From the Departments of Biochemistry and Structural Biology and.
the Second Xiangya Hospital, Central South University, Changsha 410011, China.
J Biol Chem. 2018 Feb 16;293(7):2573-2585. doi: 10.1074/jbc.RA117.001348. Epub 2018 Jan 3.
Connexin channels help maintain eye lens homeostasis and transparency. The G143R missense substitution in connexin (Cx) 46 is associated with congenital Coppock cataracts; however, the underlying molecular mechanism is largely unknown. Here, we report that compared with WT Cx46, the G143R substitution abolishes hemichannel conductance in oocytes and in HeLa cells. Moreover, this substitution is dominant-negative and inhibits conductance of WT Cx46. CD analysis indicated that the substitution greatly reduces the α-helical structure of the intracellular Cx46 loop domain. Protein pulldown assays and isothermal titration calorimetry revealed that this Cx46 domain directly interacts with calmodulin (CaM) in a Ca-dependent fashion, an observation confirmed by immunofluorescent co-localization of Cx46 with CaM. Interestingly, the G143R substitution enhanced the Cx46-CaM interaction and attenuated its abolishment by Ca depletion. Moreover, Cx46 increased dye influx, and the G143R substitution augmented this effect. Inhibition of Ca-mediated CaM activation blocked hemichannel permeability. The membrane potential plays a crucial role in Cx46 membrane permeability. We found that the activity of hemichannels is detectable under rest and hyperpolarization conditions but is eliminated with depolarization. These results suggested that the G143R substitution impairs voltage-dependent electrical conductance and alters membrane permeability mediated by Cx46 hemichannels. The latter likely is caused by the substitution-induced structural changes of the intracellular loop domain associated with the increased interaction with CaM and reduced Ca sensitivity. The data suggest that the G143R-induced enhancement of the CaM-Cx46 interaction results in altered hemichannel activities and might be related to cataract formation.
缝隙连接通道有助于维持眼睛晶状体的内稳态和透明度。连接蛋白（Cx）46 的 G143R 错义取代与先天性 Coppock 白内障有关；然而，其潜在的分子机制在很大程度上尚不清楚。在这里，我们报告与 WT Cx46 相比，G143R 取代在卵母细胞和 HeLa 细胞中消除了半通道电导。此外，这种取代是显性负性的，并抑制 WT Cx46 的电导。CD 分析表明，该取代大大降低了细胞内 Cx46 环域的α-螺旋结构。蛋白下拉测定和等温滴定量热法显示，该 Cx46 结构域以 Ca 依赖性方式直接与钙调蛋白（CaM）相互作用，这一观察结果通过 Cx46 与 CaM 的免疫荧光共定位得到证实。有趣的是，G143R 取代增强了 Cx46-CaM 相互作用，并减弱了 Ca 耗竭对其的消除作用。此外，Cx46 增加了染料内流，而 G143R 取代增强了这种作用。抑制 Ca 介导的 CaM 激活阻断了半通道通透性。膜电位在 Cx46 膜通透性中起着至关重要的作用。我们发现，在静息和超极化条件下可以检测到半通道的活性，但在去极化时则消除了。这些结果表明，G143R 取代损害了电压依赖性电导率，并改变了由 Cx46 半通道介导的膜通透性。后者可能是由与 CaM 相互作用增加和 Ca 敏感性降低相关的细胞内环结构变化引起的。数据表明，G143R 诱导的 CaM-Cx46 相互作用增强导致半通道活性改变，可能与白内障形成有关。
