Structural and Functional Characterization of a Novel Scorpion Toxin that Inhibits NaV1.8 via Interactions With the DI Voltage Sensor and DII Pore Module

FIGURE 1. Voltage gated sodium channel structure and function. (A) The channel consists of four homologous domains (DI – DIV). Each domain consists of six transmembrane helices (S1 – S6). The fourth helix (S4) in each domain contains basic amino acids that act as voltage-sensor gating charges. The re-entrant loops between S5 and S6 in each domain form the pore (black arrow points to pore in DI). The extracellular loops connecting S5 to the pore (S5 – SS1) and the pore to S6 (SS2 – S6) form the mouth of the pore. The intracellular loop connecting DIII to DIV forms the fast inactivation hinged lid. The isoleucine-phenylalanine-methionine (IFM) motif is critical for fast inactivation gating. The DII and DIV S3 – S4 linkers form the primary scorpion β and α toxin binding sites, respectively. (B) When the channel is in the resting state, the activation gate is closed. When the channel is activated, the gate opens. During activation the inactivation gate (hinged lid) is disengaged. During fast inactivation, the hinged lid occludes the intracellular side of the pore. When the channel transitions into the slow inactivated state, the pore loops change conformation to obstruct the pore.

FIGURE 2. Effects of NaTx36 on grasshopper mouse recombinant OtNaV1.8 Na+ current density. Na+ currents were elicited by a 100-ms depolarization to +20 mV from a holding potential of −80 mV. Tetrodotoxin (TTX, 500 nM) was added to the bath solution to block endogenous TTX sensitive Na+ currents. Representative Na+ current traces and current density (pA/pF) recorded from whole-cell voltage-clamped ND7/23 cells transfected with OtNaV1.8. Na+ currents were recorded before and after application of NaTx36 and AZ bark scorpion venom. (A) NaTx36 (blue symbols, 10 μg/ml, n = 9 cells), and (B) venom (red symbols, 1.0 μg/ml, n = 6 cells) inhibited tetrodotoxin resistant (TTX-R) Na+ currents. Filled circles represent current density values for individually recorded cells. Histograms represent mean current density reported as picoamps divided by picofarads (pA/pF, * p < 0.05).

FIGURE 3. NaTx36 inhibits OtNaV1.8 in a concentration dependent manner. (A) TTX-R Na+ current traces and (B) current density (pA/pF) recorded from ND7/23 cells expressing OtNav1.8 before and after application of NaTx36. Na+ currents were elicited by a 100-ms depolarization to +20 mV from a holding potential of −80 mV before and after application of NaTx36. Tetrodotoxin (TTX, 500 nM) was added to the bath solution to block endogenous TTX sensitive Na+ currents. Currents decrease in response to samples of NaTx36 ranging from 0.01 to 3.47 µM (n = 5 – 6 cells per concentration). (C) Na+ current values in response to NaTx36 were fit to a Dose-response equation with variable Hill slope to estimate the half-maximum inhibitory concentration (IC50 = 0.68 ± 0.05 µM).

FIGURE 4. Effects of NaTx36 on OtNaV1.8 current-voltage relationship (I-V curves). Cells were pretreated with 500 nM TTX to block TTX-sensitive sodium channels, and currents were elicited by 50-ms depolarizing steps to various voltages ranging from −80 to +40 mV in 5-mV increments. All Na+ currents were normalized to the maximum amplitude of control peak current. (A) Representative I-V curves before and after 100 nM NaTx36. When the holding potential was −80 mV, NaTx36 could inhibit TTX-R Na+ current recorded from ND7/23 cells transfected with recombinant OtNav1.8 (control, n = 5; NaTx36, n = 4). NaTx36 also enhanced activation by hyperpolarizing the membrane potential at which channels begin opening. (B) When the holding potential was changed to −120 mV, inhibition of OtNaV1.8 by 100 nM NaTx36 was substantially reduced (−80 mV, n = 4; −120 mV, n = 3).

