SIMILARITY OF INSECT AND MAMMALIAN RYANODINE BINDING-SITES

Ryanodine is a principal active ingredient of the botanical insecticide ryania. [H-3]Ryanodine is the most important radioligand for the Ca2+ release channel in mammalian muscle and brain. Characterization of the [H-3]ryanodine binding site in insect muscle and brain membranes may therefore help our understanding of the properties of the Ca2+ release channel in insects as a target for insecticide action. [H-3]Ryanodine undergoes 60 to 99% specific binding to membrane fractions from house fly thorax, American cockroach and cricket muscle and brain, armyworm larval head and body, and (for comparison) mouse muscle and brain. The standard assay contained 1 nM [H-3]ryanodine, 250 mu g membrane protein, 0.4 mM CaCl2 5 mM ATP, 0.75 M KCl, 0.15 M sucrose, and 0.8% bovine serum albumin (BSA) buffered at pH 7.4. Equilibrium is achieved with incubation for 2 hr at 37 degrees C. There is a single saturable high-affinity binding site for [H-3]ryanodine with similar K-D and B-max values for membrane fractions from house fly thorax, cockroach muscle, and mouse muscle, i.e., ranging for the three preparations from 4.4 to 5.6 nM for K-D and from 350 to 630 fmol/0.25 mg protein for B-max. The activity is enhanced by Ca2+, ATP, and KCl in each case and by BSA with house fly thorax membranes. The low potency of ryanodol (the naturally occurring alcohol component of ryanodine) at the house fly and cockroach [H-3]ryanodine receptors, despite its outstanding insecticidal activity, suggests that the binding site for ryanodol may be different from that for ryanodine in these insects. (C) 1994 Academic Press, Inc.