Prolyl isomerization controls activation kinetics of a cyclic nucleotide-gated ion channel

Abstract

AbstractSthK, a cyclic nucleotide-modulated ion channel fromSpirochaeta thermophila, activates slowly upon cAMP increase. This is reminiscent of the slow, cAMP-induced activation reported for the hyperpolarization-activated and cyclic nucleotide-gated channel HCN2 in the family of so-called pacemaker channels. Here, we investigate slow cAMP-induced activation in purified SthK channels using stopped-flow assays, mutagenesis, enzymatic catalysis and inhibition assays revealing that thecis/transconformation of a conserved proline in the cyclic nucleotide-binding domain determines the activation kinetics of SthK. We propose that SthK exists in two forms:transPro300 SthK with high ligand binding affinity and fast activation, andcisPro300 SthK with low affinity and slow activation. Following channel activation, thecis/transequilibrium, catalyzed by prolyl isomerases, is shifted towardstrans, while steady-state channel activity is unaffected. Our results reveal prolyl isomerization as a regulatory mechanism for SthK, and potentially eukaryotic HCN channels. This mechanism could contribute to electrical rhythmicity in cells.