Testing the specificity of allosteric modulations of muscarinic: receptorsin phylogenetically closely related histamine H-1-receptors

Gallamine, alcuronium and W84 (hexane-1,6-bis[dimethyl-3'-phthalimidopropyl -ammonium bromide]) are prototype allosteric modulators of the G-protein co upled muscarinic acetylcholine receptor family, especially of the M-2-subty pe. In order to probe the specificity of muscarinic allosteric modulation, we checked whether these agents interact with histamine H-1-receptors which have a high homology with muscarinic receptors. Binding experiments (38 mM Na2HPO4, 12 mM KH2PO4, pH 7.5) were performed with the H-1-receptor antago nist [H-3]mepyramine ([H-3]MEP) in guinea pig cerebellar homogenates. For t he sake of comparison, binding off [H-3]N-methylscopolamine ([H-3]NMS) at m uscarinic M-2-receptors was measured in porcine cardiac homogenates under i dentical conditions. The modulators retarded [H-3]NMS dissociation (t(1/2,c ontrol)=1.3 min) concentration-dependently indicating their allosteric acti on with half-maximum effects for gallamine at EC50,diss=27 mu M, for alcuro nium at EC50,diss=53 nM, and for W84 at EC50,diss=170 nM. In contrast, [H-3 ]MEP dissociation from H-1-receptors (t(1/2,control)=2.6 min) remained unch anged up to concentrations of 1 mM of the modulators. Equilibrium binding o f [H-3]NMS (K-D=0.46 nM, B-max=98 fmol/mg protein) was inhibited by gallami ne, elevated by alcuronium and left almost unchanged by W84, indicating neg ative, positive and nearly neutral cooperativity, respectively, with the ra dioligand. The ternary complex model of allosteric actions yielded the equi librium dissociation constants K-A for the binding of the allosteric modula tors to free M-2-receptors: K-A,K-gallamine=100 nM, K-A,K-alcuronium=450 nM , K-A,K-W84=69 nM. In H-1-receptors, more than 1000-fold higher concentrati ons than in M-2-receptors were required to elicit an effect on the binding of [H-3]MEP (K-D=1.2 nM, B-max=205 fmol/mg protein). Half-maximal reduction was observed at 10 mM for gallamine, 1 mM for alcuronium and 92 mu M for W 84. In conclusion, the muscarinic modulators have little effect on the hist amine H-1-receptors.