Wild-type and mutant Nav1.2 channels were transiently expressed in tsA-201 cells, and binding affinity for Css IV toxin was measured as described under “Experimental Procedures”. a, competitive binding curves for inhibition of 125I-Css IV binding to transiently expressed channels by unlabeled Css IV for transiently expressed Nav1.2 (filled circles, Kd (wt) =0.12 ± 0.01 nm) and E779Q channels (open circles, Kd (E779Q) = 0.46 ± 0.04 nm). b, competitive binding curves for inhibition of 125I-Css IV binding to transiently expressed sodium channels by unlabeled Css IV for transiently expressed NaV1.2 (filled circles), E837Q (open squares, Kd (E837Q) = 0.56 ± 0.12 nm), and L840C channels (open triangles, Kd (L840C) = 0.68 ± 0.04 nm). c, bar graph illustrating Kd values for all mutants. The dotted line denotes the Kd value for wild type.