Abstract
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.
Highlights
Tion of electrical impulses in most excitable tissues [1]
The voltage dependence of binding and the specific effect of these toxins on inactivation (19 –23) imply that membrane potential affects the structure of neurotoxin receptor site 3, that this region of the channel is important for the coupling of activation to inactivation, and that toxin binding to this site can slow or block a conformational change required for fast inactivation
Two distinct regions of the Naϩ channel ␣ subunit have been implicated in ␣-scorpion toxin binding by photoaffinity labeling of the S5-SS1 loop in domain I and inhibition of Molecular Determinants of ␣-Scorpion and Sea Anemone Toxin Binding toxin binding by site-directed antipeptide antibodies directed against peptides of the S5-SS1 and SS2-S6 loops in domain I and the S5-SS1 loop in domain IV [28, 29]
Summary
Materials—␣-Scorpion toxin (LqTx) was purified from venom (Leiurus quinquestriatus quinquestriatus, Latoxan) and iodinated as described previously [17, 29]. Cell Culture—CNaIIa-1 cells stably expressing the rIIa Naϩ channel ␣ subunit were maintained in RPMI media supplemented with 5% fetal bovine serum (Hyclone), as described previously [27]. The tsA-201 cells were maintained in Dulbecco’s modified Eagle’s medium/F-12 media supplemented with 10% fetal bovine serum, 20 g/ml penicillin, and 10 g/ml streptomycin. Both lines were maintained at 37 °C in a 5% CO2 incubator. Mutagenesis and Assembly of cDNAs Encoding Naϩ Channels with Site-directed Mutations—Three M13 constructs containing type IIa Naϩ channel ␣ subunit cDNA sequence (30 –32) were used for mutagenesis: mp18KXNC, which contained a KpnI/XbaI fragment (nt 23–540); mp19XaI, which contained a XbaI/SmaI fragment (nt 541-1897); and, mp18RVNC, which contained an EcoRV fragment (nt 4279 –5997). To be certain that undetected rearrangements did not influence interpretation of our results, multiple independently iso-
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