Identification of a domain on the beta-subunit of the rod cGMP-gated cation channel that mediates inhibition by calcium-calmodulin.
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.
Grunwald
M E
ME
Department of Neuroscience and Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185, USA.
Yu
W P
WP
Yu
H H
HH
Yau
K W
KW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology*; D002417:Cattle; D003001:Cloning, Molecular; D006152:Cyclic GMP / Q000378:metabolism* / Q000494:pharmacology; D054815:Cyclic Nucleotide-Gated Cation Channels; D004110:Diltiazem / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D007473:Ion Channels / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D002147:Calmodulin; D054815:Cyclic Nucleotide-Gated Cation Channels; D007473:Ion Channels; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D004533:Egtazic Acid; D004110:Diltiazem; D006152:Cyclic GMP; D002118:Calcium