Molecular Mechanisms For Jingzhaotoxin-Ⅰ And Ⅲ Inhibiting Fast Inactivation Of Na_v1.5

Previous work has shown that Jingzhaotoxin-Ⅲ (jingzhaotoxin-Ⅲ, JZTX-Ⅲ) could selectively inhibit cardiac sodium channel Nav1.5 by binding to DⅡ S3-S4 loop and trapping DⅡ S4 voltage sensor. The toxin belongs to the family of receptor site 4 toxins. However, although mutations of JZTX-Ⅲ decreased toxin binding affinity for hNav1.5 by 100-fold, mutations of hNav1.5 only decreased toxin binding affinity by 18-fold, suggesting that JZTX-Ⅲ might have multiple binding sites on Nav1.5. In this study, we found that in high concentrations (e.g.1 or 5μM), JZTX-Ⅲ not only inhibited channel activation but also slowed down fast inactivation of cardiac sodium channel Nav1.5. A positively charged residue at position 800(R800) is proved to play a crucial role in Nav1.5 interacting with JZTX-Ⅲ. The mutation R800A could reduce JZTX-Ⅲ binding affinity by 18-fold. On the Nav1.5 R800A mutant channel, inhibition of fast inactivation by high concentrations of JZTX-Ⅲ was detected, too. These results indicated that JZTX-Ⅲ might interact with DIV because DIVS4 is responsible for fast inactivation. Furthermore, site-directed mutagenesis analysis indicated that Ala-mutation of two residues (Asn1612 and Lsy1613) in DIV S3-S4 linker evidently reduced the ability of JZTX-Ⅲ to slow down fast inactivation. On the contrary, the reverse mutations E1589Q and T1590K in hNav1.7 greatly increased the sensitivity of DIV to JZTX-Ⅲ. Therefore, our results indicated that JZTX-Ⅲ not only docked at the hNav1.5 DⅡ S3-S4 linker, but also could bind to DIVS3-S4 linker. Our data also revealed a noval mechanism for JZTX-Ⅲ interacting with Nav1.5.Jingzhaotoxin-I(jingzhaotoxin-I, JZTX-I) is an a-like toxin that prefers to inhibit cardiac sodium channels inactivation. When hNav1.5 channels were expressed in HEK293T cells, the IC50 value for JZTX-I inhibiting fast inactivation was 339.8±0.04 nM. Site-directed mutagenesis analysis indicated that an acidic residue (Asp 1609) and a basic residue (Lysl613) played important roles in the binding of JZTX-I to hNav1.5 DIV S3-S4 linker. Mutations D1609A and K1613A decreased toxin affinity by 29.5-and 4.2-fold, respectively. Our results confirmed that JZTX-I belonged to the family of receptor site-3 toxins. JZTX-I might be a valuable tool to investigate the function and structure relationship of cardiac sodium channel DIV S4 voltage sensor.