Effects of apamin, quinine and neuromuscular blockers on calcium‐activated potassium channels in guinea‐pig hepatocytes.

Abstract

The bee venom peptide, apamin, was radiolabeled with 125I, the monoiodinated derivative purified and its binding to intact guinea pig liver cells studied. At 37.degree. C 125I-monoiodoapamin associated with, and dissociated from, guinea pig hepatocytes remarkably rapidly. The association and dissociation rate constants were 1.4 .times. 108 M-1 s-1 and 0.035 s-1, respectively. Equilibrium binding studies demonstrated a saturable binding component compatible with 1:1 binding to a single class of site and having an equilibrium dissociation constant (KL) of 390 pM. The maximal binding capacity was 1.1 fmol mg-1 dry wt of tissue. Unlabeled apamin displaced bound 125I-monoiodoapamin with a KI of 380 pM, which is consistent with the concentration of apamin required to inhibit Ca2+-activated K+ permeability (PK(Ca)) in these cells. Inhibitable binding of 125I-monoiodoapamin to rat hepatocytes was much less than to guinea-pig hepatocytes and could not be reliably quantified. Neither was there any discernible inhibitable binding to human erythrocytes. This is in keeping with the lack of apamin-sensitive Ca2+-activated K+ channels in these cell types. Various agents were tested for their ability to inhibit monoiodoapamin binding to, and Ca2+-mediated K+ efflux from, guinea-pig hepatocytes. All compounds tested which inhibited binding also blocked K+ efflux at similar concentrations. TEA [tetraethylammonium] and quinine affected hepatocytes only at high concentration (KI = 5.8 and 0.51 mM respectively). 9-Aminoacridine, quinacrine and chloroquine were slightly more effective (KI = 70-180 .mu.M). By far the most active compounds (apart from apamin) were the neuromuscular blocking agents; tubocurarine, pancuronium and atracurium (KI = 7.5, 6.8 and 4.5 .mu.M respectively). Gallamine was slightly less effective (KI = 14 .mu.M) and decamethonium and hexamethonium much less so (KI = 620 and 760 .mu.M respectively). 3,4-Diaminopyridine, .alpha.-bungarotoxin and tetrodotoxin were among several compounds which showed little or no affinity for apamin binding sites or inhibition of K+ efflux in guinea-pig hepatocytes. The saturable binding of 125I-monoiodoapamin to guinea-pig hepatocytes corresponds to .apprx. 1700 sites/cell. Binding sites apparently correspond to channels the rate of K+ loss observed following agonist action can readily be explained if these channels have unitary conductances in the range reported for PK(Ca) in other tissues.