An [3H]oxotremorine binding method reveals regulatory changes by guanine nucleotides in cholinergic muscarinic receptors of cerebral cortex.

A rapid, reliable filtration method for [3H]oxotremorine binding to membranes of the cerebral cortex that allows the direct study of regulation by guanine nucleotides of muscarinic receptors was developed. [3H]Oxotremorine binds to cerebral cortex membranes with high affinity (KD, 1.9 nM) and low capacity (Bmax, 187 pmol/g protein). These sites, which represent only about 18% of those labeled with [3H]quinuclidinyl benzilate, constitute a population of GTP-sensitive binding sites. Association and dissociation binding experiments revealed a similar value of KD (2.3 nM). Displacement studies with 1-4000 nM oxotremorine showed the existence of a second binding site of low affinity (KD, 1.2 microM) and large capacity (Bmax, 1904 pmol/g protein). Gpp(NH)p, added in vitro, produced a striking inhibition of [3H]oxotremorine binding with an IC 50 of 0.3 microM. Saturation assays, in the presence of 0.5 microM Gpp(NH)p, revealed a non-competitive inhibition of the binding with little change in affinity. These results are discussed from the viewpoint of conflicting reports in the literature about guanine nucleotide regulation of muscarinic receptors in reconstituted systems and membranes from different tissues.