Structural Basis for High-Affinity Trapping of the NaV1.7 Channel in Its Resting State by Tarantula Toxin

Abstract

Voltage-gated sodium channels initiate electrical signals and are frequently targeted by deadly gating-modifier neurotoxins, including tarantula toxins, which trap the voltage sensor in its resting state. The structural basis for tarantula-toxin action remains elusive because of the difficulty of capturing the functionally relevant form of the toxin-channel complex. Here, we engineered the model sodium channel Na_VAb with voltage-shifting mutations and the toxin-binding site of human Na_V1.7, an attractive pain target. This mutant chimera enabled us to determine the cryoelectron microscopy (cryo-EM) structure of the channel functionally arrested by tarantula toxin. Our structure reveals a high-affinity resting-state-specific toxin-channel interaction between a key lysine residue that serves as a "stinger" and penetrates a triad of carboxyl groups in the S3-S4 linker of the voltage sensor. By unveiling this high-affinity binding mode, our studies establish a high-resolution channel-docking and resting-state locking mechanism for huwentoxin-IV and provide guidance for developing future resting-state-targeted analgesic drugs.

Keywords: NaV1.7; analgesics; cryo-EM; electrophysiology; gating-modifier toxins; huwentoxin; pain; protein structure; tarantula; voltage-gated sodium channel.

Figure 1.. Design of Voltage Shifting Na_VAb/Na_V1.7-VS2 Chimera

(A) Topology diagram of substitution. Transmembrane and extracellular portion of human Na_V1.7 VS2 S1-S2 and S3-S4 helices (cyan) were grafted onto equivalent region of Na_VAb (gold). (B) Model of substitution. Cyan highlights portions of human Na_V1.7 S1-S2 and S3-S4 helices that have been grafted onto Na_VAb (gold). (C) Sequence alignment of S3-S4 region of different human Na_V subtypes. Sequence portion including the “LFLAD” motif that interacts with HwTx-IV is highlighted in yellow. (D) Representative current families of Na_VAb/Na_V1.7-VS2 chimera (cyan) and with L834A mutation (VS2A) (red). 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 × 1 nA. (E) Normalized conductance-voltage (G/V) relationships and Boltzmann fits for Na_VAb (black dash; WT (Gamal El-Din et al., 2013)), Na_VAb/Na_V1.7-VS2 chimera alone (cyan; V_1/2 = 34.6 ± 1.4 mV, k = 10.0, n = 0.1, n = 6) and with L834A mutation (VS2A) (red; V_1/2 = 83.4 ± 0.9 mV, k=9.1 ± 0.6, n = 3). 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)). See also Figure S1.

Figure 2.. Voltage Sensor Trapping of Na_VAb/Na_V1.7 VS2A by m3-HwTx-IV.

(A) Representative traces of sodium outward currents and following inward tail currents in Hi5 cells in response to 50-ms pulses from a holding potential of −160 mV in 10 mV steps from 0 mV to +160 mV (Black). Sodium currents measured from the same cell after perfusion with 10 nM m3-HwTx-IV (Red). Tail currents before (black) and after (red) treatment with different concentrations of m3-HwTx-IV (0.1, 1, 10, and 100 nM as indicated). (B) Normalized Current/Voltage (I/V) plot of Na_VAb/Na_V1.7-VS2A before (black) and after (red) treating cells with 100 nM m3-HwTx-IV. Tail currents measured after m3-HwTx-IV treatment were normalized to the one measured previously. Conductance was estimated from the tail current amplitude and normalized to the control amplitude at the +160-mV test potential. The solid black line is the fit of the Boltzmann equation to the data. The V_1/2 was 107.2 ± 0.5 mV with a slope factor K= 12.5 ± 0.3. Data are represented as mean ± SEM with n = 5 to 8. (C) Concentration-response curve of m3-HwTx-IV interaction with Na_VAb/Na_V1.7-VS2A chimera. The solid red line is the best fit using the Hill equation. IC₅₀= 2.6 ± 0.3 nM with a Hill coefficient = 0.7. Data are represented as mean ± SEM with n = 5 to 7. (D) Time course for recovery of blocked tail current following strong depolarizations in the presence of 250 nM m3-HwTx-IV. Current amplitude was measured at +80, +120, and +160 mV. Different pulse durations were used from 50-ms to 600-ms. Recovered currents were normalized to the peak current elicited from depolarizations to 600-ms. Data are represented as mean ± SEM with n = 3 to 5. Inset. Representative current traces of recovered currents at +120 mV depolarizing potentials.

