title: Mutating three residues in the bovine rod cyclic nucleotide-activated channel can switch a nucleotide from inactive to active.
abstract: Cyclic nucleotide-gated (CNG) channels, which were initially studied in retina and olfactory neurons, are activated by cytoplasmic cGMP or cAMP. Detailed comparisons of nucleotide-activated currents using nucleotide analogs and mutagenesis revealed channel-specific residues in the nucleotide-binding domain that regulate the binding and channel-activation properties. Of particular interest are N(1)-oxide cAMP, which does not activate bovine rod channels, and Rp-cGMPS, which activates bovine rod, but not catfish, olfactory channels. Previously, we showed that four residues coordinate the purine interactions in the binding domain and that three of these residues vary in the alpha subunits of the bovine rod, catfish, and rat olfactory channels. Here we show that both N(1)-oxide cAMP and Rp-cGMPS activate rat olfactory channels. A mutant of the bovine rod alpha subunit, substituted with residues from the rat olfactory channel at the three variable positions, was weakly activated by N(1)-oxide cAMP, and a catfish olfactory-like bovine rod mutant lost activation by Rp-cGMPS. These experiments underscore the functional importance of purine contacts with three residues in the cyclic nucleotide-binding domain. Molecular models of nucleotide analogs in the binding domains, constructed with AMMP, showed differences in the purine contacts among the channels that might account for activation differences.
authors.affiliation: Department of Microbiology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
authors.forename: S P
authors.initials: SP
authors.lastname: Scott
authors.forename: J
authors.initials: J
authors.lastname: Cummings
authors.forename: J C
authors.initials: JC
authors.lastname: Joe
authors.forename: J C
authors.initials: JC
authors.lastname: Tanaka
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D055592:Biophysical Phenomena; D001703:Biophysics; D002397:Catfishes; D002417:Cattle; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism; D054815:Cyclic Nucleotide-Gated Cation Channels; D006801:Humans; D066298:In Vitro Techniques; D007473:Ion Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009712:Nucleotides, Cyclic / Q000378:metabolism*; D018034:Olfactory Receptor Neurons / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism*
chemical_list: D054815:Cyclic Nucleotide-Gated Cation Channels; D007473:Ion Channels; D008024:Ligands; D009712:Nucleotides, Cyclic; D011994:Recombinant Proteins; D006152:Cyclic GMP