Structural changes during HCN channel gating defined by high affinity metal bridges

Abstract

Hyperpolarization-activated cyclic nucleotide-sensitive nonselective cation (HCN) channels are activated by membrane hyperpolarization, in contrast to the vast majority of other voltage-gated channels that are activated by depolarization. The structural basis for this unique characteristic of HCN channels is unknown. Interactions between the S4-S5 linker and post-S6/C-linker region have been implicated previously in the gating mechanism of HCN channels. We therefore introduced pairs of cysteines into these regions within the sea urchin HCN channel and performed a Cd(2+)-bridging scan to resolve their spatial relationship. We show that high affinity metal bridges between the S4-S5 linker and post-S6/C-linker region can induce either a lock-open or lock-closed phenotype, depending on the position of the bridged cysteine pair. This suggests that interactions between these regions can occur in both the open and closed states, and that these regions move relative to each other during gating. Concatenated constructs reveal that interactions of the S4-S5 linker and post-S6/C-linker can occur between neighboring subunits. A structural model based on these interactions suggests a mechanism for HCN channel gating. We propose that during voltage-dependent activation the voltage sensors, together with the S4-S5 linkers, drive movement of the lower ends of the S5 helices around the central axis of the channel. This facilitates a movement of the pore-lining S6 helices, which results in opening of the channel. This mechanism may underlie the unique voltage dependence of HCN channel gating.

Figure 1.

Schematic of the structure of spHCN channel subunits. (A) Schematic of the putative topology of an spHCN subunit. Locations of the charged S4 voltage sensor, the S4–S5 linker, the post-S6/C-linker, the CNBD, and the binding site for cyclic nucleotide monophosphate (cNMP) are shown. (B) Sequence alignments of the S4, S4–S5 linker, pore-lining S6, and post-S6/C-linker regions of spHCN1 (GenBank accession no. CAA76493), with human Kv1.2 (NCBI Protein database accession no. NP_004965), rat Kv1.2 (NCBI Protein database accession no. NP_037102), and KcsA (UniProtKB/Swiss-Prot sequence P0A334) channel subunits. Alignments were made using ClustalW2. Mutations of spHCN1 discussed in Results and Discussion are shown as bold and underlined.

Figure 2.

Metal bridges can form between the S4–S5 linker and C-linker in the open state. Representative current traces of selected single C-linker residue mutants and double mutants with F359C. Currents were elicited by 500-ms voltage steps to −120 mV from a holding potential of 0 mV, followed by a voltage step to +50 mV. Currents were recorded in the absence (black) or presence (red) of 1 µM Cd²⁺ from excised inside-out patches expressing the various mutants as indicated. Current traces recorded from the WT partial cysteine-less background, which contains C211Y, C224I, C254V, C266M, C369F, and C373G in addition to the M349I and H462Y mutations, are shown. Current traces recorded from the single F359C mutant are also shown, as well as current traces recorded from single S472C, S473C, S474C, Q476C, R478C, E479C, K482C, and E485C mutants. Current traces from double mutants containing F359C and each of the C-linker mutations are also shown.

Figure 3.

Metal bridges can form between the S4–S5 linker and C-linker in both open and closed states. Representative current traces of selected C-linker mutants with V361C and A364C. Currents were elicited by voltage steps to −120 mV from a holding potential of 0 mV, followed by a voltage step to +50 mV. Currents were recorded in the absence (black) or presence (red) of 1 µM Cd²⁺ from excised inside-out patches expressing the various mutants as indicated. For the V361CQ476C and A364CS473C mutants, currents recorded in response to a 1-s hyperpolarizing pulse from 0 to −120 mV are also shown to highlight the slowing of activation by Cd²⁺. For the A364CS474C, A364CQ476C, and A364CR478C mutants, currents recorded in response to a 2-s hyperpolarizing pulse from 0 to −120 mV are also shown to highlight the slowing of activation by Cd²⁺.

Figure 4.

