title: Effects of apamin, quinine and neuromuscular blockers on calcium-activated potassium channels in guinea-pig hepatocytes.
abstract: The bee venom peptide, apamin, has been radiolabelled with 125I, the monoiodinated derivative purified, and its binding to intact guinea-pig liver cells studied. At 37 degrees C 125I-monoiodoapamin associated with, and dissociated from, guinea-pig hepatocytes remarkably rapidly. The association and dissociation rate constants were 1.4 X 10(8) 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. Unlabelled 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 reported 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 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 microM). 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 microM respectively). Gallamine was slightly less effective (KI = 14 microM) and decamethonium and hexamethonium much less so (KI = 620 and 760 microM 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 about 1700 sites per cell. Assuming, tentatively, that binding sites 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.
authors.forename: N S
authors.initials: NS
authors.lastname: Cook
authors.forename: D G
authors.initials: DG
authors.lastname: Haylett
mesh_terms: D000818:Animals; D001030:Apamin / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D001514:Bee Venoms / Q000494:pharmacology*; D001667:Binding, Competitive / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007473:Ion Channels / Q000187:drug effects*; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D009466:Neuromuscular Blocking Agents / Q000494:pharmacology*; D011188:Potassium / Q000378:metabolism; D011803:Quinine / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D001514:Bee Venoms; D007473:Ion Channels; D009466:Neuromuscular Blocking Agents; D001030:Apamin; C033900:monoiodoapamin; D011803:Quinine; D011188:Potassium; D002118:Calcium