AaHII interacts with the rNav1.2a VSD IV paddle motif. (A) Nav channel cartoon embedded in a lipid membrane. Each domain (DI–IV) consists of six transmembrane segments (S1–S6) of which S1–S4 form the VSD and the S5–S6 segments of each domain form the pore. Paddle motif amino acid sequences are shown for VSD II (red) and IV (green). Underlined residues were mutated as reported in Results. (B) Effect of 100 nM AaHII on the rNav1.2a/Kv2.1 VSD IV chimera containing the R1629A/L1630A substitutions. K+ current (left) was elicited by a 300-ms depolarization to −100 mV (tail voltage was −150 mV) after a 500-ms step to −150 mV from a holding voltage of −10 mV (near the Nernst potential for K+). The data show a clear toxin-induced inhibition of the double mutant channel (black, control; green, 100 nM AaHII). Right panel displays normalized tail current voltage–activation relationships of the rNav1.2a/Kv2.1 VSD IV chimera without (open circles) and with the R1629A/L1630A substitutions (closed circles) where tail current amplitude (I/Imax) is plotted against test voltage before (black) and in the presence of 100 nM AaHII (green). Holding voltage for the mutant was −10 mV, followed by a 500-ms hyperpolarizing step to −150 mV to close all channels. Next, 10-mV step depolarizations of 300 ms (V) were trailed by a 300-ms tail voltage step to −150 mV (I). A Boltzmann fit of the obtained data (n = 3; error bars represent mean ± SEM) reveals a shift in midpoint (V1/2) for the double mutant (−105 ± 1 mV; slope, 17.1 ± 1.0) compared with the rNav1.2a/Kv2.1 VSD IV chimera (7 ± 2 mV; slope, 17.3 ± 1.6). Moreover, 100 nM AaHII strongly inhibits the double mutant (apparent KD = 193 ± 42 nM), whereas the rNav1.2a/Kv2.1 VSD IV chimera is influenced less (apparent KD = 1,008 ± 92 nM), with n = 3 for each value (error bars represent mean ± SEM).
