Resting-State Structure and Gating Mechanism of a Voltage-Gated Sodium Channel

Abstract

Voltage-gated sodium (Na_V) channels initiate action potentials in nerve, muscle, and other electrically excitable cells. The structural basis of voltage gating is uncertain because the resting state exists only at deeply negative membrane potentials. To stabilize the resting conformation, we inserted voltage-shifting mutations and introduced a disulfide crosslink in the VS of the ancestral bacterial sodium channel Na_VAb. Here, we present a cryo-EM structure of the resting state and a complete voltage-dependent gating mechanism. The S4 segment of the VS is drawn intracellularly, with three gating charges passing through the transmembrane electric field. This movement forms an elbow connecting S4 to the S4-S5 linker, tightens the collar around the S6 activation gate, and prevents its opening. Our structure supports the classical "sliding helix" mechanism of voltage sensing and provides a complete gating mechanism for voltage sensor function, pore opening, and activation-gate closure based on high-resolution structures of a single sodium channel protein.

Keywords: Na(V); X-ray crystallography; cryo-EM; disulfide crosslinking; electrophysiology; gating charge; ion channel; membrane protein; voltage sensor; voltage-gated sodium channel.

Figure 1. Na_VAb Double Cysteine Substitutions Reveal State-dependent Disulfide Crosslinking

(A) Design of double-cysteine substitution experiment for disulfide crosslinking. The crystal structure of Na_VAb in the activated state (Payandeh et al., 2011) was used as a starting template. The domain-swapped architecture of the Na_VAb homotetramer permits intermolecular disulfide crosslinking between one subunit (purple) and the neighboring subunit (blue) that results in a formation of a covalently linked tetramer. Cysteine substitutions that lead to structures are shown as spheres at Cα for G94 (red) and V100 (green) and Q150 (blue). (B) Close-up view for intermolecular disulfide crosslinking design in the double-cysteine substitution experiment. Twelve residues on S3-S4 loop and S4 were screened against 5 residues on S5 that yielded a total of 60 variants. Key S4 residues that showed disulfide-crosslinked tetramer with S5 residue (Q150, blue) are highlighted in red (G94) for resting state, and green (V100) for activated states. (C) Effects of KAV mutations on state-dependent disulfide crosslinking under hyperpolarization/oxidation condition. (D) Effects of KAV mutations on state-dependent disulfide crosslinking under control condition. Percent crosslink denotes the extent of crosslinking quantified from the intensity of the tetramer band divided by the total stain intensity. The bar graph (left axis) shows % crosslink of double-cysteine substitutions for Na_VAb (yellow) and Na_VAb KAV (red). The line graph shows differences (Δ) in % crosslink between the same double-cysteine substitutions for Na_VAb KAV vs. Na_VAb (black circles). Error bars represent SEM with n = 5. See also Figures S1 and S2.

Figure 2. Electrophysiological Recordings of Na_VAb/WT, Na_VAb/N49K, Na_VAb/KAV and Na_VAb/KAV Cysteine Mutants

(A) Normalized conductance-voltage (G/V) relationships and Boltzmann fits for WT and each mutant. Sf9 cells expressing Na_VAb KAV mutants (all C-terminal Δ28 truncation constructs) were stimulated with 50 ms pulses from 0–150 mV in 10 mV increments from a holding potential of −150 mV. Dashed: WT from (Gamal El-Din et al., 2013); Green, Na_VAb/N49K, V_1/2=−21.8±1.8 mV, k=7.9±1.0 mV, n=4; Gray: Na_VAb/N49K/L109A/M116V (KAV) Δ28, V_1/2=59.1±0.8 mV, k=7.7±0.8 mV, n=3; Blue: Na_VAb/KAV/Q150C Δ28, V_1/2=76.8±3.1 mV, k=10.0±0.9 mV, n=3; Red: Na_VAb/KAV/G94C Δ28, V_1/2=88.4±1.1 mV, k=10.6±0.4 mV, n=4; Purple: Na_VAb/KAV/G94C/Q150C Δ28 in 50 mM DTT, V_1/2=105.7±0.8 mV, k=10.2±0.2 mV, n=3; Black: Na_VAb/KAV/G94C/Q150C Δ28 with no DTT, V_1/2=n/a, k=n/a, n=5. Markers and error bars represent average G/G_max ± standard error of the mean (SEM). Half activation (V_1/2) and slope (k) values are averages of individual fits ± SEM. Curves = 1/(1+ê(( V_1/2−V_m)/k)). Data points within 15 mV of V_rev were omitted to reduce noise. (B) Representative current families of Na_VAb/KAV Δ28 (gray), Na_VAb/KAV/Q150C Δ28 (blue), Na_VAb/KAV/Q94C Δ28 (red), and Na_VAb/KAV/G94C/Q150C Δ28 in 50 mM DTT (purple). Transiently transfected Sf9 cells were held at −150 mV and stimulated for 50 ms to depolarized voltages in 10 mV increments. Scale bars represent 10 msec x 1 nAmp. See also Figure S3.

