Characterization of agonist and antagonist binding to muscarinic cholinergic receptors solubilized from rat cerebral cortex

Summary

The muscarinic acetylcholine receptor was solubilized from rat brain cortex by the zwitter-ionic detergent, 3-((3-cholamidopropyl)dimethylamino)1-propane sulfonate (CHAPS). The supernatant, after centrifugation at 100,000 × g, was shown to contain molecules with binding sites for both³H-pirenzepine (³H-PZ) and³H-(-) quinuclidinyl benzilate (³H-QNB). Maximum binding values for³H-PZ and³H-QNB binding to solubilized receptors were approximately 176±24 pmol/g and 370±53 pmol/g of protein, respectively. The K_d values for³H-PZ and³H-QNB binding to solubilized receptors were 27±6.3nM and 0.17±0.03 nM, respectively. The rank order of potencies of muscarinic drugs, in terms of their ability to inhibit binding of either³H-PZ or³H-QNB, was atropine > pirenzepine > oxotremorine > carabachol. Pirenzepine inhibited³H-QNB binding with a Hill coefficient of 0.77, but inhibited³H-PZ with a Hill coefficient of 0.94. Hill coefficients for agonists were less than 1. These findings indicate that muscarinic receptors solubilized from rat brain cortex retain their abilities to interact selectively with muscarinic receptor agonists and antagonists.