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 r ods has recently been shown to be inhibited by calcium-calmodulin, thr ough direct binding of the latter to the beta-subunit of the heterotet rameric channel complex, Here, we report the characterization of this inhibition and the identification of a domain crucial for this modulat ion, Heterologous expression of the alpha- and beta-subunits of the hu man rod channel in HEK 293 cells produced a cGMP-gated current that wa s highly sensitive to calcium-calmodulin, with half-maximal inhibition at approximately 4 nM, In biochemical and electrophysiological experi ments on deletion mutants of the beta-subunit, we have identified a re gion on its cytoplasmic N terminus that binds calmodulin and is necess ary for the calmodulin-mediated inhibition of the channel, However, in gel shift assays and fluorescence emission experiments, peptides deri ved from this region indicated a low calmodulin affinity, with dissoci ation 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 affinit y, 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 a re likely to be involved as well, In this respect, the rod channel app ears to differ from the olfactory cyclic nucleotide-gated channel, whi ch is also modulated by calcium-calmodulin.