Molecular Determinants of High Affinity Binding of α-Scorpion Toxin and Sea Anemone Toxin in the S3-S4 Extracellular Loop in Domain IV of the Na+ Channel α Subunit

• Published in: The Journal of biological chemistry Vol. 271; no. 27; pp. 15950 - 15962
• Main Authors: Rogers, John C., Qu, Yusheng, Tanada, Timothy N., Scheuer, Todd, Catterall, William A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 05.07.1996
• Subjects: Amino Acid Sequence; Base Sequence; Binding Sites; Binding, Competitive; Brain - metabolism; Cell Line; Cnidarian Venoms - metabolism; DNA Primers; DNA, Complementary; Humans; Kidney; Kinetics; Macromolecular Substances; Membrane Potentials; Models, Structural; Molecular Sequence Data; Mutagenesis, Site-Directed; Point Mutation; Protein Structure, Secondary; Recombinant Fusion Proteins - metabolism; Saxitoxin - metabolism; Scorpion Venoms - metabolism; Sequence Homology, Amino Acid; Sodium Channels - chemistry; Sodium Channels - metabolism; Tetrodotoxin - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.27.15950

α-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 α 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 α subunit were converted to neutral or basic amino acids using site-directed mutagenesis, and altered channels were transiently expressed in tsA-201 cells and tested for 125I-α-scorpion toxin binding. Conversion of Glu1613 at the extracellular end of transmembrane segment IVS3 to Arg or His blocked measurable α-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 α-scorpion toxin or sea anemone toxin. Moreover, chimeric Na+ channels in which amino acid residues at the extracellular end of segment IVS3 of the α subunit of cardiac Na+ channels were substituted into the type IIa channel sequence had reduced affinity for α-scorpion toxin characteristic of cardiac Na+ channels. Electrophysiological analysis showed that E1613R has 62- and 82-fold lower affinities for α-scorpion and sea anemone toxins, respectively. Dissociation of α-scorpion toxin is substantially accelerated at all potentials compared to wild-type channels. α-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 α-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 α-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.