Summary of the effects of Cd²⁺ on spHCN channels containing introduced cysteine residues. (A) The lock-open effects of 1 µM Cd²⁺, as indicated by the proportion of the nondecaying tail current, in the single C-linker controls (left-most panel) and the double mutants containing one of the F359C, V361C, or A364C mutations are shown. Gray bars indicate values for control conditions, and green bars indicate values after the addition of Cd²⁺. The mutants giving rise to significant lock-open effects are clustered in two separate regions. (B) The lock-closed effects of 1 µM Cd²⁺, as indicated by the prolongation of the rise time between 10 and 50% of the maximum current in a 500-ms pulse, in the single C-linker controls (left-most panel) and the double mutants containing F359C, V361C, or A364C mutations are shown. Gray bars indicate values for control conditions, and red bars indicate values after the addition of Cd²⁺. (C) A summary of the effects of 1 µM Cd²⁺ on the double mutants tested in this study.

Figure 5.

Use of concatenated subunits shows that in the open state metal bridges can form between A364C and S472C, possibly within the same subunit. (A) Representative current traces were recorded in the absence (black) or presence (red) of 1 µM Cd²⁺ from inside-out patches expressing one of the tandem dimers (364C472C_364C472C, WT_364C472C, or 364C_472C). Currents were elicited by 500-ms voltage steps to −120 mV from a holding potential of 0 mV, followed by a voltage step to +50 mV. (B) Effects of Cd²⁺ on the average proportions of nondecaying tail current produced by the three tandem–dimeric constructs.

Figure 6.

Intersubunit metal bridges can form between A364C and E482C in the open state. (A) Representative current traces were recorded in the absence (black) or presence (red) of 1 µM Cd²⁺ from excised inside-out patches expressing one of the tandem dimers (364C482C_364C482C, WT_364C482C, or 364C_482C). Currents were elicited by 500-ms voltage steps to −120 mV from a holding potential of 0 mV, followed by a voltage step to +50 mV. (B) Effects of Cd²⁺ on the average proportions of nondecaying tail currents produced by the three tandem dimers.

Figure 7.

Intersubunit metal bridges can form between A364C and Q476C in the closed state. (A) Representative current traces were recorded in the absence (black) or presence (red) of 1 µM Cd²⁺ from excised inside-out patches expressing one of the tandem dimers (364C476C_364C476C, WT_364C476C, or 364C_476C). Currents were elicited by voltage steps to −120 mV from a holding potential of 0 mV, followed by a voltage step to +50 mV. Currents from a 2-s (364C476C_364C476C) or 1-s (WT_364C476C and 364C_476C) hyperpolarizing pulse in the presence of Cd²⁺ are superimposed on the control current traces to highlight the slowing of the activation kinetics by Cd²⁺. (B) Effects of Cd²⁺ on the average rise time of currents produced by the intersubunit (364C_476C) and intrasubunit (WT_364C476C) tandem dimers. (C) The time course of activation was fit to a double-exponential function, and the time constants for the two components, along with the relative proportion of the slow component, are shown. The slowing of activation kinetics was mainly caused by an increase in the proportion of the slow time constant (tau).

Figure 8.

Model of the structure and gating motions of an HCN channel based on high affinity metal bridges. (A) Stereo view of a model of the cAMP-bound closed state of the spHCN channel, based on the crystal structures of KcsA (Protein Data Bank accession no. 1K4C) and the cAMP-bound CNBD of spHCN (Protein Data Bank accession no. 2PTM). Residue D471 of spHCN is in approximately the same position as KcsA-118, based on the equivalence of spHCN-468 to KcsA-115 (Rothberg et al., 2003). The uncoiled strands between the S6 helices and the A′ helices include the spHCN residues ⁴⁷²SSS⁴⁷⁴. (B) Stereo view of the locations of C-linker residues that when mutated to cysteine form metal bridges with 364C to produce lock-open (green spheres) and lock-closed (red spheres) effects. Spheres of ∼6-Å diameter are centered around the β carbon of each C-linker residue involved in bridges with 364C. (C) Model of possible motions occurring during HCN channel gating. A view from the extracellular side of the channel, showing the locations of lock-open (green) and lock-closed (red) effects with 364C, superimposed on the closed-state model. Cylinders represent each S4–S5 linker, and asterisks indicate the positions of 364C residues. The proposed motions of 364C residues (dashed arrows) and the lower ends of the S6 helices (solid arrows) during channel opening are indicated. The color of the numbers for the C-linker residues studied matches the ribbon color for the corresponding subunit.