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

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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 250000 ± 20000 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 ofMr= 33000 ± 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= 250000 ± 20000 containing an apamin‐binding subunit of Mr= 33000 ± 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 × 106 M−1 s−1 and 1.4 × 10−4 s−1 respectively. These binding characteristics are similar to those found for the membrane‐bound apamin receptor.