Connexin46 mutations linked to congenital cataract show loss of gap junction channel function
Connexin46 mutations linked to congenital cataract show loss of gap junction channel function
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DOI：
10.1152/ajpcell.2000.279.3.c596
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发表时间：
2000-09-01
影响因子：
5.5
通讯作者：
Ebihara, L
中科院分区：
文献类型：
--
作者：
Pal, JD;Liu, XQ;Ebihara, L
人类连接蛋白46(HCx46)形成连接晶状体纤维细胞的缝隙连接通道，似乎对正常晶状体功能至关重要，因为hCx46突变与先天性白内障有关。我们研究了两个hCx46突变体，N63S，第一个胞外区的错义突变，以及fs380，一种移码突变，在氨基酸残基380处移动翻译阅读框架。我们在非洲爪哇卵母细胞中表达了野生型Cx46及其两个突变体。表达产物经[S-35]蛋氨酸代谢标记或免疫印迹后，用十二烷基硫酸钠-PAGE进行鉴定。双双微电极电压钳研究表明，hCx46在非洲爪哇卵母细胞中既形成缝隙连接通道，又在单个卵母细胞中形成半缝隙连接通道。相反，两个与白内障相关的hCx46突变体都不能在成对的非洲爪哇卵母细胞中形成细胞间通道。HCx46突变体形成半缝隙连接通道的能力也受到了损害。当N63S或fs380与野生型连接蛋白共表达时，这两个突变都表现为“功能丧失”，而不是“显性负”突变，因为它们不影响野生型hCx46或野生型hCx50诱导的缝隙连接电导。
Human connexin46 (hCx46) forms gap junctional channels interconnecting lens fiber cells and appears to be critical for normal lens function, because hCx46 mutations have been linked to congenital cataracts. We studied two hCx46 mutants, N63S, a missense mutation in the first extracellular domain, and fs380, a frame-shift mutation that shifts the translational reading frame at amino acid residue 380. We expressed wild-type Cx46 and the two mutants in Xenopus oocytes. Production of the expressed proteins was verified by SDS-PAGE after metabolic labeling with [S-35]methionine or by immunoblotting. Dual two-microelectrode voltage-clamp studies showed that hCx46 formed both gap junctional channels in paired Xenopus oocytes and hemi-gap junctional channels in single oocytes. In contrast, neither of the two cataract-associated hCx46 mutants could form intercellular channels in paired Xenopus oocytes. The hCx46 mutants were also impaired in their ability to form hemi-gap-junctional channels. When N63S or fs380 was coexpressed with wild-type connexins, both mutations acted like "loss of function" rather than "dominant negative" mutations, because they did not affect the gap junctional conductance induced by either wild-type hCx46 or wild-type hCx50.