Point mutations at the local anesthetic receptor site modulate the state-dependent block of rat Nav1.4 sodium channels by pyrazoline-type insecticides

Pyrazoline-type insecticides (PTIs) selectively block sodium channels at membrane potentials that promote slow sodium channel inactivation and are proposed to interact with a site that overlaps the local anesthetic (LA) receptor site. Mutagenesis studies identified two amino acid residues in the S6 segment of homology domain IV (Phe-1579 and Tyr-1586 in the rat Nav1.4 sodium channel) as principal elements of the LA receptor. To test the hypothesis that PTIs bind to the LA receptor, we constructed mutated Nav1.4/F1579A and Nav1.4/Y1586A cDNAs, expressed native and mutated channels in Xenopus oocytes, and examined the effects of these mutations on channel block by three PTIs (indoxacarb, its bioactivation product DCJW, and RH3421) by two-electrode voltage clamp. DCJW and RH3421 had no effect on Nav1.4 channels held at −120 mV but caused a slowly developing block upon depolarization to −30 mV. Estimated IC50 values following 15 min of exposure were 1 and 4 μM for DCJW and RH3421, respectively. Indoxacarb failed to block Nav1.4 channels under all experimental conditions. Sensitivity to block by DCJW and RH3421 at −30 mV was significantly reduced in Nav1.4/F1579A channels, a finding that is consistent with the impact of this mutation on drug binding. In contrast to its effect on drug binding, the Y1586A mutation increased the sensitivity of Nav1.4 channels held at −30 mV to all three compounds, conferring modest sensitivity to indoxacarb and increasing sensitivity to DCJW and RH3421 by 58- and 16-fold, respectively. These results provide direct evidence for the action of PTIs at the LA receptor.