Molecular properties of the apamin‐binding component of the Ca2+‐dependent K+ channel Radiation‐inactivation, affinity labelling and solubilization

Abstract

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 dodecylsulfate gels following covalent cross-linking of 125I-apamin to its receptor in a synaptosomal membrane preparation from rat cortex revealed a single labeled polypeptide chain of Mr = 33,00 .+-. 2000 in the presence of protease inhibitors. The Ca2+-dependent K+ channel from rat cortex was probably 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 was reversible and saturable. The Kd of the apamin-receptor complex was 40-150 pM, the Ka and Kd being 3.2 .times. 106 M-1 s-1 and 1.4 .times. 10-4/s, respectively. These binding characteristics were similar to those found for the membrane-bound apamin receptor.