Mechanisms of cyclic amp-dependent modulation in pacemaker channels

Pacemaker (HCN) channels are hyperpolarization-activated and their activity is enhanced by cyclic AMP (cAMP), but whether cAMP-gating is coupled to voltage-gating remains unresolved. With long hyperpolarizations, HCN channels form a secondary open state after the initial opening; this briefly sustains the activation of open channels after a return to a resting voltage. CAMP effects on the formation and decay of the secondary open state remain unclear. We studied mouse HCN4 channels in excised oocyte membrane patches, and found cAMP increases sustained activation. We also studied a charge-reversing S4 mutation (K381E) and found it causes surprisingly little change in voltage-dependence. Notably, K381E dramatically increases the cAMP-dependence of sustained activation. Our results suggest cAMP and K381E synergistically enhance the formation of a secondary open state and that this state decays during deactivation through a voltage- and cAMP-dependent step. These results suggest a physical coupling of voltage-sensing and cAMP-modulatory machinery in HCN channels.