Three residues predicted by molecular modeling to interact with the purine moiety alter ligand binding and channel gating in cyclic nucleotide-gated channels.

Abstract

Cytoplasmic cAMP and cGMP are soluble cellular messengers that directly activate cyclic nucleotide-gated (CNG) channels. These channels mediate sensory transduction in photoreceptors and olfactory neurons. The closely related CNG channels in these cell types have different nucleotide activation profiles, and we have investigated the molecular basis of their nucleotide selectivity properties. Previously, we predicted that the purine moiety of the nucleotide interacts with residues F533, K596, and D604 (bovine rod alpha CNG channel subunit sequences) of the nucleotide binding domain. In this study, we replaced these three residues with the corresponding residues of the bovine olfactory CNG channel. Mutations at each position altered the nucleotide activation of the rod CNG channels. In a mutant where K596 was replaced with arginine, cAMP-activated currents were enhanced 8-12-fold, suggesting that residue 596 influences channel gating. Thermodynamic cycle analysis of the data showed that (1) the residues are energetically coupled and (2) energetic coupling exists between the potentiating effects of Ni2+ and the replacement of F533 with tyrosine. These data suggest that changes in one of the residues alter the purine contacts with the other residues and that F533 communicates with the C-linker region of the channel involved in Ni2+ potentiation.