Mutations in the gene for connexin 26 (GJB2) that cause hearing loss have a dominant negative effect on connexin 30
Mutations in the gene for connexin 26 (GJB2) that cause hearing loss have a dominant negative effect on connexin 30
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DOI：
10.1093/hmg/ddg076
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发表时间：
2003-04-15
影响因子：
3.5
通讯作者：
Forge, A
中科院分区：
文献类型：
--
作者：
Marziano, NK;Casalotti, SO;Forge, A
编码连接蛋白26 （cx26）的基因（GJB2）突变与感觉神经性听力损失有关，无论是单独的还是作为综合征的一部分。在这里，我们比较了四种cx26突变体的特性，这些突变体来源于主要遗传性听力损失相关的点突变，包括非综合征性（W44S, R75W）或各种皮肤疾病（G59A, D66H, R75W）。由于cx26和cx30在内耳内共定位，因此确定了显性cx26突变对这两种野生型蛋白的影响。缺乏通讯的HeLa细胞。通过微量注射瞬时转染各种cDNA构建物。使用间隙连接渗透示踪剂Cascade Blue的染料转移研究表明，所有四种突变蛋白的细胞间偶联都受到破坏。转染细胞的免疫染色显示，对于G59A和D66H突变体，这与细胞内运输和质膜靶向受损相关，因为这两种蛋白都有核周定位。cx26和cx30的寡聚化修复了受损的转运，表明cx26和cx30可以形成异质连接子。与单独表达野生型蛋白相比，cx26或cx30与W44S或R75W共表达的细胞之间的染料转移率显著降低。G59A和D66H突变体分别只在cx30和cx26上起显性作用。我们认为cx26和cx30在体内内耳内形成异质连接子，具有听力所必需的特殊特性。某些突变对这些异质通道的破坏可能是耳聋的非综合征性质的基础。
Mutations in the gene (GJB2) encoding connexin 26 (cx26) have been linked to sensorineural hearing loss either alone or as part of a syndrome. Here we compare the properties of four cx26 mutants derived from point mutations associated with dominantly inherited hearing loss, either non-syndromic (W44S, R75W) or with various skin disorders (G59A, D66H, R75W). Since cx26 and cx30 are co-localized within the inner ear the effect of the dominant cx26 mutations on both of these wild-type proteins was determined. Communication-deficient HeLa cells. were transiently transfected with the various cDNA constructs by microinjection. Dye transfer studies using the gap junction permeant tracer Cascade Blue demonstrated a disruption to the intercellular coupling for all four of the mutant proteins. Immunostaining of the transfected cells revealed that for the G59A and D66H mutants this correlated with impaired intracellular trafficking and targeting to the plasma membrane, as both proteins had a perinuclear localization. The impaired trafficking was rescued by oligomerization both with cx26 and with cx30, suggesting that cx26 and cx30 can form heteromeric connexons. Significantly reduced dye transfer rates were observed between cells co-expressing either cx26 or cx30 together with W44S or R75W compared with the wild-type proteins alone. The dominant actions of the G59A and D66H mutants were only on cx30 and cx26, respectively. We suggest that cx26 and cx30 form heteromeric connexons in vivo, within the inner ear, with particular properties essential for hearing. Disruption of these heteromeric channels by certain mutations may underlie the non-syndromic nature of the deafness.