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

Native cylic nucleotide-gated (CNG) channels are composed of a and β 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 α and β subunits. Dimers contained three subunit types: α(wt), β(wt), and α(m). The α(m) 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 α(wt)ω(wt) dimers and from α(wt) monomers were indistinguishable. Channels from injection of α(wt)a(m) dimers had apparent affinities 3-fold lower than α(wt) homomultimers, suggesting a channel with two α(wt) and two αm subunits. Channels from coinjection of α(wt)α(wt) and ββ dimers were indistinguishable from those composed of α and β monomers and shared all of the characteristics of the α+β phenotype of heteromeric channels. Coinjection of α(wt)α(m) and ββ dimers yielded channels also of the a+β phenotype but with an apparent affinity 3-fold lower, indicating the presence of α(m) in the tetramer and that α+β channels have adjacent α-subunits. To distinguish between an α- α-α-β and an α-α-bβ-a- arrangement, we compared apparent affinities for channels from coinjection of α(wt)α(wt) and βα(wt) or α(wt)α(wt) and βα(m) dimers. These channels were indistinguishable. To further argue against an α-α-α-β arrangement, we quantitatively compared dose-response data for channels from coinjection of α(wt)α(m) and ββ dimers to those from α and β monomers. Taken together, our results are most consistent with an α-α-β-β arrangement for the heteromeric olfactory CNG channel.