Molecular properties of the apamin-binding component of the Ca2+-dependent K+ channel. Radiation-inactivation, affinity labelling and solubilization.
Radiation-inactivation was used to assess the functional size of the apamin-binding component of the Ca2+-dependent K+ channel. The amount of specific binding of 125I-apamin to receptors in synaptic membranes of rat cortex decayed exponentially with increasing doses of ionizing radiation and target size analysis was consistent with a relative molecular mass of 250 000 +/- 20 000 for the 125I-apamin receptor. Analysis on sodium dodecyl sulfate gels following covalent cross-linking of 125I-apamin to its receptor in a synaptosomal membrane preparation from rat cortex revealed a single labelled polypeptide chain of Mr = 33 000 +/- 2000 in the presence of protease inhibitors. Our results suggest that the Ca2+-dependent K+ channel from rat cortex is an oligomeric structure of Mr = 250 000 +/- 20 000 containing an apamin-binding subunit of Mr = 33 000 +/- 2000. The apamin-binding component of the Ca2+-dependent K+ channel from rat synaptosomes was solubilized using detergents such as sodium cholate or 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate. Phospholipids did not increase the stability of the apamin-binding component during the solubilization. Binding of apamin to its solubilized receptor is reversible and saturable. The dissociation constant of the apamin-receptor complex is 40-150 pM, the rates constants of association and dissociation being 3.2 X 10(6) M-1s-1 and 1.4 X 10(-4)s-1 respectively. These binding characteristics are similar to those found for the membrane-bound apamin receptor.
D000345:Affinity Labels; D000818:Animals; D001030:Apamin / Q000378:metabolism*; D001514:Bee Venoms / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D007473:Ion Channels / Q000378:metabolism* / Q000528:radiation effects; D007700:Kinetics; D011188:Potassium / Q000378:metabolism*; D015221:Potassium Channels; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D012995:Solubility; D013570:Synaptic Membranes / Q000378:metabolism* / Q000528:radiation effects
D000345:Affinity Labels; D001514:Bee Venoms; D003432:Cross-Linking Reagents; D007473:Ion Channels; D015221:Potassium Channels; D011956:Receptors, Cell Surface; C033799:apamin receptor; D001030:Apamin; D012964:Sodium; D011188:Potassium