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 cl ones in Xenopus oocytes and studied in inside out patches. Using tande m dimers composed of linked subunits, we investigated the stoichiometr y and arrangement of the alpha and beta subunits. Dimers contained thr ee subunit types: alpha(wt), beta(wt), and alpha(m). The alpha(m) subu nit lacks an amino-terminal domain that greatly influences gating, dec reasing the apparent affinity of the channel for ligand by 9-fold, mak ing it a reporter for inclusion in the tetramer. Homomeric channels fr om injection of alpha(wt)alpha(wt) dimers and from alpha(wt) monomers were indistinguishable. Channels from injection of alpha(wt)alpha(m) d imers had apparent affinities 3-fold lower than alpha(wt) homomultimer s, suggesting a channel with two alpha(wt) and two alpha(m) subunits. Channels from coinjection of alpha(wt)alpha(wt) and beta beta dimers w ere indistinguishable from those composed of alpha and beta monomers a nd shared all of the characteristics of the alpha+beta phenotype of he teromeric channels. Coinjection of alpha(wt)alpha(m), and beta beta di mers yielded channels also of the alpha+beta phenotype but with an app arent affinity 3-fold lower, indicating the presence of alpha(m) in th e tetramer and that alpha+beta channels have adjacent alpha-subunits. To distinguish between an alpha-alpha-alpha-beta and an alpha-alpha-be ta-beta arrangement, we compared apparent affinities for channels from coinjection of alpha(wt)alpha(wt) and beta alpha(wt) or alpha(wt)alph a(wt) and beta alpha(m) dimers. These channels were indistinguishable. To further argue against an alpha-alpha-alpha-beta arrangement, we qu antitatively compared dose-response data for channels from coinjection of alpha(wt)alpha(m) and beta beta dimers to those from alpha and bet a monomers. Taken together, our results are mast consistent with an al pha-alpha-beta-beta arrangement for the heteromeric olfactory CNG chan nel.