INFLUENCE OF MONOVALENT CATIONS ON THE BINDING OF A CHARGED AND AN UNCHARGED (CARBO-)MUSCARINIC ANTAGONIST TO MUSCARINIC RECEPTORS

1 The effect of the buffer concentration on binding of [H-3]-N-methyls copolamine to muscarinic receptors M(2) was tested in rat heart. Trace r binding was of low affinity in a 20 mM imidazole buffer (pK(D) 8.3), inhibited by an increase from 10 to 100 mM of the sodium phosphate bu ffer concentration (pK(D) 9.92 to 9.22), slightly inhibited by an incr ease of the Tris/HCl buffer concentration from 20 to 100 mM (pK(D) 9.7 0 to 9.47) and unaffected by an increase of the histidine/HCl buffer c oncentration from 20 to 100 mM (pK(D) 9.90 to 9.82). We chose the last buffer to analyse the effect of ions on antagonists binding to cardia c M(2) receptors and to transiently expressed wild-type and (Y533-->F) mutant m3 muscarinic receptors in COS-7 cells. 2 Equilibrium [H-3]-N- methylscopolamine binding to cardiac M(2) receptors was inhibited, app arently competitively, by monovalent salts (LiCl greater than or equal to NaCl greater than or equal to KCl). In contrast, binding of the un charged 3,3-dimethylbutan-1-ol ester of diphenylglycolic acid (BS-6181 ) was facilitated by addition of monovalent salts (LiCl greater than o r equal to NaCl greater than or equal to KCl) to the binding buffer. T his cation binding pattern is consistent with interaction with a large , negative field strength binding site, such as, for instance, a carbo xylic acid. 3 In the presence of 100 mM NaCl, [H-3]-N-methylscopolamin e had a similar affinity for the wild-type m3 receptor (pK(D) 9.85) an d for a (Y533-->F) mutant m3 receptor (pK(D) 9.68). However, in the ab sence of added salts, the tracer had a significantly lower affinity fo r the mutated (pK(D) 10.19) as compared to the wild-type (pK(D) 10.70) m3 receptor. BS-6181 had a significantly lower affinity for the (Y533 -->F) mutant m3 muscarinic receptor, as compared to the wild-type m3 r eceptor, both in the absence (pK(D) 6.19-6.72) in the presence (pK(D) 6.48-7.40) of 100 mM NaCl. The effects of NaCl on binding of the uncha rged ester and of [H-3]-N-methylscopolamine to the m3 receptor were de creased by the mutation. 4 Taken together, these results support the h ypothesis that monovalent cations from the buffer may interact with th e cation binding site of the receptors (an aspartate residue in the th ird transmembrane helix of muscarinic receptors). Buffer cations may i nhibit competitively the binding of (charged) muscarinic ligands havin g a tertiary amine or ammonium group, while facilitating the receptor recognition by uncharged, isosteric 'carbo-analogues'. Mutation of the (Y533-->F) of the m3 receptor decreased the affinity of the receptor for positive charges, including the sodium ion.