Purification and Characterization of JZTx-14, a Potent Antagonist of Mammalian and Prokaryotic Voltage-Gated Sodium Channels

Abstract

Exploring the interaction of ligands with voltage-gated sodium channels (Na_Vs) has advanced our understanding of their pharmacology. Herein, we report the purification and characterization of a novel non-selective mammalian and bacterial Na_Vs toxin, JZTx-14, from the venom of the spider Chilobrachys jingzhao. This toxin potently inhibited the peak currents of mammalian Na_V1.2⁻1.8 channels and the bacterial NaChBac channel with low IC₅₀ values (<1 µM), and it mainly inhibited the fast inactivation of the Na_V1.9 channel. Analysis of Na_V1.5/Na_V1.9 chimeric channel showed that the Na_V1.5 domain II S3⁻4 loop is involved in toxin association. Kinetics data obtained from studying toxin⁻Na_V1.2 channel interaction showed that JZTx-14 was a gating modifier that possibly trapped the channel in resting state; however, it differed from site 4 toxin HNTx-III by irreversibly blocking Na_V currents and showing state-independent binding with the channel. JZTx-14 might stably bind to a conserved toxin pocket deep within the Na_V1.2⁻1.8 domain II voltage sensor regardless of channel conformation change, and its effect on Na_Vs requires the toxin to trap the S3⁻4 loop in its resting state. For the NaChBac channel, JZTx-14 positively shifted its conductance-voltage (G⁻V) and steady-state inactivation relationships. An alanine scan analysis of the NaChBac S3⁻4 loop revealed that the 108th phenylalanine (F108) was the key residue determining the JZTx-14⁻NaChBac interaction. In summary, this study provided JZTx-14 with potent but promiscuous inhibitory activity on both the ancestor bacterial Na_Vs and the highly evolved descendant mammalian Na_Vs, and it is a useful probe to understand the pharmacology of Na_Vs.

Keywords: NaChBac; mammalian NaVs; peptide toxin; pharmacology.

Figure 1

Purification and characterization of JZTx-14. (A) RP-HPLC purification profile of Chilobrachys jingzhao venom. The peaks containing JZTx-14, JZTx-2, and JZTx-27 are labeled by asterisk and arrows, respectively. (B) JZTx-14 was purified to homogeneity by analytical RP-HPLC. (C) MALDI-TOF MS analysis of purified JZTx-14. The average molecular mass of JZTx-14 was determined as 3422.17 Da (M + H⁺). (D) The mature peptide sequences of JZTx-14 and Jingzhaotoxin F4-32.60. The C-terminal amidation signals G in Jingzhaotoxin F4-32.60 and GR in JZTx-14 were boxed. (E) Chymotrypsin digestion combined with LC-MS analysis determined the toxin to be JZTx-14. The MS/MS spectrum of the C-terminal fragment is shown (sequence: TEICIL). (F) Sequence alignment of JZTx-14 with similar toxins in database. The bacterial Na_V toxin JZTx-27 is included.

Figure 2

Activity of JZTx-14 on mammalian Na_Vs. (A–H) Representative current traces showing that JZTx-14 blocked the currents of Na_V1.2–1.8 and slowed the fast inactivation of Na_V1.9 (black traces: control; red traces: after toxin application; blue traces: after 2- to 3-min bath solution perfusion). The insets in (A) and (B) show that the toxin slowed the fast inactivation of Na_V1.2 and Na_V1.3. (I) Dose–response curves for JZTx-14 inhibiting Na_V1.2–1.8 currents. The IC₅₀ values were 194.0 ± 10.3 nM, 426.3 ± 48.8 nM, 290.1 ± 23.2 nM, 478.0 ± 32.0 nM, 158.6 ± 29.4 nM, 188.9 ± 46.3 nM, and 824.0 ± 68.7 nM for Na_V1.2, Na_V1.3, Na_V1.4, Na_V1.5, Na_V1.6, Na_V1.7, and Na_V1.8, respectively (n = 4–6). (J) The comparison of JZTx-14 affinities to Na_V1.2–1.8 channels. The IC₅₀ value of JZTx-14 for each Na_V subtype was normalized to that of the Na_V1.6 channel. (K) The effect of 2 µM JZTx-14 on the Na_V1.5/1.9DIIS3–4 chimeric channel constructed by substituting the Na_V1.5 domain II S3–4 loop with that of Na_V1.9 (n = 4).

