameshift mutation causing oculodentodigital dysplasia and palmoplantar keratoderma
Functional characterization of a GJA1 frameshift mutation causing oculodentodigital dysplasia and palmoplantar keratoderma
复制标题
DOI：
10.1074/jbc.m605961200
复制
发表时间：
2006-10-20
影响因子：
4.8
通讯作者：
Laird, Dale W.
中科院分区：
文献类型：
--
作者：
Gong, Xiang-Qun;Shao, Qing;Laird, Dale W.
由编码Cx43的GJA1基因的二核苷酸缺失(780-781del)产生的移码突变导致移码，在259个残基后产生46个异常氨基酸，与野生型Cx43的382个残基相比，蛋白质缩短了305个残基。该移码突变(FS260)导致眼齿-指发育不良(ODDD)，包括增加的掌足角皮病。当在各种细胞系中表达时，FS260突变体通常定位于内质网和其他细胞内隔室。FS260突变体，而不是G138R奇数连锁Cx43突变体或残基259(T259)截短的Cx43突变体，减少了内源性Cx43在正常大鼠肾脏细胞或角质形成细胞中形成的明显缝隙连接斑块的数量。有趣的是，编码在46个异常氨基酸结构域的假定的FF内质网保留基序的突变未能恢复FS260突变体到缝隙连接的有效组装。双全细胞膜片钳记录显示，与野生型Cx43和突变体T259相比，表达FS260的N2A细胞表现出严重的电耦合减少，而单膜片电容记录显示FS260也能明显抑制野生型Cx43的功能。共表达研究进一步表明，FS260对野生型Cx43的显性负效应是剂量依赖的，在预测的1：1表达比率下，FS260突变体使野生型Cx43介导的缝隙连接电导降低了60%以上。这些结果表明，与移码突变相关的46个氨基酸残基的异常至少部分地导致了掌跖角化病症状的出现。
A frameshift mutation generated from a dinucleotide deletion (780-781del) in the GJA1 gene encoding Cx43 results in a frameshift yielding 46 aberrant amino acids after residue 259 and a shortened protein of 305 residues compared with the 382 in wild-type Cx43. This frameshift mutant (fs260) causes oculodentodigital dysplasia (ODDD) that includes the added condition of palmoplantar keratoderma. When expressed in a variety of cell lines, the fs260 mutant was typically localized to the endoplasmic reticulum and other intracellular compartments. The fs260 mutant, but not the G138R ODDD-linked Cx43 mutant or a Cx43 mutant truncated at residue 259 (T259), reduced the number of apparent gap junction plaques formed from endogenous Cx43 in normal rat kidney cells or keratinocytes. Interestingly, mutation of a putative FF endoplasmic reticulum retention motif encoded within the 46 aberrant amino acid domain failed to restore efficient assembly of the fs260 mutant into gap junctions. Dual whole cell patch-clamp recording revealed that fs260-expressing N2A cells exerted severely reduced electrical coupling in comparison to wild-type Cx43 or the T259 mutant, whereas single patch capacitance recordings showed that fs260 could also dominantly inhibit the function of wild-type Cx43. Co-expression studies further revealed that the dominant negative effect of fs260 on wild-type Cx43 was dose-dependent, and at a predicted 1:1 expression ratio the fs260 mutant reduced wild-type Cx43-mediated gap junctional conductance by over 60%. These results suggest that the 46 aberrant amino acid residues associated with the frameshift mutant are, at least in part, responsible for the manifestation of palmoplantar keratoderma symptoms.