LOCALIZATION AND PHARMACOLOGICAL CHARACTERIZATION OF NICOTINIC CHOLINERGIC BINDING-SITES IN COCKROACH BRAIN USING ALPHA-BUNGAROTOXIN AND NEURONAL BUNGAROTOXIN

[125I]α-Bungarotoxinisusedasaprobetostudythenicotinic-cholinergicreceptorinmembrane preparations of the cockroach brain. Binding is restricted mainly to particulate fractions of brain homogenates, is time dependent and is saturable above 2 nM with very low non-specific binding. Scatchard analysis indicates that binding is associated with a single affinity site (Kd = 1.09 nM) having a Bmax of 8926 fmol/mg protein which is the highest concentration of binding sites yet reported in insects. Association kinetics are best fit by a mono-exponential model with a kobs = 4.37 × 10-3 s-1. Dissociation is best described by a bi-exponential model giving dissociation constants of 1.18 × 10-5 and 9.94 × 10-5 s-1. The Kds calculated from kinetic data are 0.029 and 0.25 nM suggesting the possibility of heterogeneous binding sites not detected by saturation studies. Displacement studies indicate that binding follows a nicotinic pharmacology and demonstrate the high affinity of methyllycaconitine and the anthelmintics, morantel and pyrantel. Displacement by neuronal bungarotoxin shows the presence of two distinct binding sites not differentiated by α-bungarotoxin. Autoradiographic studies show α-bungarotoxin to be binding to neuropile regions of the brain, to be displaced from these regions by agents effective in binding studies and demonstrate that the neuronal bungarotoxin binding sites can be regionally localized. © 1990.