CHARACTERIZATION OF MUSCARINIC BINDING-SITES IN THE CENTRAL-NERVOUS-SYSTEM OF LARVAL MANDUCA-SEXTA

Putative muscarinic receptors (mAChRs) were characterized in the CNS o f larval Manduca sexta through the binding of H-3-quinuclidyl benzilat e ([H-3]-QNB). Specific binding isotherms revealed a high affinity bin ding site in both crude homogenates (Ka=3.22+/-0.62 nM(-1) (Kd 0.311 n M), Bmax=65.4+/-9.8 fmoles/mg protein) and in purified membrane prepar ations (Ka=7.61+/-1.78 nM(-1) (Kd 0.130 nM), Bmax=22.8+/-2.15 fmoles/m g protein), In purified membrane preparations the binding was complex, consisting of at least two sites (Hill coefficient =0.514+/-0.041). B ecause of the high proportion of non-specific binding at QNB concentra tions greater than 10 nhl, the binding parameters for the additional l ow affinity site could not be determined accurately but the Kd was est imated to be greater than 8 nM. Complex binding was also exhibited in the kinetics of [H-3]-QNB association and dissociation in purified mem branes, Analysis resolved two high affinity sites (dissociation rate c onstants =80.4 and 1.2 min, Kds=0.272 and 0.909 nM, consisting of 65.9 +/-3.7 and 26+/-4.9% of the sites respectively). Presumably, the high affinity site identified in saturation studies consists of these two c omponents, The primary high affinity site was isolated using a dissoci ative method and its pharmacology determined in competition studies, [ H-3]-QNB binding at this site was displaced by pirenzepine and methoct ramine (Kis=248 and 707 nM) closely matching the pharmacology of the c loned Drosophila mAChR. Competition data for 4-DAMP were better fitted by a two site model (Kis=37 and 547 nM). These results demonstrate un expected complexity in muscarinic ligand binding and are consistent wi th mAChR heterogeneity in the CNS of Manduca. Copyright (C) 1996 Elsev ier Science Ltd.