Localization of the receptor site for alpha-scorpion toxins by antibody mapping: implications for sodium channel topology.
Site-directed and monoclonal antibodies recognizing different extracellular regions of the RII sodium channel alpha subunit have been used to determine the sequences that comprise the receptor for alpha-scorpion toxins by evaluating the effect of antibody on voltage-dependent binding of radio-labeled toxin isolated from Leiurus quinquestriatus to both reconstituted rat brain sodium channel and rat brain synaptosomes. Of six antibodies tested, two recognizing amino acid residues 355-371 and 382-400 located on an extracellular loop between transmembrane segments S5 and S6 of domain I and one recognizing residues 1686-1703 of a similar loop of domain IV inhibit binding by 30-55%. Inhibition is concentration-(EC50 = 0.4-2 microM) and time- (t1/2 = 40-80 min) dependent. Five different monoclonal antibodies recognizing the same extracellular loop in domain I inhibit binding completely with similar EC50 values as observed for site-directed antibodies. Kinetic studies of the antibody effect are consistent with a slowly reversible competition for the toxin receptor site. Our results suggest that the extracellular loops between segments S5 and S6 of domains I and IV comprise at least part of the alpha-scorpion toxin receptor site and support the membrane topology models in which domains I and IV are adjacent in the tertiary structure of the channel protein and six transmembrane sequences are contained in each of the four homologous domains.
Thomsen
W J
WJ
Department of Pharmacology, School of Medicine, University of Washington, Seattle 98195.
Catterall
W A
WA
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex / Q000032:analysis; D001921:Brain / Q000378:metabolism*; D007700:Kinetics; D008961:Models, Structural; D051381:Rats; D011950:Receptors, Cholinergic / Q000032:analysis / Q000378:metabolism*; D012604:Scorpion Venoms / Q000378:metabolism; D015222:Sodium Channels / Q000378:metabolism* / Q000648:ultrastructure; D013574:Synaptosomes / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D011950:Receptors, Cholinergic; D012604:Scorpion Venoms; D015222:Sodium Channels; C029574:scorpion toxin receptor