Properties of a quinuclidinyl benzilate binding component in the bulb mite

1. 1. About 95% of the specific binding (defined by atropine) of the muscarinic ACh receptor antagonist [ 3 H]-quinuclidinyl benzilate occurred in the low speed pellet (5500 g , 10 min) of whole bulb mite homogenates and was heat sensitive, linear with tissue concentration and saturable. Rosenthal analysis indicated that [ 3 H]QNB was binding with high affinity ( K d 474 pM) to a single class of low density ( B max 202 fmol g −1 ) binding sites; the Hill coefficient was 1.0. 2. 2. Kinetic studies revealed that binding was rapid and reversible, with association ( K +1 ) and dissociation ( k −1 ) rate constants of 7.8 × 10 5 sec −1 M −1 and 3.1 × 10 −4 sec −1 , respectively. The K d from kinetic data ( k −1 k −1 ) was 414 pM. 3. 3. Muscarinic drugs generally were much more potent than were nicotinic drugs as inhibitors of [ 3 H]QNB binding, and muscarinic antagonists generally were more potent than were agonists. 4. 4. Binding was stereoselective because R (−)QNB was about 10,000 times more potent than S( + )-QNB, and dexetimide was about 1000 times more potent than its enantiomer levetimide as inhibitors of [ 3 H]QNB binding. 5. 5. Pirenzepine, a selective antagonist of mammalian M-1 muscarinic receptors was very effective in protecting against [ 3 H]QNB binding, whereas methoctramine, a selective antagonist of M-2 muscarinic receptors, was much less effective. 6. 6. Altogether these data suggest that the specific binding of [ 3 H]QNB is most likely to a popoulation of putative muscarinic ACh receptors, probably of the M-1 subtype.