Discovery of a Novel Nav1.7 Inhibitor From Cyriopagopus albostriatus Venom With Potent Analgesic Efficacy

Abstract

Spider venoms contain a vast array of bioactive peptides targeting ion channels. A large number of peptides have high potency and selectivity toward sodium channels. Na_v1.7 contributes to action potential generation and propagation and participates in pain signaling pathway. In this study, we describe the identification of μ-TRTX-Ca2a (Ca2a), a novel 35-residue peptide from the venom of Vietnam spider Cyriopagopus albostriatus (C. albostriatus) that potently inhibits Na_v1.7 (IC₅₀ = 98.1 ± 3.3 nM) with high selectivity against skeletal muscle isoform Na_v1.4 (IC₅₀ > 10 μM) and cardiac muscle isoform Na_v1.5 (IC₅₀ > 10 μM). Ca2a did not significantly alter the voltage-dependent activation or fast inactivation of Na_v1.7, but it hyperpolarized the slow inactivation. Site-directed mutagenesis analysis indicated that Ca2a bound with Na_v1.7 at the extracellular S3-S4 linker of domain II. Meanwhile, Ca2a dose-dependently attenuated pain behaviors in rodent models of formalin-induced paw licking, hot plate test, and acetic acid-induced writhing. This study indicates that Ca2a is a potential lead molecule for drug development of novel analgesics.

Keywords: Nav1.7; analgesic activity; electrophysiology; peptide toxin; sodium channel; tarantula spider.

FIGURE 1

Identification of μ-TRTX-Ca2a from the venom of spider C. albostriatus. (A) Isolation of native Ca2a from the pooled protein fraction by a C18 RP-HPLC column. (B) Ca2a was purified to homogeneity by analytical RP-HPLC. (C) MALDI-TOF MS analysis showing a single predominant mass of 3905.08 Da. (D) Cysteines shaded in cyan formed disulfide bonds. Sequence alignment of Ca2a with related toxins. N-terminal sequencing analysis revealed a peptide with 35 residues containing 6 cysteines.

FIGURE 2

Effects of μ-TRTX-Ca2a on Na_v1.2–Na_v1.9 channels. (A–H) Representative Na_v1.2–Na_v1.9 current traces before (black) and after (red) addition of Ca2a. Ca2a at 1 μM inhibited Na_v1.2–Na_v1.3, Na_v1.6, and Na_v1.7. 10 μM Ca2a showed no obvious effect on Na_v1.4–Na_v1.5 or Na_v1.8–Na_v1.9 current. Inset above panel (A) shows the pulse protocol for recording Na_v1.2–Na_v1.7 channel currents. Inset above panel (G) shows the pulse protocol for recording Na_v1.8 channel current. Inset above panel (H) shows the pulse protocol for recording Na_v1.9 channel current. (I) Concentration-response curves of Ca2a at Na_v1.2–Na_v1.3, Na_v1.6, and Na_v1.7 assessed by whole-cell patch-clamp experiments. Data are mean ± SEM, with n = 4–7 cells per data point.

FIGURE 3

Effects of μ-TRTX-Ca2a on the voltage dependence of Na_v1.7 activation and inactivation gating. (A) Representative current traces of Na_v1.7 channel inhibited by 0.2 μM Ca2a. (B) I–V curves before (black) and after (red) treatment of Ca2a (n = 10). Inset shows the pulse protocol for measuring current–voltage (I–V) relationships. (C) G–V curves before (black) and after (red) treatment of Ca2a (n = 10). (D) Voltage-dependence of steady-state fast inactivation curves before (black) and (red) after treatment of Ca2a (n = 10). Inset shows the pulse protocol for measuring steady-state fast inactivation.

FIGURE 4

Na_v1.7 kinetic parameters affected by the addition of μ-TRTX-Ca2a. (A) Voltage-dependence of fast inactivation time constants of Na_v1.7 before (black) and after (red) addition of 0.2 μM Ca2a. (B) Kinetics of current recovery from fast inactivation of Na_v1.7 before (black) and after (red) addition of 0.2 μM Ca2a at –100 mV. Inset shows the pulse protocol for measuring recovery from fast-inactivation. (C) Voltage-dependence of steady-state slow inactivation of Na_v1.7 before (black) and after (red) addition of 0.2 μM Ca2a. Inset shows the pulse protocol for measuring steady-state slow inactivation. (D) Time course of dissociation of 1 μM Ca2a from Na_v1.7 at 100, 80, and 60 mV. Data are mean ± SEM, with n = 6–11 cells per data point. Inset shows the pulse protocol for measuring the rate of toxin dissociation.

FIGURE 5

Effect of Ca2a on WT and mutant Na_v1.4 expressed in HEK293T cells. (A) Amino acids of potential binding sites in the sequence were shaded in cyan. Sequence alignment of DIIS3-S4 of Na_v1.2–Na_v1.7. (B) Dose-response curves of Q657E and N655D/Q657E. (C–F) Representative current traces for WT and mutant channels (N655D, Q657E, and N655D/Q657E) inhibited by 1 μM Ca2a. Data are mean ± SEM, with n = 4–5 cells per data point.

FIGURE 6

Effect of Ca2a on WT and mutant Na_v1.7 expressed in HEK293T cells. (A) Representative current traces for WT inhibited by 1 μM Ca2a. (B–D) Representative current traces for mutant channels (D816N, E818Q, and D816N/E818Q) inhibited by 1 μM Ca2a. (E) Dose-response curves of Na_v1.7 and D816N. Data are mean ± SEM, with n = 5 cells per data point.

FIGURE 7

Analgesic effect of Ca2a. (A) Time course of the antinociceptive effect of Ca2a in the formalin test. Evaluation of the antinociceptive effect of Ca2a on phase I (B) or phase II (C). (D) Time course of the antinociceptive effect of Ca2a in hot plate test. (E) Analgesic effect was assessed after 30 min of Ca2a injection. (F) The antinociceptive effect of Ca2a in the abdominal constriction test. The data are shown as mean ± SEM, with n = 6–8; ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 vs. vehicle.