Molecular determinants of high affinity binding of alpha-scorpion toxin and sea anemone toxin in the S3-S4 extracellular loop in domain IV of the Na+ channel alpha subunit

alpha-Scorpion toxins and sea anemone toxins bind to a common extracellular site on the Na+ channel and inhibit fast inactivation, Basic amino acids of the toxins and domains I and IV of the Na+ channel alpha subunit have been previously implicated in toxin binding, To identify acidic residues required for toxin binding, extracellular acidic amino acids in domains I and IV of the type IIa Na+ channel alpha subunit mere converted to neutral or basic amino acids using site-directed mutagenesis, and altered channels were transiently expressed in tsA-201 cells and tested for I-125-alpha scorpion toxin binding, Conversion of Glu(1613) at the extracellular end of transmembrane segment IVS3 to Arg or His blocked measurable alpha-scorpion toxin binding, but did not affect the level of expression or saxitoxin binding affinity, Conversion of individual residues in the IVS3-S4 extracellular loop to differently charged residues or to Ala identified seven additional residues whose mutation caused significant effects on binding of alpha-scorpion toxin or sea anemone toxin, Moreover, chimeric Na+ channels in which amino acid residues at the extracellular end of segment IVS3 of the alpha subunit of cardiac Na+ channels were substituted into the type IIa channel sequence had reduced affinity for alpha-scorpion toxin characteristic of cardiac Na+ channels, Electrophysiological analysis showed that E1613R has 62- and 82-fold lower affinities for alpha-scorpion and sea anemone toxins, respectively, Dissociation of alpha-scorpion toxin is substantially accelerated at all potentials compared to wild-type channels, alpha-Scorpion toxin binding to wild type and E1613R had similar voltage dependence, which was slightly more positive and steeper than the voltage dependence of steady-state inactivation, These results indicate that nonidentical amino acids of the IVS3-S4 loop participate in alpha-scorpion toxin and sea anemone toxin binding to overlapping sites and that neighboring amino acid residues in the IVS3 segment contribute to the difference in alpha-scorpion toxin binding affinity between cardiac and neuronal Na+ channels, The results also support the hypothesis that this region of the Na channel is important for coupling channel activation to fast inactivation.