Identification of a domain on the β-subunit of the rod cGMP-gated cation channel that mediates inhibition by calcium-calmodulin

被引:84
作者
Grunwald, ME
Yu, WP
Yu, HH
Yau, KW
机构
[1] Johns Hopkins Univ, Sch Med, Howard Hughes Med Inst, Baltimore, MD 21205 USA
[2] Johns Hopkins Univ, Sch Med, Dept Neurosci, Baltimore, MD 21205 USA
关键词
D O I
10.1074/jbc.273.15.9148
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 mu M 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.
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页码:9148 / 9157
页数:10
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