Abstract
The ligand binding approach to receptor characterization is a convenient way of monitoring receptor numbers and distribution, assessing pharmacological specificity and following the receptor during purification procedures. Ligand binding assays using the high-affinity, pseudo-irreversible antagonist a-bungarotoxin have made an enormous contribution to the understanding of the muscle nicotinic acetylcholine receptor (nAChR) and its counterpart in Torpedo electroplax [l]. The success of a-bungarotoxin in this respect prompted its use to seek out and characterize nAChR in the nervous system. Despite the clearcut identification of specific binding sites for a-bungarotoxin in brain and in autonomic ganglia, this toxin fails to act as a nicotinic antagonist in the majority of instances in which it has been examined [2]. This was surprising since a-bungarotoxin binding sites in nervous tissue are clearly nicotinic in character, binding being displaced by nicotine, acetylcholine and other competitive nicotinic drugs. In 1980, the first substantial characterization of ligand binding sites for []nicotine in brain membranes was documented [ 31. Subsequent studies with []nicotine or other tritiated nicotinic agonists (N-methylcarbamylcholine, or acetylcholine in the presence of atropine) have established the nicotinic character of these binding sites [4]. [ 'HINicotine binding sites and '1-a-bungarotoxin binding sites do not co-purify [ 51, they have quite different anatomical distributions in the brain [6] and give distinctive subunit patterns on SDS/ PAGE [7]. But given the uncertainty about the receptor status of neuronal a-bungarotoxin sites, it would be imprudent to elevate the tritiated agonist binding sites to nAChR status in the absence of corroborative evidence. Thus it was deemed imperative to complement ligand binding assays with measures of nicotinic function in the brain. This objective is not easily realized. The ligand-gated ion channel nature of nAChR requires intact neurons to demonstrate function, since this depends on ion flux across the plasma membrane. The low density of nicotinic binding sites in the brain (50-100 fmol/ mg of protein for both []nicotineand I2'I-abungarotoxin-labelled sites) results in weak
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