Structural Determinants of the Hyperpolarization-Dependent Gating of HCN Channels

Abstract

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels contain conserved voltage-sensing domains (VSD), but how hyperpolarization-induced movements of the charged S4 helix in the VSD lead to pore opening remains unclear. With a non-domain-swapped architecture, all cyclic nucleotide-binding domain (CNBD) family channels, including HCN channels, have a VSD closely packed to the S5 helix of the same subunit and in close proximity to the S6 helix and the following C-linker region of the adjacent subunit. Despite these similarities, HCN channels are activated with membrane hyperpolarization while KCNH channels are activated with depolarization. Furthermore, sea urchin HCN (spHCN) channels inactivate with hyperpolarization in the absence of cyclic nucleotides. To examine the structural determinants of the hyperpolarization-dependent gating in spHCN channels, we measured distances of the S4 helix and the S5 helix — at their intracellular sides — to the C-linker regions in the adjacent subunit. To achieve these distance measurements, we used transition metal ion FRET (tmFRET) along with the incorporation of a fluorescent noncanonical amino acid L-Anap. Patch-clamp fluorometry was used to measure simultaneously the ionic current and the quenching of the Anap fluorescence by transition metals introduced in the C-linker. We compared these distances in the absence versus the presence of cAMP at 0 mV. Applying cAMP increased the relative distance between the carboxyl-terminal end of the S4 helix to the A’ helix of the adjacent C-linker. In contrast, cAMP binding shortened the distance between the amino-terminal part of the S5 helix and the A’ helix. Importantly, gating current measurements revealed that the hyperpolarization-dependent charge movement depended on cAMP binding. These results suggest that small resting-state rearrangements between the VSD and the S6/C-linker gate can have profound effects on the voltage-dependent gating of HCN channels.