Cyclic nucleotide-gated (CNG) ion channels mediate sensory signal transduction in photoreceptors and olfactory cells. Structurally, CNG channels are heterotetramers composed of either two or three homologue subunits. Although it is well established that activation is a cooperative process of these subunits, it remains unknown whether the cooperativity is generated by the ligand binding, the gating, or both, and how the subunits interact. In this study, the action of homotetrameric olfactory-type CNGA2 channels was studied in inside-out membrane patches by simultaneously determining channel activation and ligand binding, using the fluorescent cGMP analogue 8-DY547-cGMP as the ligand. At concentrations of 8-DY547-cGMP?