In vertebrate olfactory receptors, cAMP produced by odorants opens cyclic nucleotide-gated (CNG) channels, which allow Ca2+ entry and depolarization of the cell. These CNG channels are composed of α subunits and at least two types of β subunits that are required for increased cAMP selectivity. We studied the molecular basis for the altered cAMP selectivity produced by one of the β subunits (CNG5, CNCα4, OCNC2) using cloned rat olfactory CNG channels expressed in Xenopus oocytes. Compared with α subunit homomultimers (α channels), channels composed of α and β subunits (α+β channels) were half-activated (K(1/2)) by eightfold less cAMP and fivefold less cIMP, but similar concentrations of cGMP. The K(1/2) values for heteromultimers of the α subunit and a chimeric β subunit with the α subunit cyclic nucleotide-binding region (CNBR) (α+β-CNBRα channels) were restored to near the values for α channels. Furthermore, a single residue in the CNBR could account for the altered ligand selectivity. Mutation of the methionine residue at position 475 in the β subunit to a glutamic acid as in the α subunit (β-M475E) reverted the K(1/2,cAMP)/K(1/2.cGMP) and K(1/2,cIMP)/K(1/2.cGMP) ratios of α+β-M475E channels to be very similar to those of α channels. In addition, comparison of α+β-CNBRα channels with α+β-M475E channels suggests that the CNBR of the β subunit contains amino acid differences at positions other than 475 that produce an increase in the apparent affinity for each ligand. Like the wild-type β subunit, the chimeric β/α subunits conferred a shallow slope to the dose-response curves, increased voltage dependence, and caused desensitization. In addition, as for α+β channels, block of α+βCNBRα channels by internal Mg2+ was not steeply voltage-dependent (zδ ~ 1e-) as compared to block of α channels (zδ 2.7e-). Thus, the ligand-independent effects localize outside of the CNBR. We propose a molecular model to explain how the β subunit alters ligand selectivity of the heteromeric channels.
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