Insights into the molecular mechanism for hyperpolarization-dependent activation of HCN channels

Abstract

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels are both voltage- and ligand-activated membrane proteins that contribute to electrical excitability and pace-making activity in cardiac and neuronal cells. These channels are members of the voltage-gated Kv channel superfamily and cyclic nucleotide-binding domain subfamily of ion channels. HCN channels have a unique feature that distinguishes them from other voltage-gated channels: the HCN channel pore opens in response to hyperpolarizing voltages instead of depolarizing voltages. In the canonical model of electromechanical coupling, based on Kv channels, a change in membrane voltage activates the voltage-sensing domains (VSD) and the activation energy passes to the pore domain (PD) through a covalent linker that connects the VSD to the PD. In this investigation, the covalent linkage between the VSD and PD, the S4-S5 linker, and nearby regions of spHCN channels were mutated to determine the functional role each plays in hyperpolarization-dependent activation. The results show that: (i) the S4-S5 linker is not required for hyperpolarization-dependent activation or ligand-dependent gating; (ii) the S4 C-terminal region (S4_C-term) is not necessary for ligand-dependent gating but is required for hyperpolarization-dependent activation and acts like an autoinhibitory domain on the PD; (iii) the S5_N-term region is involved in VSD-PD coupling and holding the pore closed; and (iv) spHCN channels have two voltage-dependent processes, a hyperpolarization-dependent activation and a depolarization-dependent recovery from inactivation. These results are inconsistent with the canonical model of VSD-PD coupling in Kv channels and elucidate the mechanism for hyperpolarization-dependent activation of HCN channels.

Keywords: SpIH; allostery; cyclic nucleotide-gated; patch-clamp; voltage-dependent gating.

Fig. 1.

HCN channels: structure and function. (A) Top view of Kv1.2-2.1 (PDB ID code 2R9R) and hHCN1 (PDB ID code 5U6O) with subunits in different colors. (B) A subunit of spHCN homology model labeled with VSD (blue), PD (orange), C-linker (purple), CNBD (green), and HCND (red). Also shown is part of the neighboring subunit (gray). (C and D) ZD7288-subtracted currents measured in the absence of ligand (black, triangles), 1 mM cGMP (green, squares), or 1 mM cAMP (red, circles) (Materials and Methods). (C) SpHCN_WT currents (D) SpHCN_∆Cterm currents (diamonds). (E) GV curves with smooth lines that represent Boltzmann fits (SI Appendix, Table S1).

Fig. 2.

The HA modular gating scheme used to describe hyperpolarization-dependent activation. (A) Schematic of the HA model showing each functional domain PD, VSD, and CNBD (Materials and Methods). The equilibrium constants are L, H(V), and K. The coupling factors are C, F, and E. (B) Popen vs. voltage predictions of the HA model for spHCN_WT channels. Also shown are predictions when VSD equilibrium constant H(V) is increased, the VSD–PD coupling factor F is decreased, or the PD equilibrium constant L is increased (WT: H₀ = 0.024, V_H = −9.63 mV, F = 10.1, L = 5.91, α_cAMP = 1, α_cGMP = 0.091, α_NocNMP = 0.0021; Increase H: H₀ = 1; decrease F = 1.8; increase L = 100).

Fig. 3.

The S4–S5 linker is not required for hyperpolarization-dependent activation or ligand-dependent gating. (A) Sequence alignment and homology model showing the S4 through S5 regions (#344–370) of spHCN_WT along with sequences defining split channels. Highlighted in blue are the S4–S5 linker residues. (B–D) ZD7288-subtracted currents and GV curves: (B) spHCN_WT, (C) split spHCN_(359:360), and (D) split spHCN_(359:363). Fits of the Boltzmann equation (smooth lines) (SI Appendix, Table S1) and the HA model (dashed lines) (Table 1).

Fig. 4.

The S4_C-term region is important for hyperpolarization-dependent activation. (A) Sequence alignment and homology model with S4_C-term residues in blue. (B–D) ZD7288-subtracted currents and GV curves: (B) split spHCN_(359:360), (C) split spHCN_(355:360), and (D) split spHCN_(350:360).

Fig. 5.

The S5_N-term region helps keep spHCN channels closed. (A) Sequence alignment and homology model with S4_C-term and S5_N-term residues in blue. (B–D) ZD7288-subtracted currents and GV curves: (B) split spHCN_(350:360), (C) split spHCN_(350:363), and (D) split spHCN_(350:367).

Fig. 6.

The S5_N-term region is involved in VSD–PD coupling. (A) Sequence alignment and homology model showing S4–S5 linker and S5_N-term residues in blue. (B and C) ZD7288-subtracted currents and GV curves: (B) split spHCN_(359:363) and (C) split spHCN_(359:367).

Fig. 7.

Mutations in the S4_C-term region reveal two voltage-dependent processes. (A) Sequence alignment and homology model with S4_C-term residues highlighted in orange or blue. (B–D) ZD7288-subtracted currents and GV curves: (B) intact spHCN_WT, (C) spHCN-A4, and (D) spHCN-A6.

Fig. 8.

Functional effects of individual mutations of conserved residues in the S4_C-term. (A) Sequence alignment with some conserved S4_C-term and S5_N-term residues in blue. The homology model showing residues with side-chains and putative interactions (A, Lower). (B–H) ZD7288-subtracted currents and GV curves: (B) R350A, (C) F351A, (D) Q354A, (E) W355A, (F) E356A, (G) R367A. Note: these currents were not leak-subtracted with ZD7288. (H) D471A.

Fig. 9.

Deletion of QAF reveals a depolarization-dependent activation. (A) Sequence alignment and homology model with S4_C-term residues highlighted in blue or orange. Voltage protocol included a 1.5-s prepulse (−100 mV), 500-ms test pulses (−100 to +100 mV), and tail pulse (−100 mV). (B–D) ZD7288-subtracted currents and GV curves: (B) spHCN_WT, (C) spHCN_(∆QWE), and (D) spHCN_(∆QAF) channels. (Inset) Enlargement of tail currents.

Fig. 10.

Model of hyperpolarization-dependent activation for HCN channels. Hyperpolarization causes movement of the VSD (blue) which relieves autoinhibition of the PD (orange). The binding of cyclic nucleotide (red) to the CNBD (green) removes autoinhibition of the PD.