Efficient coupling of ligand binding to channel opening by the binding domain of a modulatory (β) subunit of the olfactory cyclic nucleotide-gated channel

CNG channels in vivo are heteromers of homologous alpha and beta subunits that each contain a six-transmembrane segment domain and a COOH-terminal cytoplasmic cyclic nucleotide binding domain (BD). In heterologous expression systems, heteromeric cep channels activate with greater sensitivity to ligand than do homomeric alpha channels; however, ligand-gating of channels containing only beta subunit BDs has never been studied because beta subunits cannot form functional homomeric CNG channels. To characterize directly the contribution of the beta subunit BD to ligand-gating, we constructed a chimeric subunit, X-beta, whose BD sequence was that of the beta subunit CNG5 from rat, but whose sequence outside the BD was derived from a subunits. For comparison, we constructed another chimera, X-alpha, whose sequence outside the BD was identical to that of X-beta, but whose BD sequence was that of the a subunit CNG2 from catfish. When expressed in Xenopus oocytes, X-beta and X-alpha each formed functional homomeric channels activated by both cAMP and cGMP This is the first demonstration that the beta subunit BD can couple ligand binding to activation in the absence of a subunit BD residues. Notably; both agonists activate X-beta more effectively than X-alpha (higher opening efficacy and lower K-1/2). The BD is believed to comprise two functionally distinct subdomains: (1) the roll subdomain (beta -roll and flanking A- and B-helices) and (2) the C-helix subdomain. Opening efficacy was previously believed to be controlled primarily by the C-helix, but when we made additional chimeras by exchanging the subdomains between X-beta and X-alpha, we found that both subdomains contain significant determinants of efficacy and agonist selectivity. In particular, only channels containing the roll subdomain of the beta subunit had high efficacy. Thermodynamic linkage analysis shows that interaction between the two subdomains accounts for a significant portion of their contribution to activation energetics.