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 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-alpha-scorpion toxin binding. Conversion of Glu1613 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.
Rogers
J C
JC
Department of Pharmacology, University of Washington, Seattle, Washington 98195, USA.
Qu
Y
Y
Tanada
T N
TN
Scheuer
T
T
Catterall
W A
WA
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D003064:Cnidarian Venoms / Q000378:metabolism*; D017931:DNA Primers; D018076:DNA, Complementary; D006801:Humans; D007668:Kidney; D007700:Kinetics; D046911:Macromolecular Substances; D008564:Membrane Potentials; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012530:Saxitoxin / Q000378:metabolism; D012604:Scorpion Venoms / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015222:Sodium Channels / Q000737:chemistry* / Q000378:metabolism*; D013779:Tetrodotoxin / Q000378:metabolism
D003064:Cnidarian Venoms; D017931:DNA Primers; D018076:DNA, Complementary; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D012604:Scorpion Venoms; D015222:Sodium Channels; D012530:Saxitoxin; D013779:Tetrodotoxin