Functional characterization of a naturally occurring Cx50 truncation
Functional characterization of a naturally occurring Cx50 truncation
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DOI：
10.1167/iovs.05-1582
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发表时间：
2006-10-01
影响因子：
4.4
通讯作者：
White, Thomas W.
中科院分区：
文献类型：
--
作者：
DeRosa, Adam M.;Mui, Rickie;White, Thomas W.
目的。晶状体连接蛋白在纤维成熟过程中C端发生蛋白水解裂解。虽然这在功能上的意义尚不清楚，但解理与通道门控特性的变化有关。本研究通过表征c端截断的Cx50蛋白的特性来评估这种内源性截断的功能后果。小鼠和人的Cx50分别在290和294个氨基酸处被截断，然后在配对的爪蟾卵母细胞或哺乳动物细胞中表达。免疫细胞化学检测蛋白表达。双全细胞电压钳技术用于分析宏观和单通道电导、电压门控特性和动力学；pH门控灵敏度采用100% co2饱和介质进行复配。与全长Cx50相比，Cx50tr290通道的平均宏观电导降低了86%至89%。异型通道形成功能性的间隙连接，表现出中等水平的耦合，并表现出不变的电压门控特性。c端截断不改变单通道门控特性或统一电导。有趣的是，截断的和全长的Cx50通道电导被细胞质酸化可逆地阻断。Cx50的c端截断不抑制同型或异型通道的形成。然而，对于截短的通道，观察到电导率显著下降，这一现象与电压门控灵敏度、动力学或化学门控的变化无关。这些结果为晶状体纤维成熟过程中观察到的连接耦合减少50%提供了合理的解释。
PURPOSE. Lens connexins undergo proteolytic cleavage of their C termini during fiber maturation. Although the functional significance of this is unknown, cleavage has been correlated with changes in channel-gating properties. This study evaluates the functional consequences of this endogenous truncation by characterizing the properties of a C-terminal truncated Cx50 protein.METHODS. Murine and human Cx50 were truncated at amino acids 290 and 294, respectively, before expression in paired Xenopus oocytes or mammalian cells. Protein expression was evaluated by immunocytochemistry. Dual whole-cell voltage clamp techniques were used to analyze macroscopic and single-channel conductance, voltage-gating properties, and kinetics; pH gating sensitivity was measured by superfusion with 100% CO2-saturated media.RESULTS. Cx50tr290 channels exhibited an 86% to 89% reduction in mean macroscopic conductance compared with full-length Cx50. Heterotypic channels formed functional gap junctions, displayed an intermediate level of coupling, and exhibited unaltered voltage-gating properties. C-terminal truncation did not alter single-channel gating characteristics or unitary conductance. Interestingly, truncated and full-length Cx50 channel conductances were reversibly blocked by cytoplasmic acidification.CONCLUSIONS. C-terminal truncation of Cx50 did not inhibit the formation of homotypic or heterotypic channels. However, a significant decrease in conductance was observed for truncated channels, a phenomenon independent of alterations in voltage-gating sensitivity, kinetics, or chemical gating. These results provide a plausible explanation for the 50% decrease in junctional coupling observed during lens fiber maturation.