Interaction of Mg2+ with the allosteric site of muscarinic M2 receptors

Abstract

Mg²⁺-ions have been suspected to attenuate the inhibitory effect of allosteric modulators on the dissociation of orthosteric ligands from muscarinic M₂ receptors. It was aimed to gain more insight into the molecular events underlying the effect of Mg²⁺. The interaction of Mg²⁺ with the allosteric model compounds W84 (hexane-1,6-bis [dimethyl-3’-phthalimidopropylammonium bromide]) and Chin3/6 (hexane-1,6-bis[dimethyl-3’-{4-oxo-2-phenyl-3,4-dihydro-2H-quinazolin-1-yl}propylammonium bromide]) was studied in porcine heart muscarinic receptors, the primary binding site of which was occupied by the ligand [³H]N-methylscopolamine ([³H]NMS). The incubation buffer was composed of 4 mM Na₂HPO₄ and 1 mM KH₂PO₄ (pH 7.4, 23°C). The retardation of [³H]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 contribution to the ionic strength of the buffer solution. Concentration-effect curves for the allosteric retardation of [³H]NMS dissociation by W84 (half maximal effective concentration EC_0.5 = 24 nM in the absence of Mg²⁺) and by Chin3/6 (EC_0.5 = 28 nM) were shifted by Mg²⁺ to the right in a parallel fashion. The curve-shift was compatible with a competitive interplay between Mg²⁺ and the modulators. The pK _b-values 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 [³H]NMS; the maximal retardation of [³H]NMS dissociation was about 3fold, the concentration-effect relationship was compatible with a two-site model using the above-mentioned pK _b-values as affinity constants. Since the equilibrium-binding of [³H]NMS remained unchanged up to a Mg²⁺-concentration of 3 mM, the cation appears to inhibit the association and dissociation of [³H]NMS to the same extent in this concentration range. Taken together, the findings indicate that Mg²⁺ may bind to the allosteric region of muscarinic M₂ receptors and that more than one site is involved in this interaction. The sites of action may represent divalent cation binding sites.