Grunwald M E, Yu W P, Yu H H, Yau K W
Department of Neuroscience and Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185, USA.
J Biol Chem. 1998 Apr 10;273(15):9148-57. doi: 10.1074/jbc.273.15.9148.
The cGMP-gated cation channel mediating phototransduction in retinal rods has recently been shown to be inhibited by calcium-calmodulin, through direct binding of the latter to the beta-subunit of the heterotetrameric channel complex. Here, we report the characterization of this inhibition and the identification of a domain crucial for this modulation. Heterologous expression of the alpha- and beta-subunits of the human rod channel in HEK 293 cells produced a cGMP-gated current that was highly sensitive to calcium-calmodulin, with half-maximal inhibition at approximately 4 nM. In biochemical and electrophysiological experiments on deletion mutants of the beta-subunit, we have identified a region on its cytoplasmic N terminus that binds calmodulin and is necessary for the calmodulin-mediated inhibition of the channel. However, in gel shift assays and fluorescence emission experiments, peptides derived from this region indicated a low calmodulin affinity, with dissociation constants of approximately 3-10 microM. On the C terminus, a region was also found to bind calmodulin, but it was likewise of low affinity, and its deletion did not abolish the calmodulin-mediated inhibition. We suggest that although the identified region on the N terminus of the beta-subunit is crucial for the calmodulin effect, other regions are likely to be involved as well. In this respect, the rod channel appears to differ from the olfactory cyclic nucleotide-gated channel, which is also modulated by calcium-calmodulin.
最近研究表明，介导视网膜视杆细胞光转导的环磷酸鸟苷（cGMP）门控阳离子通道可被钙调蛋白抑制，其机制是钙调蛋白直接与异源四聚体通道复合物的β亚基结合。在此，我们报告了这种抑制作用的特征以及对该调节至关重要的结构域的鉴定。在HEK 293细胞中异源表达人视杆通道的α亚基和β亚基，产生了对钙调蛋白高度敏感的cGMP门控电流，在约4 nM时抑制作用达到半数最大效应。在对β亚基缺失突变体进行的生化和电生理实验中，我们在其胞质N端鉴定出一个与钙调蛋白结合的区域，该区域是钙调蛋白介导的通道抑制所必需的。然而，在凝胶迁移实验和荧光发射实验中，源自该区域的肽显示出较低的钙调蛋白亲和力，解离常数约为3 - 10 μM。在C端，也发现一个区域可结合钙调蛋白，但亲和力同样较低，其缺失并未消除钙调蛋白介导的抑制作用。我们认为，尽管在β亚基N端鉴定出的区域对钙调蛋白效应至关重要，但可能还有其他区域也参与其中。在这方面，视杆通道似乎与嗅觉环核苷酸门控通道不同，后者也受钙调蛋白调节。
