Agonist-Induced Conformational Changes in the Ligand Binding Domain of Cyclic Nucleotide-Regulated Channels

Abstract

Cyclic nucleotide-gated (CNG) channels and hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels are opened by the direct binding of cyclic nucleotides (e.g. adenosine 3’,5’-cyclic monophosphate, cAMP). The ligand binding domain is primarily formed by an eight-stranded β-roll followed by two helices (B and C helices). Following agonist binding to residues in the β-roll, the C helix is thought to initiate the opening conformational change by moving towards the binding pocket and transitioning from a less ordered coil to a stable helix. To further investigate this hypothesis, we expressed the soluble C-terminal domain of the HCN2 channel, which contains the cyclic nucleotide binding domain, and introduced cysteines into the C helix, which were labeled with the fluorophore bimane. Into the same helix, we placed pairs of histidines in helical register. Colored transition metal ions can bind to these histidines and quench the fluorophore by a FRET mechanism. The amount of quenching was used to estimate the distance between the fluorophore and the metal binding site in the presence and absence of cAMP. For all of the constructs tested, cAMP binding induced a large increase in metal affinity at the di-histidine binding sites, indicating a stabilization of the helical structure. We also observed a change in the total amount of quenching in some constructs indicating a change in distance between the fluorophore and metal binding site. These data suggest a conformational change within the C helix induced by ligand binding. When a similar di-histidine binding site was introduced into the C helix of intact CNG channels, binding of Ni2+ increased the efficacy of the partial agonist inosine 3’,5’-cyclic monophosphate (cIMP), consistent with the hypothesis that stabilization of the secondary structure of the C helix is part of the gating conformational change.