[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 brain synaptic plasma membrane vesicles with a dissociation constant of 0.1 pM under equilibrium binding conditions. This K-d value was confirmed by kinetic experiments (K-d of 0.07 pM), competition assays employing 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 affinity, reversible radioligand for any membrane-bound receptor or ion channel described to date. [I-125]MgTX binding in this system is modulated by charybdotoxin (K-i of 5 pM), kaliotoxin (K-i of 1.5 pM), and the agitoxins I and II (K-i's of 0.1 and 0.3 pM, respectively), in a noncompetitive manner. Moreover, alpha-dendrotoxin displayed a K-i value of 0.5 pM. Iberiotoxin was without any effect, suggesting that the receptor site is likely to be associated with a voltage-gated K+ channel complex. [I-125]MgTX binding is inhibited by cations that are established blockers of voltage-dependent K+ channels (Ba2+, Ca2+, Cs+). The monovalent cations Na+ and K+ stimulate binding at low concentrations before producing complete inhibition as their concentrations are increased. Stimulation of binding results from an allosteric interaction that decreases K-d, whereas inhibition is due to an ionic strength effect. Affinity labeling of the binding site in rat brain synaptic plasma membranes employing [I-125]MgTX and the bifunctional cross-linking reagent, disuccinimidyl suberate, causes specific and covalent incorporation of toxin into a glycoprotein of an apparent molecular weight (M(r)) of 74 000. Deglycosylation studies reveal an M(r) for the core polypeptide of the MgTX receptor of 63 000. Immunoprecipitation studies, employing 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.