Differential potency of dominant negative connexin43 mutants in oculodentodigital dysplasia
Differential potency of dominant negative connexin43 mutants in oculodentodigital dysplasia
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DOI：
10.1074/jbc.m609653200
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发表时间：
2007-06-29
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.
眼齿指发育不良（ODDD）是一种表型变异的先天性常染色体显性遗传病，与编码连接蛋白43（Cx43）的GJA 1基因突变有关。考虑到Cx43突变体被认为与ODDD患者中的野生型Cx43同等共表达，因此必须在反映该摩尔比的模型系统中检查这些突变体的结果。为此，我们使用突变体和野生型Cx43的差异荧光蛋白标记来定量监测相同推定的间隙连接斑块内的突变体/野生型的比率，并通过免疫共沉淀来确定突变体是否与野生型Cx43相互作用。将基于荧光的测定与膜片钳分析结合在一起以评估Cx43突变体的显性负性效力。我们的研究结果表明，ODDD连接的Cx43突变体，G21 R和G138 R，以及氨基末端绿色荧光蛋白标记的Cx43，能够与野生型Cx43共定位在差距连接斑块样结构，并与野生型Cx43共免疫沉淀。所有Cx43突变体表现出显性负作用的野生型Cx43，但不是Cx 32的间隙连接电导。更有趣的是，这些Cx43突变体在抑制野生型Cx43的功能方面表现出不同的效力，其中G21 R突变体的效力是G138 R突变体的两倍。ODDD连锁的Cx43突变体的显性阴性特性的效力差异可能对ODDD患者中观察到的各种症状和疾病严重程度具有临床意义。
Oculodentodigital dysplasia (ODDD) is a congenital autosomal dominant disorder with phenotypic variability, which has been associated with mutations in the GJA1 gene encoding connexin43 (Cx43). Given that Cx43 mutants are thought to be equally co-expressed with wild-type Cx43 in ODDD patients, it is imperative to examine the consequence of these mutants in model systems that reflect this molar ratio. To that end, we used differential fluorescent protein tagging of mutant and wild-type Cx43 to quantitatively monitor the ratio of mutant/wild-type within the same putative gap junction plaques and co-immunoprecipitation to determine if the mutants interact with wildtype Cx43. Together the fluorescence-based assay was combined with patch clamp analysis to assess the dominant negative potency of Cx43 mutants. Our results revealed that the ODDD-linked Cx43 mutants, G21R and G138R, as well as amino terminus green fluorescent protein-tagged Cx43, were able to co-localize with wild-type Cx43 at the gap junction plaque-like structures and to co-immunoprecipitate with wild-type Cx43. All Cx43 mutants demonstrated dominant negative action on gap junctional conductance of wild-type Cx43 but not that of Cx32. More interestingly, these Cx43 mutants demonstrated different potencies in inhibiting the function of wild-type Cx43 with the G21R mutant being two times more potent than the G138R mutant. The potency difference in the dominant negative properties of ODDD-linked Cx43 mutants may have clinical implications for the various symptoms and disease severity observed in ODDD patients.