Figure 3.. Cryo-EM Structure of Na_VAb/Na_V1.7 VS2A Chimera in the Resting State Trapped by m3-HwTx-IV.

(A) Cryo-EM density map colored by local resolution in side and top views. Local resolution was evaluated using ResMap (Kucukelbir et al., 2014). The right panel shows resolution spectrum bar. (B) Overall structure of m3-HwTx-IV:Na_VAb/Na_V1.7-VS2A chimera. 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 teal and S4 in magenta. The S4-S5 linker is highlighted in blue and the pore module in yellow. m3-HwTx-IV is colored in red superimposed with transparent van der Waals surface. See also Figures S2 and S3, and Table S1.

Figure 4.. Structure of Na_VAb/Na_V1.7-VS2A Trapped by m3-HwTx-IV in the Resting State and Comparison to Na_V1.7 VS2 Trapped by HwTx-IV in a Partially Activated State.

(Left panels) Na_VAb/Na_V1.7-VS2A in the resting state; (Right panels) Na_V1.7 VS2 in a partially activated state bound to HwTx-IV + saxitoxin (PDB 6J8G) (A) Structures of the VS as backbone cartoon superimposed with the molecular surface. S0 to S3 are shown in light teal and S4 in magenta. An aqueous cleft between S1-S2 and S3-S4 helix-loop-helix is indicated. (B) Gating charge movement. Four Arg gating charges R1-R4 (blue), extracellular negative charge (ENC) cluster of E753 on S1 and N769 on S2, intracellular negative charge (INC) cluster of E779 on S2 and D801 on S3 (red), and F776 in the hydrophobic constriction site (HCS) (green) are shown in sticks. S4 (magenta) moves outward from resting state to partially activated state by ∼7.3 Å, passing nearly two gating charges through the HCS on S2. Part of S3 is omitted for clarity. (C) Side view of the structures with the S0 to S3 segments shown in gray, S4 in magenta, the S4-S5 linker in blue, and the pore module in yellow. The S4 segment moves outward across the membrane from the resting to the partially 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. Three VS and two pore modules are omitted for clarity. (D) Bottom (intracellular) view of the structures in (C) with complete pore modules. 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 partially activated state. S0 to S3 are omitted for clarity. See also Figures S4 and S6.

Figure 5.. Molecular Interactions of HwTx-IV with Neurotoxin Receptor Site 4 in Resting and Partially Activated Na_V1.7-VS2

(A) Binding position of m3-HwTx-IV to the resting state of neurotoxin receptor site 4 of Na_VAb/Na_V1.7-VS2A in side and top views. Cryo-EM density map filtered to 4.5 Å resolution for m3-HwTx-IV (red) is shown superimposed with the atomic model. (B) Binding position of HwTx-IV to the partially activated state of neurotoxin receptor site 4 of Na_V1.7 in side and top views. Cryo-EM density map (EMD-9781) filtered to 4.5 Å resolution for HwTx-IV (green) is shown in comparison to (A). (C) Detailed molecular interactions of m3-HwTx-IV with Na_VAb/Na1.7-VS2A chimera in the resting state. Residues making contacts within 5 Å are shown as sticks models and highlighted in yellow for residues from VS2 chimera as in Figure 1C. Some parts of the interactions were only partially resolved in the experimentally determined density and were modeled based on rigid-body docking and refinement of the known NMR structure of the toxin. See also Figure S5.

Figure 6.. Detailed Map of Interactions Between m3-HwTx-IV and VS2

(A) Amino acid sequence of m3-HwTx-IV. Cysteine residues that form disulfide bonds are highlighted in gray. Positive charge and negative charge residues are colored in blue and red letters, respectively. Residues of m3-HwTx-IV that when mutated to alanine resulted in a loss of affinity for Na_V1.7 by less than 2-fold are highlighted in yellow, 2–5-fold are in pink, and more than 5-fold in red, or positive gain in affinity in green according to (Revell et al., 2013). The C-terminal “²⁶RKxRWCK³²” motif important for binding is underlined. (B) Close up top view as in Figure 5C right panel with m3-HwTx-IV removed. Surface residues are colored based on changes in affinity when mutated according to (Xiao et al., 2011). (C) Close up view as in Figure 5C right panel with m3-HwTx-IV turned 180° open face up to show resides making interactions with the Na_VAb/Na1.7-VS2A. Surface residues are colored based on changes in affinity when mutated to Ala as in (A).