A Second S4 Movement Opens Hyperpolarization-Activated HCN Channels

Abstract

Rhythmic activity in pacemaker cells, such as in sino-atrial node cardiomyocytes, depends on the activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. As in depolarization-activated K+ channels, the fourth transmembrane segment S4 is positively charged and functions as the voltage sensor in hyperpolarization-activated HCN channels. But how the inward movement of S4 at negative voltages couples to channel opening in HCN channels is not understood. In the cryo-EM structure of the human HCN1 channel with S4 in the up/resting state and the gate closed, there is an interaction between a glutamate in S4 and an asparagine in S5. We measured the effects of mutations of the homologous residues (E356 and N370) in the sea urchin HCN channel on S4 and gate movements using Voltage Clamp Fluorometry. Our results show that a hydrogen bond between E356 and N370 stabilizes S4 in its up state. Breaking this hydrogen bond by inserting the E356A mutation shifts the main S4 movement to positive voltages, but channel opening remains at negative voltages. Instead, E356A reveals a second S4 movement at negative voltages that correlates with gate opening. Our data suggest a model in which a hydrogen bond between S4 and S5 stabilizes S4 in the up/resting state. Upon hyperpolarization, this bond is broken by the initial inward S4 movement, whereas a subsequent S4 movement triggers channel opening. Our molecular models suggest that the second S4 movement opens a hydrophilic intracellular crevice between S4 and S5 that would allow radial movement of the intracellular ends of S5 and S6 to open the pore of HCN channels.