Mapping the receptor site for alpha-scorpion toxins on a Na+ channel voltage sensor

Abstract

The α-scorpions toxins bind to the resting state of Na(+) channels and inhibit fast inactivation by interaction with a receptor site formed by domains I and IV. Mutants T1560A, F1610A, and E1613A in domain IV had lower affinities for Leiurus quinquestriatus hebraeus toxin II (LqhII), and mutant E1613R had ~73-fold lower affinity. Toxin dissociation was accelerated by depolarization and increased by these mutations, whereas association rates at negative membrane potentials were not changed. These results indicate that Thr1560 in the S1-S2 loop, Phe1610 in the S3 segment, and Glu1613 in the S3-S4 loop in domain IV participate in toxin binding. T393A in the SS2-S6 loop in domain I also had lower affinity for LqhII, indicating that this extracellular loop may form a secondary component of the receptor site. Analysis with the Rosetta-Membrane algorithm resulted in a model of LqhII binding to the voltage sensor in a resting state, in which amino acid residues in an extracellular cleft formed by the S1-S2 and S3-S4 loops in domain IV interact with two faces of the wedge-shaped LqhII molecule. The conserved gating charges in the S4 segment are in an inward position and form ion pairs with negatively charged amino acid residues in the S2 and S3 segments of the voltage sensor. This model defines the structure of the resting state of a voltage sensor of Na(+) channels and reveals its mode of interaction with a gating modifier toxin.

Fig. 1.

Electrophysiological effect of LqhII on WT and mutant Na_V1.2 channels. (A, and C–F). Normalized voltage-clamp current traces from tsA-201 cells expressing WT and mutant channels in the absence (Control, black) and in the presence of 1 nM (red), 10 nM (green), or 100 nM LqhII (blue). Cells expressing Na_V1.2a channel were held at −100 mV and Na currents were elicited with a 30-ms step to 0 mV. (A) WT, (C) E1613A, (D) E1613R, (E) F1610A. (F) T1560A. (B and G–H). Concentration-response relations for LqhII block of fast inactivation from cells expressing WT and mutant channels. (B) WT (K_d = 0.47 ± 0.05 nM, n = 3–8). (G) E1613A (K_d = 1.8 ± 0.4 nM, n = 3–5) and E1613R (K_d = 34.3 ± 5.7 nM, n = 3–4). (H) F1610A (K_d = 5.0 ± 1.8 nM, n = 3–6) and T1560A (K_d = 2.8 ± 0.8 nM, n = 4–8).

Fig. 2.

Voltage-dependent dissociation rates of LqhII. (A) Traces demonstrating time-dependent dissociation of LqhII at 100 mV. Cells expressing WT Na_V1.2 channels were incubated in 100 nM LqhII for 6 min at a holding potential of −100 mV to allow binding. The rate of toxin dissociation was determined with the illustrated pulse paradigm by stepping to a depolarizing pulse of 100, 80, or 60 mV, for 1 to 1,024 ms, returning to −100 mV for 20 ms to allow recovery from fast inactivation and then assessing the effect of the depolarizing pulse with a 10-ms test pulse to 0 mV (test). (B) Time course of dissociation of 100 nM LqhII from cells expressing WT channels at 100 mV (τ = 95 ± 3 ms, n = 3), 80 mV (τ = 135 ± 17 ms, n = 3), and 60 mV (τ = 289 ± 53 ms, n = 3). Time constants were determined by fits of monoexponential functions to the data. (C and D) Time constants of dissociation as a function of potential for WT, E1613A, F1610A, and T1560A channels. (C) 100 nM LqhII. (D) 30 nM LqhII.

Fig. 3.

Rates of LqhII association after repolarization. (A) Current traces demonstrating time-dependent reassociation of LqhII at −100 mV. Cells expressing WT channels were incubated in 100 nM LqhII. Toxin was dissociated from the channel using a 200-ms depolarization to 100 mV. The rate of toxin association was measured by hyperpolarizing to −120, −100, or −80 mV for increasing times. The effect of repolarization on toxin action was assessed with a 10-ms test pulse to 0 mV (test). (B) The time course of association of 100 nM LqhII from cells expressing WT channel was determined at −120 mV (τ = 1.6 ± 0.01 s, n = 3), −100 mV (τ = 1.2 ± 0.2 s, n = 3), and −80 mV (τ = 1.2 ± 0.3 s, n = 3). (C and D) Time constants of association as a function of voltage for WT, E1613A, F1610A, and T1560A channels. (C) 100 nM LqhII. (D) 30 nM LqhII.

Fig. 4.

Characterization of mutation T393A in domain ISSII-S6. (A) Normalized current traces during steps to 0 mV from cells expressing T393A channels in the absence (control) and in the presence of 1 nM and 100 nM LqhII. (B) Concentration-response relations for LqhII removal of fast inactivation in cells expressing T393A channels (K_d = 1.6 ± 0.5 nM, n = 2–5). (C) Time constants for LqhII dissociation for T393A and WT channels as a function of potential in the presence of 30 and 100 nM LqhII (n = 3). (D) Time constants of association for T393A and WT channels in the presence of 30 or 100 nM LqhII (n = 3).

Fig. 5.

Summary of effects of mutations in domains I and IV on LqhII affinity. Fold-changes in K_d measured either by displacement of bound ¹²⁵I-LqTxV with unlabeled LqTxV adapted from our previous work (open bars) (14) or fold-changes in K_d, as determined using electrophysiology experiments described in Results (filled bars, n = 2–13). Different substitutions for E1613 are shown as stacked bars of different colors. Ala and Gly residues (A396, A397, G398, G1608, and A1612) were not studied. R395Q and R395H gave no Na⁺ current.

Fig. 6.

Full atom and molecular surface representation of LqhII binding to the voltage-sensing domain IV of Na_V1.2. Segments S1 through S4 of the voltage-sensing domain colored individually and labeled. (A and B) Side view of the structural model with the voltage-sensing domain segments S1 and S4 on the front. (C and D) Side view of the structural model with the voltage-sensing domain segments and with S2 and S3 on the front (rotated 180° when viewed from the extracellular side of the membrane compared with orientation shown in A and B). Side chains of key residues for LqhII-Na_V1.2 interaction are shown in space-filling representation and all other side chains shown in stick representation. A probe radius of 1.4 Å was used to scan the molecular surface of each structural model. This figure was generated using Chimera (43).