The HCN channel voltage sensor undergoes a large downward motion during hyperpolarization

Abstract

Voltage-gated ion channels (VGICs) contain positively charged residues within the S4 helix of the voltage-sensing domain (VSD) that are displaced in response to changes in transmembrane voltage, promoting conformational changes that open the pore. Pacemaker hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are unique among VGICs because their open probability is increased by membrane hyperpolarization rather than depolarization. Here we measured the precise movement of the S4 helix of a sea urchin HCN channel using transition metal ion fluorescence resonance energy transfer (tmFRET). We show that the S4 undergoes a substantial (~10 Å) downward movement in response to membrane hyperpolarization. Furthermore, by applying distance constraints determined from tmFRET experiments to Rosetta modeling, we reveal that the carboxy-terminal part of the S4 helix exhibits an unexpected tilting motion during hyperpolarization activation. These data provide a long-sought glimpse of the hyperpolarized state of a functioning VSD and also a framework for understanding the dynamics of reverse gating in HCN channels.

Figure 1 |. Patch-clamp fluorometry (PCF) simultaneously measures current and Anap fluorescence in HCN channels.

a, Cartoon showing the architecture of an HCN channel subunit (only two subunits are shown). Each subunit contains a cyclic nucleotide-binding domain (CNBD) in the carboxy-terminal region that is connected to the pore region via a gating ring formed by the C-linker of each subunit. b, Structure of L-Anap. c, Cartoon illustrating amber stop-codon suppression strategy for incorporating noncanonical amino acids. d, Representative PCF images showing a giant inside-out patch expressing spHCN-W355Anap channels in bright field (top left) and epifluorescence (heat map, bottom left) from the same patch; the dashed area in both images is an exemplar region of interest used for quantifying the patch fluorescence. Top right, channel current elicited by a series of hyperpolarizing voltages from 0 mV to −120 mV with 10 mV steps (Bottom right, the intensity of Anap fluorescence is proportional to the magnitude of the peak tail current (bottom right). e, Sequence alignment of the S4 helix of spHCN, human HCN1, and HCN2 channels. L-Anap sites are indicated with arrows, positively-charged arginines on the S4 close to the L-Anap sites are highlighted in black boxes, and the 3₁₀-helical segment of the S4 is indicated with a black line. f, Positions of Anap sites within S4 shown in the homology model of spHCN based on human HCN1 (PDB 5U6O). g, Top, map of spHCN constructs showing eYFP fused to the C-terminal end (size of eYFP is not to scale). Bottom, exemplar PCF images showing the specific incorporation of L-Anap at the spHCN-W355 site. h, G-V relationships of spHCN constructs in the presence of 1 mM cAMP with L-Anap incorporated into different sites in the S4, compared to wild-type spHCN (WT) (also see Supplementary Table 1). Current traces are in Supplementary Fig. 1.

Figure 2 |. Hyperpolarization-dependent change in Anap fluorescence of spHCN channels with L-Anap incorporated into the S4 voltage sensor.

a, Representative epifluorescence images and spectra of the S346Anap fluorescence at 0 mV and −100 mV. b, Two-dimensional S4 topology and the Anap fluorescence changes at different S4 sites in response to a −100 mV pulse in the presence of 1 mM cAMP. c, Summary of the percent change of the Anap fluorescence for different sites within the S4 helix due to a −100 mV pulse. Data shown are mean ± s.e.m., n = 6 patches for S346, n = 4 for L348 and S353, n = 7 for W355 and n = 4 for F359. d, Simultaneous current and fluorescence measurements from spHCN-S346Anap channels in response to a family of hyperpolarizing voltage pulses in 1 mM cAMP. e, Fluorescence change-voltage (F-V) and conductance-voltage (G-V) relationships for the spHCN-S346Anap channels. Data shown are mean ± s.e.m., n = 3 patches.

Figure 3 |. tmFRET detects a hyperpolarization-dependent downward movement of S4 in spHCN-S346Anap channels.

a, Spectral properties of free L-Anap emission and transition metal ion absorption. Emission spectra from L-Anap in different solvents (black, SBT buffer; red, EtOH; green, DMSO) are overlaid on the absorption spectra of Cu²⁺-TETAC (dark blue ) and Co²⁺-HH (magenta) b, Cartoon showing FRET between the S346Anap site and the diHis site in the HCN domain (L182H, L186H). c, Time course of Anap fluorescence in spHCN-S346Anap, L182H, L186H channels in response to a −100 mV pulse in the presence of cAMP before and after Co²⁺ application and EDTA to sequester Co²⁺. The same experiment for spHCN-S346Anap channels without the introduced diHis in the presence of Co²⁺ is also shown as a control. d, Voltage dependence of the apparent FRET efficiency for the spHCN-S346Anap, L182H-L186H channels in the presence of 1 mM cAMP. Data shown are mean ± s.e.m, n = 3 patches.

Figure 4 |. The distance change measured by ACCuRET decreases as Anap is positioned closer to the C-terminal end of S4.

a, Cartoon showing ACCuRET between S346Anap and Cu²⁺-TETAC attached to L186C in the HCN domain. Also shown is the reaction of Cu²⁺-TETAC with a cysteine in a protein. b, Representative spectra of the Anap emission at 0 mV and −100 mV, before and after quenching by Cu²⁺-TETAC, for spHCN-S346Anap, L186C channels. c, Summary of the fraction of Anap fluorescence quenched by Cu²⁺-TETAC for the four Anap sites in S4, without (left, n = 4 for S346, n = 3 for L348, S353, and W355) and with (right, n = 5 for S346, n = 4 for L348 and S353, n = 7 for W355) the introduced cysteine L186C. Data shown are mean ± s.e.m., n is number of patches, * p < 0.05 by two-tailed Student’s t-test. d, Distance dependence of the measured FRET efficiency at 0 mV for four L-Anap sites (circle markers) in S4 and the W218Anap site in S1 versus the β-carbon distances between each site and L186C (based on the spHCN homology model). Also shown are the predicted distance dependencies of the Förster equation (black) and the Förster Convolved with Gaussian (FCG) equation (green), using a Gaussian distribution with a standard deviation σ of 7.5 Å (inset). The distances of these 5 pairs at −100 mV estimated using the FCG equation are also shown with square markers. e, Summary of the distances of each FRET pair at 0 mV and −100 mV, calculated from the FCG equation in panel d, using the FRET efficiencies in Supplementary Fig. 5a. f. Helical-wheel display of the C-terminal α-helical part of S4 (S346 to F359) viewed from the extracellular side, highlighting the magnitude of the hyperpolarization-induced distance changes for the four Anap sites in the S4.

Figure 5 |. Rosetta model of S4 movement in HCN channels based on experimentally-determined distance constraints.

a, Model structures of the voltage-sensor domain of spHCN at 0 mV and −100 mV. Top: side view parallel to the membrane. Bottom: view from the intracellular side. b, Comparison of the measured distance changes using tmFRET from Fig. 4 and the distance changes in the Rosetta models. c, A vacuum electrostatic surface illustration of the S1-S3 helices and the HCN domain, showing a negative charged surface (red) facing the S4 helix. d, Structural diagrams showing the ion pair partners between arginines or lysine (blue) within S4 and aspartic acids (red) within S1-S3 at 0 mV and −100 mV in the Rosetta models. The “phenylalanine cap” F260 in the hydrophobic constriction site (HCS) of the charge transfer center is highlighted in magenta. The α-carbon distance changes between K1, R3-R6 residues at 0 mV and −100 mV are illustrated.