Localization of receptor sites for insect-selective toxins on sodium channels by site-directed antibodies.
Site-directed antibodies corresponding to conserved putative extracellular segments of sodium channels, coupled with binding studies of radiolabeled insect-selective scorpion neurotoxins, were employed to clarify the relationship between the toxins' receptor sites and the insect sodium channel. (1) The depressant insect toxin LqhIT2 was shown to possess two noninteracting binding sites in locust neuronal membranes: a high-affinity (KD1 = 0.9 +/- 0.6 nM) and low-capacity (Bmax1 = 0.1 +/- 0.07 pmol/mg) binding site as well as a low-affinity (KD2 = 185 +/- 13 nM) and high-capacity (Bmax2 = 10.0 +/- 0.6 pmol/mg) binding site. (2) The high-affinity site serves as a target for binding competition by the excitatory insect toxin AaIT. (3) The binding of LqhIT2 was significantly inhibited in a dose-dependent manner by each of four site-directed antibodies. The binding inhibition resulted from reduction in the number of binding sites. (4) The antibody-mediated inhibition of [125I]AaIT binding differs from that of LqhIT2: three out of the four antibodies which inhibited LqhIT2 binding only partially affected AaIT binding. Two antibodies, one corresponding to extracellular and one to intracellular segments of the channel, did not affect the binding of either toxin. These data suggest that the receptors to the depressant and excitatory insect toxins (a) comprise an integral part of the insect sodium channel, (b) are formed by segments of external loops in domains I, III, and IV of the sodium channel, and (c) are localized in close proximity but are not identical in spite of the competitive interaction between these toxins.
Gordon
D
D
Department of Cell and Animal Biology, Hebrew University, Jerusalem, Israel.
Moskowitz
H
H
Eitan
M
M
Warner
C
C
Catterall
W A
WA
Zlotkin
E
E
D000818:Animals; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D006110:Grasshoppers; D007700:Kinetics; D008961:Models, Structural; D009474:Neurons / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D011950:Receptors, Cholinergic / Q000276:immunology / Q000378:metabolism*; D012604:Scorpion Venoms / Q000378:metabolism*; D015222:Sodium Channels / Q000378:metabolism*; D013574:Synaptosomes / Q000378:metabolism
C076349:LqhIT2 insect toxin; D011950:Receptors, Cholinergic; D012604:Scorpion Venoms; D015222:Sodium Channels; C029574:scorpion toxin receptor; C065634:AaIT neurotoxin, Androctonus australis