FIGURE 5. Effects of NaTx36 on OtNav1.8 steady-state fast and slow inactivation. (A,B) Representative steady-state fast inactivation and slow inactivation curves before and after 100 nM NaTx36 application to ND7/23 cells transfected with recombinant OtNav1.8. Cells were pretreated with 500 nM TTX to block TTX-sensitive sodium channels. All Na+ currents were normalized to the maximum amplitude of control peak current. (A) Using a double-pulse protocol, Na+ currents were induced by a 20-ms depolarizing potential of +30 mV following a 500-ms prepulse at voltages ranging from −120 to +10 mV. Currents were plotted as a fraction of the maximum peak current. NaTx36 shifted the voltage dependence of steady-state fast inactivation to hyperpolarized membrane potentials (control, n = 5 cells; NaTx36, n = 3 cells). (B) Using a standard stimulus protocol, slow inactivation was induced with 5 s pre-pulses ranging from −130 to +10 mV, followed by 10 ms pulses to −80 mV to allow recovery from fast inactivation. A 20 ms test pulse to +30 mV was then used to determine the fraction of current available. Currents were plotted as a fraction of the maximum peak current. NaTx36 shifted the voltage dependence of…

FIGURE 6. Effects of AZ bark scorpion venom on DI inward gating pore currents. Gating pore currents were generated by glycine mutation of the first and second gating charges (two outermost arginine residues) on OtNaV1.8 DI S4 (R215G/R218G). (A–C) Schematic diagram depicting channel central pore current (A), generation of inward gating-pore current (B), and block of inward gating pore current by venom (C). (D) A typical protocol was used to elicit inward gating pore current. Cells held at −80 mV were stimulated by 50-ms hyperpolarizing steps to various potentials that ranged from –200 to +40 mV in 10-mV increments. (E) 100 μg/ml venom completely inhibited central pore current of wild type OtNaV1.8 channels but failed to affect background leak currents. (F) 100 μg/ml venom only partially inhibited central pore currents recorded from OtNaV1.8 mutant channels, suggesting that the double mutation (R215G/R218G) on DI S4 could reduce venom binding affinity towards OtNaV1.8 channels. Venom also depressed inward gating pore currents through the DI voltage sensor domain (VSD). Blue and red traces represent typical leak current (gating pore current) and central pore current at −200 and −10 mV,…

FIGURE 7. Effects of AZ bark scorpion venom on Na+ currents recorded from a DI S4 voltage sensor mutant. (A) Venom inhibits wildtype OtNav1.8 Na+ currents. (B) The first GC arginine (R215) in the DI S4 voltage sensor was exchanged for a glycine (R215G). TTX (500 nM) was added to the bath solution to block TTX-sensitive currents. Na+ currents were elicited by a 50 ms depolarization to +20 mV from a holding potential of −80 mV before and after application of venom (10 μg/ml). Venom inhibited wildtype OtNav1.8, but not the DI S4 voltage sensor mutant (R215G) channels expressed in ND7/23 cells (n = 3).

FIGURE 8. Effects of AZ bark scorpion venom on DII inward gating pore currents. Gating pore currents were generated by glycine mutation of the first and second gating charges (two outermost arginine residues) on OtNaV1.8 DII S4 (R756G/R759G). A typical protocol was used to elicit inward gating pore current (see schematic, Figure 6D). Cells held at −80 mV were stimulated by 50-ms hyperpolarizing steps to various potentials that ranged from –200 to +40 mV in 10-mV increments. As observed for wildtype OtNaV1.8 channels, venom completely inhibited OtNaV1.8 DII S4 R756G/R759G mutant channels, suggesting that the mutations did not affect venom binding affinity. Blue and red traces represent typical leak current (gating pore current) and central pore current at −200 and −10 mV, respectively.

