Stoichiometry and arrangement of heteromeric olfactory cyclic nucleotide-gated ion channels

Native cylic nucleotide-gated (CNG) channels are composed of alpha and beta subunits. Olfactory CNG channels were expressed from rat cDNA clones in Xenopus oocytes and studied in inside-out patches. Using tandem dimers composed of linked subunits, we investigated the stoichiometry and arrangement of the alpha and beta subunits. Dimers contained three subunit types: alphawt, betawt, and alpham. The alpham subunit lacks an amino-terminal domain that greatly influences gating, decreasing the apparent affinity of the channel for ligand by 9-fold, making it a reporter for inclusion in the tetramer. Homomeric channels from injection of alphawtalphawt dimers and from alphawt monomers were indistinguishable. Channels from injection of alphawtalpham dimers had apparent affinities 3-fold lower than alphawt homomultimers, suggesting a channel with two alphawt and two alpham subunits. Channels from coinjection of alphawtalphawt and betabeta dimers were indistinguishable from those composed of alpha and beta monomers and shared all of the characteristics of the alpha+beta phenotype of heteromeric channels. Coinjection of alphawtalpham and beta beta dimers yielded channels also of the alpha+beta phenotype but with an apparent affinity 3-fold lower, indicating the presence of alpham in the tetramer and that alpha+beta channels have adjacent alpha-subunits. To distinguish between an alpha-alpha-alpha-beta and an alpha-alpha-beta-beta arrangement, we compared apparent affinities for channels from coinjection of alphawtalphawt and betaalphawt or alphawtalphawt and betaalpham dimers. These channels were indistinguishable. To further argue against an alpha-alpha-alpha-beta arrangement, we quantitatively compared dose-response data for channels from coinjection of alphawtalpham and beta beta dimers to those from alpha and beta monomers. Taken together, our results are most consistent with an alpha-alpha-beta-beta arrangement for the heteromeric olfactory CNG channel.