Figure 3. Structures of Na_VAb G94C/Q150C and Disulfide-crosslinked Na_VAb V100C/Q150C in the Activated State

(A) Overall structure of Na_VAb/G94C/Q150C Δ28 activated state. The VS S0 to S3 segments are colored in orange and S4 in magenta. The S4-S5 linker is shown in blue and the pore module S5 to S6 in yellow. Ordered DMPC phospholipid molecules are highlighted in green for acyl chains and orange/red for phosphate head groups. (B) Superposition of Na_VAb/G94C/Q150C Δ28 structure with Na_VAb/WT structure (PDB 4EKW). Cα backbone structures are shown with Na_VAb/WT colored in gray with Cα r.m.s.d. of ~0.9 Å. (C) Overall structure of Na_VAb/V100C/Q150C Δ28 disulfide-crosslinked activated state. The structure is rendered as in (A). Difference in the C-terminal tail is due to crystallization pH condition (pH 4.8 vs. pH 5.6–6.0 for Na_VAb/G94C/Q150C Δ28). (D) Electron density map for V100C-Q150C disulfide bond. The F_O−F_C difference map is shown for V100C and Q150C side chains at 3.5σ (green) and 7σ (red) contour levels. Sulfur atoms on cysteine side chains are shown in gold. See also Figure S4 and Table S1.

Figure 4. Cryo-EM Structure of Na_VAb Resting State at 4.0 Å Resolution

(A) Cryo-EM density map of MBP-Na_VAb/KAV/G94C/Q150C disulfide-crosslinked resting state in digitonin detergent. Side view (left) and top (extracellular) view (right) are shown. The density map is colored by local resolution according to the MonoRes color code (side bar). Density map for MBP regions is blurry and not displayed for clarity but can be viewed in Figure S5D. (B) Overall structure of Na_VAb/KAV/G94C/Q150C disulfide-crosslinked resting state. Side view (left) and top view (right) are shown in the same orientation as in (A). S0 to S3 segments of the VS are colored in orange, S3-S4 loop in red, and S4 in magenta. The S4-S5 linker is highlighted in blue and the pore module in yellow. Major structural changes are observed in the S3-S4 loop, S4 segment, and the S4-S5 linker. See also Figures S5–S7 and Table S2.

Figure 5. Comparison of Na_VAb Disulfide-crosslinked Structures in the Resting State and the Activated State

(A) Structures of the VS are shown as backbone cartoon superimposed with the solvent accessibility surface. S0 to S3 are shown in gray, S3-S4 loop in red, and S4 in magenta. A wider and shallower aqueous cleft between S1-S2 and S3-S4 helix-loop-helix is present in the resting state compared to a deeper cleft in the activated state. (B) Gating charge movement. Four Arg gating charges R1-R4 (blue), extracellular negative charge (ENC) cluster of E32 and N49(K) and intracellular negative charge (INC) cluster of E59, E80 (red), Phe in the hydrophobic constriction site (HCS) (green), conserved W76 (gray) and E96 (yellow) are shown in sticks. S4 (magenta) moves outward by 11.5 Å, passing two gating charges through the HCS on S2. Part of S3 is omitted for clarity. (C) Side view of the structures focusing on S4 (magenta) and the S4-S5 linker (blue), with the S0 to S3 segments shown in gray and the pore module in yellow. The S4 segment moves outward across the membrane from the resting to the activated states while the S1 to S3 segments remain relatively unchanged with respect to the membrane. The S4-S5 linker acts as an elbow that connects the S4 movement to modulate the pore. (D) Bottom (intracellular) view of the structures in (C) with S0 to S3 omitted for clarity. The S4-S5 linker (blue) undergoes a large conformational change that tightens the collar around the S5 and S6 segments (yellow) of the pore in the resting state and loosens the collar in the activated state.

Figure 6. Gating Mechanism for Voltage-gated Sodium Channels

(A) Superposition of structures of Na_VAb in the resting/closed state and the activated/open state (PDB 5VB8) viewed from the intracellular side. Major structural changes are observed in the S4-S5 linker, and the S6 activation gate (red for the resting/closed state vs. green for the activated/open state). (B) Interactions between the S4-S5 linker and S6 near the activation gate. The elbow movement of the S4-S5 linker from the resting/closed state (left) to the activated/open state (right) causes an exchange in the interactions between the S4-S5 linker residues (blue) and the S6 residues (red for the resting/closed state vs. green for the activated/open state) surrounding the activation gate of the pore. (C) Solvent accessibility surface of Na_VAb in the resting closed state (left) and the activated/open state (right). The pore is tightly closed at the activation gate in the resting state (red) but wide open in the activated open state (green). (D) Structural transition between resting and activated states. Images of structures in the resting (left), intermediate (middle), and activated/open states (right) are captured from Video S1 and rendered as in Figure 5. Part of S3 is omitted for clarity. See also Video S2.

Figure 7. Implications of the Resting State Structure for Sodium Channel Pharmacology.

(A) Drug access to the pore of Na_VAb via the fenestrations in the resting state. Top view cutaway section below the selectivity filter of PM shows fenestration and hydrophobic access to central cavity of the pore. (B) Phospholipid binding to a new drug target site in Na_VAb G94C/Q150C. Electron density map for phospholipids near the S4-S5 linker. The 2F_O−F_C electron density is contoured at 1σ level (black mesh). Residues W76 on S2, and S121 and P128 on the S4-S5 linker interact with phosphate head groups, and V120, I124 and I127 interact with acyl chains of the lipids (not rendered). (C) Conformational change between resting and activated states at the receptor site for gating-modifier toxins and drugs in the VS.