Electromechanical coupling mechanism for activation and inactivation of an HCN channel

Gucan Dai,Frank Dimaio,Teresa K Aman,William N Zagotta

doi:10.1038/s41467-021-23062-7

Abstract

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 inactivation with hyperpolarization which occurs only in the absence of cyclic nucleotide. Here we applied transition metal ion FRET, patch-clamp fluorometry and Rosetta modeling to measure differences in the structural rearrangements between activation and inactivation of spHCN channels. We found that removing cAMP produced a largely rigid-body rotation of the C-linker relative to the transmembrane domain, bringing the A’ helix of the C-linker 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 the presence of cAMP. These results suggest that — in contrast to electromechanical coupling for channel activation — the A’ helix serves to couple the S4-helix movement for channel inactivation, which is likely a conserved mechanism for CNBD-family channels.

Highlights

Pacemaker hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels exhibit a reversed voltage-dependent gating, activating by membrane hyperpolarization instead of depolarization
In the absence of cyclic nucleotide, FRET efficiency increased substantially at −100 mV, similar to the increase seen in cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) (Fig. 1f). These results indicate that the Ser[346] position in the S4 helix moved downward–closer to the HCN domain (HCND)—with hyperpolarization in the absence of cyclic nucleotide, similar to the movement with cAMP or cGMP
Unlike mammalian HCN channels, sperm HCN (spHCN) channels undergo hyperpolarization-dependent inactivation in the absence of cyclic nucleotide which is eliminated in the presence of cAMP

Summary

Introduction

Pacemaker hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels exhibit a reversed voltage-dependent gating, activating by membrane hyperpolarization instead of depolarization. Rotation of the C-linker was elicited by hyperpolarization in the absence but not the presence of cAMP These results suggest that — in contrast to electromechanical coupling for channel activation — the A’ helix serves to couple the S4-helix movement for channel inactivation, which is likely a conserved mechanism for CNBD-family channels. For these domain-swapped channels, the coupling is thought to occur primarily through the covalent linkage between the S4 helix of the VSD and the S5 helix of the PD, the S4–S5 linker[5,20,23] This S4–S5 linker-dependent pathway is considered the canonical electromechanical coupling for VGICs. a covalent linkage between the S4 and S5 segment is not required for the voltage-dependent gating of the non-domainswapped channels EAG1, hERG1, and spHCN14,24,25. For these non-domain-swapped channels, the electromechanical coupling has been proposed to involve either a noncovalent interaction between the S4 and S5 helices of the same subunit or via the Clinker in the adjacent subunit[22,26]

Methods

Results

Conclusion