Solubilization of the apamin receptor associated with a calcium-activated potassium channel from rat brain.

The apamin binding protein was solubilized from rat brain synaptic membranes using sodium cholate. Receptor yield and stability depended closely on the detergent/protein ratio. In optimum conditions the receptor retained high affinity for mono 125I-iodoapamin with Kd = 40 pM at pH 7.5 and 1 degree C and a binding capacity of 17 fmol/mg protein. 125I-apamin binding was stimulated by K+ ions with a K0.5 = 0.6 mM, demonstrating that the regulatory K+ site is also part of the soluble complex. Other ions could be substituted for K+ with an affinity sequence Tl+ = K+ = Rb+ greater than Cs+ greater than NH4+ greater than Li+ or Na+. Binding was inhibited by the neuromuscular blockers gallamine and tubocurarine and by the K+ channel blockers quinidine and tetraethylammonium chloride but not by 4-aminopyridine, in agreement with known pharmacological profile for inhibition of apamin-sensitive K+ permeability. Increasing the K+ concentration did not reverse inhibition by tetraethylammonium ions demonstrating that it does not bind competitively to the regulatory cationic site. Analysis of the covalently labeled apamin binding protein/sodium cholate complex by density gradient centrifugation indicated a high molecular weight with S20,w = 20 S.