Molecular Mechanism Of The Tarantula Toxin Jingzhaotoxin-Ⅱ Interacting With Voltage-gated Sodium Channel Subtype Nav1.5

Jingzhaotoxin-II(JZTX-II) is a 32-residue peptide toxin isolated from Chinese tarantula Chilobrachys jingzhao venom with a molecular weight of 3561.13 Da. It contains six cysteines which form three disulfide bond and belongs to the ICK motif. The amino acid sequence of JZTX-II was determined to be GCGTMWSPCSTE-KPCCDNFSCQPAIKWCIWSP by Edman degradation.In our previous study, we showed that JZTX-II preferentially inhibits rapid inactivation of TTX-resistant(TTX-R) VGSC on cardiac myocytes and has weaker effect to subtypes on DRG neurons with almost no effect to those on brain. However,the effects of JZTX-II on specific VGSC subtypes are unclear and little is known about the molecular interaction mechanism of them. Among the tested four VGSC subtypes,h Nav1.5 was the most sensitive to JZTX-II(EC50 = 125 ± 4 n M),which was 10 fold sensitive compared with r Nav1.3,and the toxin had very low inhibitory effect on r Nav1.4 and h Nav.7. The related experimental results indicated that JZTX-II had little or no effect on steady-state inactivation of the channel,despite it has capacity to inhibit the rapid inactivation of h Nav1.5. The toxin treatment(0.5 μΜ) resulted in a 10 m V hyperpolarization shift of the maximum activation voltage without evidently altering the initial activation and reversal potentials. These data indicated that it might be easier to activate Nav1.5 current in the presence of JZTX-II by a weak depolarization voltage and it didn’t change the ion selectivity. Moreover,JZTX-II increased the rate of recovery of Nav1.5 channels,which lead to decrease of the amount of channel inactivation and caused a shorter transition from the inactivation to closed state. The results also showed that JZTX-II dissociation from the toxin-Nav1.5 complex was voltage-dependent,with more rapid dissociation during stronger depolarization. However,toxin rebinding to the resting state of sodium channels was voltage-independent,different repolarization pulses didn’t have obvious effect. The analysis of chimeric channels showed that the Nav1.5 domain IV(DIV) voltage-sensor domain(VSD) was critical for JZTX-II binding to Nav1.5 and site-directed mutagenesis analysis indicated that some mutations located in S1-S2 and S3-S4 extracellular loops of Nav1.5 DIV(T1548A,D1549 A,D1609A,Y1614 A,F1615A,S1617A) additively reduced the toxin sensitivity of Nav1.5. Our data identify the molecular mechanism of JZTX-II inhibiting the fast inactivation of Nav1.5,similar to scorpion α-toxins,involving binding to neurotoxin receptor site 3.