Selective Closed-State Nav1.7 Blocker JZTX-34 Exhibits Analgesic Effects against Pain

Figure 1. Effects of synthetic JZTX-34 on TTX-S and TTX-R sodium channels from rat DRG neurons. Inward currents traces were induced by a 50 ms depolarizing potential to −10 mV from a holding potential of −80 mV every 5 s. (A) Effect of 1μM synthetic JZTX-34 on TTX-R currents. (B) Effect of 1 μM synthetic JZTX-34 on TTX-S currents. (C) Dose-dependent inhibition of synthetic JZTX-34 on TTX-S sodium channels (n = 5). Data points (mean ± S.E.) were fitted according to the standard Hill equation.

Figure 2. Selectivity of JZTX-34 on sodium channel subtypes. All inward sodium channels were elicited by a 50 ms depolarizing potential to −10 mV from a holding potential of −80 mV every 5 s. One μM JZTX-34 was added to rNav1.3. (A) hNav1.7. (B,C) Dose-dependent inhibition of JZTX-34 on rNav1, rNav3, and hNav1.7 (n = 5). Data points (mean ± S.E.) were fitted according to the standard Hill equation.

Figure 3. JZTX-34 inhibited sodium channel subtypes Nav1.7 by binding to DII S3-S4. (A) Inhibition of 1 μM and 10 μM toxins on chimeric Nav1.5 (Nav1.7-DII-S3-S4). (B) Inhibition of 1 and 10 μM toxins on chimeric currents of Nav1.7 (Nav1.5-DIV-S3-S4) sodium currents. (C) Dose-dependent inhibition of synthetic JZTX-34 on Nav1.7 (Nav1.5-DIV-S3-S4) and (Nav1.7-DII-S3-S4) (n = 5). Data (mean ± S.E.) were fitted according to the standard Hill equation.

Figure 4. Effects of JZTX-34 on mutants of Nav1.7 and Nav1.5. (A) Amino acid sequence alignment of DII S3-S4 linker of Nav1.5 and Nav1.7 and schematic diagram of sodium channel α subunit. The positions of amino acids of interest are shaded in bold black. (B) Inhibition of WT Nav1.7, WT Nav1.5, Nav1.7 (816R) and Nav1.5 (R800D) by 1 µM JZTX-34. (C) Dose-dependent inhibition of JZTX-34 on Nav1.7 (D816R) and Nav1.5 (R800D) (n = 5). Data points (mean ± S.E.) were fitted according to the standard Hill equation.

Figure 5. JZTX-34 traps the domain II voltage sensor in closed configuration. (A) Nav1.7 current traces were elicited by a protocol as following. First, an inward sodium currents were elicited by a 50 ms depolarizing potential to 0 mV from a holding potential of −80 mV every 5 s. Second, 10 μM JZTX-34 were added to bath solution. Finally, the same pulse protocol was applied to elicit inward currents after a 5 min toxin treatment. (B) The current traces 0, 15 ms, 100 ms, and 5 min after toxin treatment are shown, which were elicited by a protocol as shown in C. (C) This pulse protocol was included three phases and applied every 5 s. In the first pulse, a 15 ms moderate depolarizing potential to 0 mV from a holding potential of −80 mV was used to induce sodium currents. After 61.2 ms of first pulse, a 1 ms strong conditioning depolarizing potential to 50 mV from a holding potential of −80 mV was applied. In the third phase, a second period of 61.2 ms at the holding potential of −80 mV, a 15ms test pulse to −65 mV was given again. (D) The current traces after toxin treatment were elicited by the third pulse (black arrow) of protocol C.

Figure 6. Effects of JZTX-34 on pain. (A) Effects of JZTX-34 on the early phase (0–5 min) and late phase (15–30 min) of formalin-induced paw licking response in mice. (B) Effects of JZTX-34 on the acetic acid-induced writhing response in mice. (C)Effects of JZTX-34 on the hot plate test in mice. All data points are shown as mean ± S.E. Six animals were used for each separate group experiment. * p < 0.05, ** p < 0.01 significantly different results compared to the saline group.