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.
D000818:Animals; D001030:Apamin / Q000378:metabolism; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D005703:Gallamine Triethiodide / Q000378:metabolism; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D011188:Potassium / Q000378:metabolism*; D015221:Potassium Channels; D011802:Quinidine / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D017981:Receptors, Neurotransmitter / Q000302:isolation & purification* / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000378:metabolism; D014403:Tubocurarine / Q000378:metabolism
D007473:Ion Channels; D015221:Potassium Channels; D017981:Receptors, Neurotransmitter; D013757:Tetraethylammonium Compounds; C033799:apamin receptor; D001030:Apamin; D019789:Tetraethylammonium; D011802:Quinidine; D005703:Gallamine Triethiodide; D011188:Potassium; D002118:Calcium; D014403:Tubocurarine