Figure 3

Kinetics of JZTx-14 interacting with Na_V1.2. (A) Representative Na_V1.2 current traces before and after a subsaturating concentration (200 nM) of JZTx-14 treatment. Currents were elicited by a cluster of depolarizations from −100 mV to +100 mV (in 10-mV increments) from the holding potential of −100 mV. For simplicity, currents in 20-mV increments were shown. (B) I–V relationships of the Na_V1.2 channel before and after 200 nM JZTx-14 treatment (black and red solid lines). Currents after toxin treatment were normalized to 1 (blue dashed line) to compare the I–V shape with that before toxin application (n = 10). (C,D) Representative Na_V1.2 current traces before and after saturating concentration (1 µM) of JZTx-14 or TTX treatment. Currents were elicited as described in Figure 3A. (E) I–V relationship of the Na_V1.2 channel before and after 1 µM JZTx-14 or TTX treatment. TTX and JZTx-14 almost fully inhibited Na_V1.2 inward currents, and TTX, but not JZTx-14, fully blocked Na_V1.2 outward currents (n = 5). (F–H) The protocol in Figure 3F was used to measure toxin dissociation from Na_V1.2 in response to a +150 mV/500 ms strong depolarization by testing the currents in test pulse 2 (t2), and 1 µM JZTx-14, HNTX-III, or TTX were used to fully block Na_V1.2 currents in test pulse 1 (t1). A large current recovery was observed in t2 in the HNTX-III group, but not in the JZTx-14 or TTX groups (n = 3–5).

Figure 4

Activity of JZTx-14 on bacterial Na_Vs. (A,B) Representative traces showing the inhibitory effect of JZTx-14 on the NaChBac and NsvBa channels. (C) Dose–response curves for JZTx-14 inhibiting the NaChBac and NsvBa currents. The IC₅₀ values were 320 ± 38 nM and 1400 ± 200 nM for NaChBac and NsvBa, respectively (n = 5–7). (D,E) The inhibitory effect of 1 μM JZTx-14 on NaVPz and NaVSp currents (n = 3).

Figure 5

Kinetics of JZTx-14 interacting with NaChBac. (A) Representative traces showing that 300 nM JZTx-14 inhibited NaChBac currents at all of the voltages tested. Currents were elicited by depolarizations from −100 mV to +100 mV from the holding potential of −100 mV (in 10-mV increments). (B) I–V relationships of NaChBac before and after 300 nM JZTx-14 treatment. Currents were normalized to that before toxin application (red solid traces). The blue dashed line shows the normalization of the currents after toxin treatment to 1 (n = 5). (C,D) Steady-state activation (G–V) and steady-state inactivation (SSI) relationships of NaChBac before and after 300 nM JZTx-14 treatment (V_a = −33.8 ± 0.41 mV and −23.7 ± 0.98 mV, K_a = 6.2 ± 0.35 mV and 9.9 ± 0.87 mV, for control and toxin treated channels, respectively, n = 5; V_h = –41.3 ± 0.55 mV and –30.9 ± 0.56 mV, K_h = −7.8 ± 0.48 mV and −7.7 ± 0.48 mV, for control and toxin treated channels, respectively; n = 5). The curves were fitted by Equation (1).

Figure 6

Effect of JZTx-14 on NaChBac mutants. (A) Sequence alignment of NaChBac with several bacterial Na_Vs determined the S3–4 extracellular loops. The mutation sites are labeled by red arrows. (B–G) Representative traces showing the inhibitory effect of 1 µM JZTx-14 on wild-type NaChBac and NaChBac mutants. (H) Dose–response curves for JZTx-14 inhibiting the currents of NaChBac mutants. The IC₅₀ values were 320.0 ± 38.0 nM, 832.6 ± 42.1 nM, 653.3 ± 92.1 nM, 809.8 ± 87.5 nM, 3472.3 ± 195.7 nM, and 1367.3 ± 129.3 nM for wild-type NaChBac, F103A, G105A, Q107A, F108A, and V109A, respectively (n = 5–7). (I) The bars show the fold changes of the IC₅₀ values of mutant channels when compared with wild-type NaChBac. (J,K) Representative traces showing the inhibitory effect of 300 nM JZTx-27 on wild-type NaChBac and F108A mutant channel. (L) Dose–response curves for JZTx-27 inhibiting the currents of the wild-type NaChBac and F108A mutant channels (n = 6–7).