[I-125] MARGATOXIN, AN EXTRAORDINARILY HIGH-AFFINITY LIGAND FOR VOLTAGE-GATED POTASSIUM CHANNELS IN MAMMALIAN BRAIN

Monoiodotyrosine margatoxin ([I-125]MgTX) specifically and reversibly labels a maximum of 0.8 pmol of sites/mg of protein in purified rat br ain synaptic plasma membrane vesicles with a dissociation constant of 0.1 pM under equilibrium binding conditions. This K-d value was confir med by kinetic experiments (K-d of 0.07 pM), competition assays employ ing native margatoxin (MgTX) (K-i of 0.15 pM), and receptor saturation studies (K-d of 0.18 pM). Thus, this toxin represents the highest aff inity, reversible radioligand for any membrane-bound receptor or ion c hannel described to date. [I-125]MgTX binding in this system is modula ted by charybdotoxin (K-i of 5 pM), kaliotoxin (K-i of 1.5 pM), and th e agitoxins I and II (K-i's of 0.1 and 0.3 pM, respectively), in a non competitive manner. Moreover, alpha-dendrotoxin displayed a K-i value of 0.5 pM. Iberiotoxin was without any effect, suggesting that the rec eptor site is likely to be associated with a voltage-gated K+ channel complex. [I-125]MgTX binding is inhibited by cations that are establis hed blockers of voltage-dependent K+ channels (Ba2+, Ca2+, Cs+). The m onovalent cations Na+ and K+ stimulate binding at low concentrations b efore producing complete inhibition as their concentrations are increa sed. Stimulation of binding results from an allosteric interaction tha t decreases K-d, whereas inhibition is due to an ionic strength effect . Affinity labeling of the binding site in rat brain synaptic plasma m embranes employing [I-125]MgTX and the bifunctional cross-linking reag ent, disuccinimidyl suberate, causes specific and covalent incorporati on of toxin into a glycoprotein of an apparent molecular weight (M(r)) of 74 000. Deglycosylation studies reveal an M(r) for the core polype ptide of the MgTX receptor of 63 000. Immunoprecipitation studies, emp loying sequence-directed antibodies indicate that at least K(v)1.2 and K(v)1.3 are integral constituents of the rat brain MgTX receptor.