Sialic acid residues as catalysts for M2-muscarinic agonist-receptor interactions

The role of sialic acid residues in the interactions of muscarinic agonists with the cardiac M2 muscarinic receptor was investigated by competitive binding experiments using the lipophilic radioligand (-)-[benzilic-4,4-3H]quinuclidinyl benzilate ([3H]QNB) and the hydrophilic ligand [N-methyl-3H]scopolamine methyl chloride ([3H]NMS). Direct labeling of the agonist binding sites was performed with the radiolabeled agonist [methyl-3H]oxotremorine M acetate ([3H]oxo-M). Neuraminidase decreased the affinity of the M2-selective agonist carbamylcholine in competitive binding experiments performed with [3H]QNB and [3H]NMS. The binding of the M1-selective agonist (4hydroxy-2-butynyl)trimethylammonium chloride m-chlorocarbanilate (McN-A-343), of the M1-selective antagonist pirenzepine, and of the M2-selective antagonist 11-([2-[(diethylamino)methyl]-1 piperidinyl]acetyl)-5,11-dihydro-6H-pyrido(2,3b)(1,4)benzodiazepin -6-on (AF-DX-116) were not affected by neuraminidase. Neuraminidase did not modify the binding parameters of 3H-antagonists but reduced the number of agonist binding sites revealed by [3H]oxo-M. The removal of sialic acid decreased the half-life of the receptor-agonist complex. The present results suggest that removal of sialic acid reduces the formation of super-high affinity agonist-receptor complexes. Sialic acid may catalyze macroscopic binding by enhancing accumulation of the agonist at the membrane surface.