Comparison of cyclic AMP binding activity within HCN channel subfamily using atomistic molecular dynamics simulations of isolated cyclic nucleotide binding domains.

Abstract

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are a subfamily of homotetrameric, voltage gated potassium/sodium channels whose activity is modulated by cyclic AMP (cAMP). The binding of cAMP to an intracellular cyclic nucleotide binding domain (CNBD) promotes an increase in channel activity through a depolarizing shift in the voltage threshold necessary for channel activation, as well as kinetically favoring the active channel conformation, leading to improved conductance. While the precise mechanisms of this change are still being investigated, the shift in the CNDB region which is induced by the binding of cAMP is able to be propagated to the remainder of the channel through an adjacent C-linker, causing an alteration in the channel that affects conductance. Although various static structures of the four HCN isoforms with and without bound cyclic nucleotide have been solved and deposited on the Protein Data Bank, there is still a need to understand the precise binding interactions which lead to different sensitivities and selectivities for cyclic nucleotides within the subfamily. Using atomistic molecular dynamics, we have simulated the isolated CNBD of HCN1-4 bound to cAMP and have used both equilibrium simulations and alchemical free energy perturbation to characterize and compare the binding activities of the different isoforms. These simulations highlight the differential cyclic nucleotide binding behavior between HCN channels.