Binding Sites for Ca2+‐Channel Effectors and Ryanodine in Periplaneta americana—Possible Targets for New Insecticides

The calcium channel and the 'calcium release channel' of muscle membrane of the cockroach Periplaneta americana have been characterized. Biological assays with calcium channel blockers and ryanodine on different insects and acari revealed pronounced insecticidal effects with ryanodine, but not with calcium channel blockers, at concentrations between 0.1 and 300 μg ml -1 . Skeletal muscle membranes derived either from the tubular network or from the sarcoplasmatic reticulum of P. americana were characterized with respect to the binding of the dihydropyridine (DHP) [ 3 H]isradipine (PN 200-110), the phenylalkylamine [ 3 H]verapamil and the alkaloid [ 3 H]ryanodine. Preliminary binding studies with the benzothiazepine [ 3 H]diltiazem suggest a low-affinity binding site with a IC 50 value of 3.3 μM. All binding sites tested were sensitive to treatment with proteinase K. Optimal conditions for binding of the radioligand ryanodine revealed the highest specific binding at pH 8 and at calcium chloride concentrations between 100 and 500 μM. EGTA at 10 μM abolished 95% of the ryanodine binding. Binding studies with calcium channel binding sites revealed a pronounced effect of low Ca 2+ concentrations on specific isradipine binding, whereas verapamil and diltiazem binding were only reduced by the presence of 200 μM EGTA. With respect to high Ca 2+ concentrations, specific binding of diltiazem, isradipine and verapamil was reduced by 73, 40 and 20%, respectively, at 5 mM Ca 2+ . Radioligand binding experiments showed high-affinity binding sites for ryanodine and isradipine. K D values of 0.95 nM (B max = 550 fmol mg -1 protein) and 0.75 nM (B max = 213 fmol mg -1 protein) were determined respectively. A lower-affinity binding site was identified in binding studies with verapamil (K D = 7.4 nM and B max = 27 fmol mg -1 protein). [ 3 H]isradipine displacement studies with several dihydropyridines revealed the following ranking of affinity : nitrendipine > isradipine > Bay K8664 >> nicardipine. Displacement of [ 3 H]verapamil binding by effectors of the phenylalkylamine binding site showed that bepridil and S(-)verapamil had the highest affinities of the compounds tested followed by (±)verapamil, nor-methylverapamil and R(+)verapamil.