SUBUNIT INTERACTIONS IN THE ACTIVATION OF CYCLIC NUCLEOTIDE-GATED IONCHANNELS

Cyclic nucleotide-gated (CNG) ion channels of retinal photoreceptors a nd olfactory neurons are multimeric proteins of unknown stoichiometry. To investigate the subunit interactions that occur during CNG channel activation, we have used tandem cDNA constructs of the rod CNG channe l to generate heteromultimeric channels composed of wild-type and muta nt subunits. We introduced point mutations that affect channel activat ion: 1) D604M, which alters the relative ability of agonists to promot e the allosteric conformational change(s) associated with channel open ing, and 2) T560A, which primarily affects the initial binding affinit y for cGMP, and to a lesser extent, the allosteric transition. At satu rating concentrations of agonist, heteromultimeric channels were inter mediate between wild-type and mutant homomultimers in agonist efficacy and apparent affinity for cGMP, cIMP, and cAMP, consistent with a mod el for the allosteric transition involving a concerted conformational change in all of the channel subunits. Results were also consistent wi th a model involving independent transitions in two or three, but not one or four, of the channel subunits. The behavior of the heterodimers implies that the channel stoichiometry is some multiple of 2 and is c onsistent with a tetrameric quaternary structure for the functional ch annel complex. Steady-state dose-response relations for homomultimeric and heteromultimeric channels were well fit by a Monod, Wyman, and Ch angeux model with a concerted allosteric opening transition stabilized by binding of agonist.