The blocking actions of the K+ channel toxins charybdotoxin, dendrotoxin and mast cell degranulating peptide were studied in B82 mouse fibroblast cells transformed to express NGK1 (Kvl.2) K+ channels. All three toxins were potent blockers of the K+ current in these cells, with KD values of 1.7, 2.8 and 185nM, respectively. The toxin block exhibited a weak voltage-dependence with the degree of inhibition decreasing at positive membrane potentials. For charybdotoxin and dendrotoxin, reducing [K+]i did not increase the fractional block, demonstrating that the relief of block at positive membrane potentials is not due to displacement of the toxin molecules by outward flow of K+ ions. A voltage-jump protocol was used to determine the rates of binding and unbinding of dendrotoxin and mast cell degranulating peptide; binding of charybdotoxin was too rapid to be quantitatively evaluated in this manner. The binding rates (dendrotoxin), ∼5 × 107/M per s; mast cell degranulating peptide, ∼0.8 × 107/M per s) were largely voltage-independent, suggesting that association of the toxin molecules with the channel is diffusion limited. The rates of unbinding (dendrotoxin, ∼0.3/s; mast cell degranulating peptide, ∼3/s at +60mV) of both toxins increased e-fold per ∼40mV change in membrane potential, thus accounting for the voltage-dependence of the equilibrium block. Internal perfusion with the three toxins failed to affect the K+ current (in contrast to internal tetraethylammonium which strongly blocked the current), indicating that the toxins exert their blocking action by binding to extracellular sites.
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