INTERACTION OF MG2-2 RECEPTORS( WITH THE ALLOSTERIC SITE OF MUSCARINIC M)

Mg2+-ions have been suspected to attenuate the inhibitor effect of all osteric modulators on the dissociation of orthosteric ligands from mus carinic M-2 receptors. It was aimed to gain more insight into the mole cular events underlying the effect of Mg2+. The interaction of Mg2+ wi th the allosteric model compounds W84 (hexane-1,6-bis [dimethyl-3'-pht halimidopropylammonium bromide]) and Chin3/6 (hexane-1,6-bis enyl-3,4- dihydro-2H-quinazolin-1-yl}propylammonium bromide]) was studied in por cine heart muscarinic receptors, the primary binding site of which was occupied by the ligand [H-3]N-methylscopolamine ([H-3]NMS). The incub ation buffer was composed of 4 mM Na2HPO4 and 1 mM KH2PO4 (pH 7.4, 23 degrees C). The retardation of [K-3]NMS dissociation (control t(1/2) = 5.6 min) induced by the allosteric test compounds was diminished by 3 mM Mg2+ to a greater extent than to be expected with regard to its co ntribution to the ionic strength of the buffer solution. Concentration -effect curves for the allosteric retardation of [H-3]NMS dissociation by W84 (half maximal effective concentration EC0.5 = 24 nM in the abs ence of Mg2+) and by Chin3/6 (EC0.5 = 28 nM) were shifted by Mg2+ to t he sight in a parallel fashion. The curve-shift was compatible with a competitive interplay between Mg2+ and the modulators. The pK(b)-value s as a measure of the antagonistic potency of Mg2+, however, differed depending on the modulator, i.e. pK(b) = 3.4 with W84 and pK(b) = 2.8 with Chin3/6. Mg2+ itself was capable of slowing the dissociation of [ H-3]NMS; the maximal retardation of [H-3]NMS dissociation was about 3f old, the concentration-effect relationship was compatible with a two-s ite model using the above-mentioned pK(b)-values as affinity constants . Since the equilibrium-binding of [H-3]NMS remained unchanged up to a Mg2+-concentration of 3 mM, the cation appears to inhibit the associa tion and dissociation of [H-3]NMS to the same extent in this concentra tion range. Taken together, the findings indicate that Mg2+ may bind t o the allosteric region of muscarinic M-2 receptors and tha more than one site is involved in this reaction. The sites of action may represe nt divalent cation binding sites.