FIGURE 9. Effects of NaTx36 on DI S4 voltage sensor and DII SS2 – S6 pore loop mutants. (A) Representative Na+ current traces were elicited by a 50-ms depolarization to +20 mV from a holding potential of −80 mV before and after application of NaTx36 to ND7/23 cells transfected with either DI or DII mutant OtNav1.8. (B,C) To determine the fraction of current after toxin treatment, Na+ currents were elicited by 50-ms depolarizing steps to various voltages ranging from −80 to +40 mV in 5-mV increments. All Na+ currents were normalized to the maximum amplitude of control peak current. (B) NaTx36 (100 nM) did not inhibit the OtNaV1.8 DI voltage sensor S4 R215G mutant (control, n = 3; NaTx36, n = 3; p = 0.23). (C) In addition, NaTx36 (100 nM) had no effect on the DII SS2 – S6 pore loop Q859E/E862Q mutant (control, n = 5; NaTx36, n = 5; p = 0.08). All cells were pretreated with 500 nM TTX to block TTX-sensitive sodium channels.

FIGURE 10. Effects of DI S4 mutation R215G on steady-state fast inactivation and slow inactivation. (A,B) Representative steady-state fast inactivation and slow inactivation curves before and after 100 nM NaTx36 application to ND7/23 cells transfected with OtNaV1.8 DI S4 first GC R215G mutant channels. Cells were pretreated with 500 nM TTX to block TTX-sensitive sodium channels. All Na+ currents were normalized to the maximum amplitude of control peak current. (A) Steady-state fast inactivation was measured by a standard double-pulse protocol in which sodium currents were elicited by a 20-ms depolarizing potential of +30 mV following a 500-ms prepulse at voltages ranging from −120 to +10 mV. 100 nM NaTx36 did not affect steady-state fast inactivation of the R215G mutant channels (control, n = 8; NaTx36, n = 5). (B) Steady-state slow inactivation was measured with 5 s pre-pulses ranging from −130 to +10 mV, followed by 10 ms pulses to −80 mV to allow recovery from fast inactivation followed by a 20-ms depolarizing potential of +30 mV. 100 nM NaTx36 did not affect steady-state fast inactivation of the R215G mutant channels (control, n = 5 cells; NaTx36, n = 4 cells).

FIGURE 11. Computational modeling of OtNaV1.8 and NaTx36 structures. (A) Representative ribbon structures for the five top models of OtNaV1.8 generated by AlphaFold; dashed lines represent approximate membrane boundaries, and the black arrow points to the DII S5 – SS1 loop. (B) Position of gating charges (GC) within the DI voltage sensor S4 segment of the OtNaV1.8 model. DI S3 – S4 segments from the OtNaV1.8 model are compared with experimental structures of activated and deactivated voltage sensor segments to illustrate that the OtNaV1.8 DI S3 – S4 was modelled in the activated (outward) state. In the activated state, the first GC R215 is exposed to the extracellular solvent. (C) Sequence alignment of NaTx36 with two other scorpion toxins, Lqh3 and AaH2, whose biological actions on voltage-gated sodium channels are well-described; rounded rectangles indicate sequence segments corresponding to alpha-helices, and pentagons indicate beta strands. (D) NaTx36 models generated by AlphaFold and RosettaFold are superimposed onto the experimental structures of scorpion toxins Lqh3 and AaH2.

FIGURE 12. Alignment of NaV1.8 from grasshopper mice, house mice, and humans comparing predicted interaction regions between NaV1.8 and NaTx36. Sequence from grasshopper mouse (OtNaV1.8) DI voltage sensing module (S1 – S2 and S3 – S4) and DII pore module (S5 – SS1, SS2 – S6) was aligned with house mouse (mNav1.8) and human (hNav1.8) sequences to identify motifs uniquely present in OtNaV1.8. The alignment color coding is based on sequence identity (high, green; medium, purple; low, white). Regions of the channel that were identified by docking simulations to interact with the toxin are highlighted with pink dashed segments above the corresponding channel sequence. (A) Sequence alignment DI voltage sensing module; Orange asterisk at the bottom of the alignment highlights the first gating charge within the S4 segment. (B) Sequence alignment corresponding to the second Pore module. The orange bar above the sequence highlights the QVSE motif necessary for NaTx36 inhibitory effect on OtNaV1.8.