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HCN ion channels belong to the voltage-gated K-channel superfamily, but their voltage-sensing (S4) helices produce an unusual hyperpolarization-activation mechanism. This hyperpolarization-activation can be enhanced by (a) cyclic AMP (cAMP) binding to a cytoplasmic C-terminal region, and (b) a voltage-independent “mode-shift” after gate-opening that forms a secondary open state (“Mode II”). We previously [Biophys. J. 94, 1400-Pos.] found that a mutation (K381E) within S4 produced a cAMP-dependent “ultra-sustained activation” phenotype without disrupting voltage-activation. Cyclic AMP applied to K381E channels in excised inside-out patches greatly slows deactivation kinetics, increasing decay time constants beyond 6 s at −40mV. In this study, we activated K381E channels with hyperpolarizing pulses too short for significant mode shift to occur, thus isolating open channels predominantly in the Mode I state. Deactivation transients were well-described with a sum of three exponential components, consistent with three subpopulations of open states. The three time constants (τfast, τmedium, and τslow) were widely separated in the absence of cAMP, enabling the rapidly deactivating Mode I kinetics (τfast ∼50 ms) to be clearly delineated from those of the more stable Mode II states (τmedium ∼300 ms and τslow ∼900 ms). Slowing of deactivation by cAMP was slight for Mode I (∼2-fold increase in τfast) in contrast with the ultra-sustained activation properties of Mode II (∼9-fold increase in τslow to ∼8 s). The voltage-dependence of Mode I versus Mode II deactivation was also markedly different (accelerating |
