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Nat Struct Mol Biol
2019 Aug 01;268:686-694. doi: 10.1038/s41594-019-0259-1.
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The HCN channel voltage sensor undergoes a large downward motion during hyperpolarization.
Dai G
,
Aman TK
,
DiMaio F
,
Zagotta WN
.
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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 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 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.
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