Molecular mechanism of cAMP modulation of HCN pacemaker channels

Hyperpolarization-activated cation channels of the HCN gene family1,2,3,4,5,6 contribute to spontaneous rhythmic activity in both heart7 and brain5,6,8. All four family members contain both a core transmembrane segment domain, homologous to the S1–S6 regions of voltage-gated K+ channels, and a carboxy-terminal 120 amino-acid cyclic nucleotide-binding domain (CNBD) motif. Homologous CNBDs are responsible for the direct activation of cyclic nucleotide-gated channels and for modulation of the HERG voltage-gated K+ channel—important for visual and olfactory signalling9 and for cardiac repolarization10, respectively. The direct binding of cyclic AMP to the cytoplasmic site on HCN channels permits the channels to open more rapidly and completely after repolarization of the action potential1,2,11, thereby accelerating rhythmogenesis6,7,8. However, the mechanism by which cAMP binding modulates HCN channel gating and the basis for functional differences between HCN isoforms remain unknown. Here we demonstrate by constructing truncation mutants that the CNBD inhibits activation of the core transmembrane domain. cAMP binding relieves this inhibition. Differences in activation gating and extent of cAMP modulation between the HCN1 and HCN2 isoforms result largely from differences in the efficacy of CNBD inhibition.