Pacemaker hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels exhibit a reversed voltage-dependent gating, activating by membrane hyperpolarization instead of depolarization. Sea urchin HCN (spHCN) channels also undergo an inactivation with hyperpolarization which occurs only in the absence of cyclic nucleotide. Here we applied transition metal ion FRET and Rosetta modeling to measure differences in the structural rearrangements between activation and inactivation of spHCN

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... nnels. We found that removal of cAMP produced a largely rigid-body rotation of the C-linker relative to transmembrane domain, bringing the A′ helix in close proximity to the voltage-sensing S4 helix. In addition, rotation of the C-linker was elicited by hyperpolarization in the absence but not in the presence of cAMP. These results suggest the A′ helix functions like a mechanical clutch to engage inactivation. cAMP binding disengages the clutch, permitting the hyperpolarization-dependent activation mediated by the S5 helix. Furthermore, rearrangement of A′ helix during recovery from inactivation of spHCN channels was similar to that for the activation of KCNH channels, suggesting a conserved mechanism for this noncanonical electromechanical coupling in the CNBD channel family.