Chen T Y, Illing M, Molday L L, Hsu Y T, Yau K W, Molday R S
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11757-61. doi: 10.1073/pnas.91.24.11757.
The cGMP-gated cation channel mediating visual transduction in retinal rods was recently found to comprise at least two subunits, 1 and 2 (or alpha and beta). SDS gels of the purified channel show, in addition to a 63-kDa protein band (subunit 1), a 240-kDa protein band that binds Ca(2+)-calmodulin, a modulator of the channel. To examine any connection between subunit 2 and the 240-kDa protein, cGMP-gated channels formed from the expressed cloned subunits in human embryonic kidney (HEK) 293 cells were tested for Ca(2+)-calmodulin effect. Homooligomeric channels formed by subunit 1 alone showed no sensitivity to Ca(2+)-calmodulin, and neither did heterooligomeric channels formed by subunit 1 and the short alternatively spliced form of subunit 2 (2a). By contrast, the cGMP half-activation constant (K1/2) for heterooligomeric channels formed from subunit 1 and the long form of subunit 2 (2b) was increased 1.5- to 2-fold by Ca(2+)-calmodulin, similar to the increase observed for the native channel. In Western blots of rod outer segment membranes, a subunit 2-specific antibody also recognized the 240-kDa protein. Finally, amino acid sequences derived from peptide fragments of the bovine 240-kDa protein showed approximately 80% identity to regions of subunit 2b of the human channel. These results together suggest that subunit 2b of the rod channel is a component of the 240-kDa protein and that it mediates the Ca(2+)-calmodulin modulation of the channel.
最近发现，介导视网膜视杆细胞视觉转导的环磷酸鸟苷（cGMP）门控阳离子通道至少由两个亚基组成，即亚基1和亚基2（或α和β）。纯化通道的十二烷基硫酸钠（SDS）凝胶显示，除了一条63 kDa的蛋白条带（亚基1）外，还有一条240 kDa的蛋白条带，该条带可结合通道的调节剂钙调蛋白（Ca²⁺-钙调蛋白）。为了研究亚基2与240 kDa蛋白之间的联系，对在人胚肾（HEK）293细胞中由表达的克隆亚基形成的cGMP门控通道进行了钙调蛋白效应测试。仅由亚基1形成的同聚体通道对钙调蛋白不敏感，由亚基1和亚基2的短可变剪接形式（2a）形成的异聚体通道也是如此。相比之下，由亚基1和亚基2的长形式（2b）形成的异聚体通道的cGMP半激活常数（K1/2）因钙调蛋白而增加了1.5至2倍，这与天然通道观察到的增加情况相似。在视杆细胞外段膜的蛋白质免疫印迹中，一种亚基2特异性抗体也识别出240 kDa的蛋白。最后，源自牛240 kDa蛋白肽片段的氨基酸序列与人通道亚基2b区域的序列一致性约为80%。这些结果共同表明，视杆细胞通道的亚基2b是240 kDa蛋白的一个组成部分，并且它介导了通道的钙调蛋白调节作用。
