Allosteric Regulation of the Cyclic Nucleotide-Binding Domain in HCN Channels

Abstract

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels play an important role in regulating pacemaking activity in the heart and brain. They are regulated by the binding of cyclic nucleotides to a conserved, intracellular cyclic nucleotide-binding domain (CNBD). Binding of cyclic nucleotides increases the rate and extent of activation of the channels and shifts channel activation to less hyperpolarized voltages. In intact channels, cAMP and cGMP are full agonists and cCMP is a partial agonist. We use double electron-electron resonance (DEER) to study the conformational change associated with the binding of these three different cyclic nucleotide species to the CNBD. We find that conformational changes associated with ligand binding vary depending on the cyclic nucleotide bound and the location in the CNBD. We use the restrained ensemble MD (re-MD) simulations method to generate structural models integrating the complete set of experimentally measured data from DEER distance distribution histograms that describe the separation between pairs of spin labels attached to the CNBD. Our results indicate that, in the B-helix, binding of cGMP or cCMP has an effect intermediate to that of cAMP. However, in the C-helix cAMP and cCMP exhibit similar effects, but cGMP produces an intermediate effect between the apo and bound cAMP/cCMP DEER distance distributions. These data provide an interesting lens for studying allostery in HCN channels and indicate that the mechanism of protein allostery in the CNBD varies for different cyclic nucleotides.