JZTX-V Targets the Voltage Sensor in Kv4.2 to Inhibit I(to) Potassium Channels in Cardiomyocytes

Kv4 potassium channels are responsible for transient outward K(+) currents in the cardiac action potential (AP). Previous experiments by our group demonstrated that Jingzhaotoxin-V (JZTX-V) selectively inhibits A-type potassium channels. However, the specific effects of JZTX-V on the transient outward (I(to)) current of cardiomyocytes and underlying mechanism of action remain unclear. In the current study, 100 nM JZTX-V effectively inhibited the I(to) current and extended the action potential duration (APD) of neonatal rat ventricular myocytes (NRVM). We further analyzed the effects of JZTX-V on Kv4.2, a cloned channel believed to underlie the I(to) current in rat cardiomyocytes. JZTX-V inhibited the Kv4.2 current with a half-maximal inhibitory concentration (IC(50)) of 13 ± 1.7 nM. To establish the molecular mechanism underlying the inhibitory action of JZTX-V on Kv4.2, we performed alanine scanning mutagenesis of Kv4.2 and JZTX-V and assessed the effects of the mutations on binding activities of the proteins. Interestingly, the Kv4.2 mutations V285A, F289A, and V290A reduced the affinity for JZTX-V while I275A and L277A increased the affinity for JZTX-V. Moreover, mutation of positively charged residues (R20 and K22) of JZTX-V and the hydrophobic patch (formed by W5, M6, and W7) led to a significant reduction in toxin sensitivity, indicating that the hydrophobic patch and electrostatic interactions played key roles in the binding of JZTX-V with Kv4.2. Data from our study have shed light on the specific roles and molecular mechanisms of JZTX-V in the regulation of I(to) potassium channels and supported its utility as a potential novel antiarrhythmic drug.