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 membra ne of the cockroach Periplaneta americana have been characterized. Bio logical assays with calcium channel blockers and ryanodine on differen t insects and acari revealed pronounced insecticidal effects with ryan odine, but not with calcium channel blockers, at concentrations betwee n 0.1 and 300 mu g ml(-1). Skeletal muscle membranes derived either fr om the tubular network or from the sarcoplasmatic reticulum of P. amer icana were characterized with respect to the binding of the dihydropyr idine (DHP) [H-3]isradipine (PN 200-110), the phenylalkylamine [H-3]ve rapamil and the alkaloid [H-3]ryanodine. Preliminary binding studies w ith the benzothiazepine [H-3]diltiazem suggest a low-affinity binding site with a IC50 value of 3.3 mu M. All binding sites tested were sens itive to treatment with proteinase K. Optimal conditions for binding o f the radioligand ryanodine revealed the highest specific binding at p H 8 and at calcium chloride concentrations between 100 and 500 mu M. E GTA at 10 mu M abolished 95% of the ryanodine binding. Binding studies with calcium channel binding sites revealed a pronounced effect of lo w Ca2+ concentrations on specific isradipine binding whereas verapamil and diltiazem binding were only reduced by the presence of 200 mu M E GTA. With respect to high Ca2+ concentrations, specific binding of dil tiazem, isradipine and verapamil was reduced by 73, 40 and 20%, respec tively, at 5 mM Ca2+. Radioligand binding experiments showed high-affi nity binding sites for ryanodine and isradipine. K-D values of 0.95 ru n (B-max = 550 fmol mg(-1) protein) and 0.75 run (B-max = 213 fmol mg( -1) protein) were determined respectively. A lower-affinity binding si te was identified in binding studies with verapamil (K-D = 7.4 run and B-max = 27 fmol mg(-1) protein). [H-3]isradipine displacement studies with several dihydropyridines revealed the following ranking of affin ity: nitrendipine > isradipine > Bay K8664 much greater than nicardipi ne. Displacement of [H-3]verapamil binding by effecters of the phenyla lkylamine binding site showed that bepridil and S(-)verapamil had the highest affinities of the compounds tested followed by(+/-)verapamil, nor-methylverapamil and R(+)verapamil.