Biophys J, May 2000, p. 2321-2333, Vol. 78, No. 5

Mutating Three Residues in the Bovine Rod Cyclic Nucleotide-Activated Channel Can Switch a Nucleotide from Inactive to Active

Sean-Patrick Scott,^* Jim Cummings, Jason C. Joe, and Jacqueline C. Tanaka

 ^*Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, and  Department of Biology, School of Science and Technology, Temple University, Philadelphia, Pennsylvania 19122 USA

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 N¹-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  subunits of the bovine rod, catfish, and rat olfactory channels. Here we show that both N¹-oxide cAMP and Rp-cGMPS activate rat olfactory channels. A mutant of the bovine rod  subunit, substituted with residues from the rat olfactory channel at the three variable positions, was weakly activated by N¹-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.

Biophys J, May 2000, p. 2321-2333, Vol. 78, No. 5
© 2000 by the Biophysical Society   0006-3495/00/05/2321/13  $2.00

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