An 125I-labeled binding probe for the muscarinic cholinergic receptor

We describe the synthesis and characterization of [125I]3-quinuclidinyl-(3-iodo-4-hydroxy-benzilate), a binding probe for the muscarinic cholinergic receptor of high specific radioactivity. The binding isotherm of this 125I-labeled compound to a rat synaptic plasma membrane-enriched fraction consists of two components: a 'specific' component which is saturable and closely fits hyperbolic binding to a single class of sites as evaluated by Scatchard analysis (Kd = 1.5 to 3 nM), and a linear component which may be measured directly by preincubating membranes in 0.1 microM 3-quinuclidinyl-benzilate, the most potent muscarinic antagonist known. The specific binding of [3-3H]3-quinuclidinyl-benzilate and [125I]3-quinuclidinyl-(3-iodo-4-hydroxy-benzilate) to rat brain subcellular fractions is parallel in myelin, synaptic plasma membrane and mitochondrial fractions with a 3--4-fold enrichment observed in synaptic plasma membrane over crude mitochondrial fractions. The concentrations of muscarinic antagonists required to block [125I]3-quinuclidinyl-(3-iodo-4-hydroxy-benzilate) binding parallel that reported for tritiated binding probes and are consistent with physiological measurements of their dissociation constants. Because of the high specific radioactivity of [125I]3-quinuclidinyl-(3-iodo-4-hydroxy-benzilate), this iodinated binding probe should prove useful in assaying low levels of muscarinic receptor in tissue culture and other biological sources.