Modes of allosteric interactions with free and [H-3]N-methylscopolamine-occupied muscarinic M-2 receptors as deduced from buffer-dependent potency shifts

Muscarinic M-2 acetylcholine receptors contain an allosteric site that is p robably located at the entrance of the ligand binding pocket above the orth osteric binding site. With the orthosteric area not occupied, allosteric ag ents might gain access to this site. The interaction of allosteric agents w ith orthoster-occupied receptors is known to depend on the buffer condition s in an alloster-specific fashion. Utilizing the buffer-dependent potency s hift as an indicator, we aimed to find out for two rod-liked shaped and fle xible allosteric agents whether or not there is evidence for a switch in th e site of attachment in free compared with [H-3]N-methylscopolamine ([H-3]N MS)-occupied porcine heart M-2 receptors. These agents are the bispyridiniu m compounds WDuo3 (1,3-bis[4-(phthalimidomethoxyimino-methyl)-pyridinium-1- yl]propane dibromide) and Duo3 (4,4'-bis-[2,6-dichloro-benzyloxy-imino-meth yl]-1,1'-propane-1,3-diyl-bis-pyridinium dibromide). The prototype alloster ic agents gallamine and alcuronium were included. Inhibition of [H-3]NMS as sociation was taken to reflect alloster interaction with free receptors, in hibition of [H-3]NMS dissociation indicated binding to [H-3]NMS-occupied re ceptors. In Na,K,P-i buffer (4 mM Na2HPO4, 1 mM KH2PO4, pH 7.4 at 23 degree sC) compared with Mg,Tris,Cl,P-i buffer (45 mM Tris-HCl, 2.6 mM MgHPO4, pH 7.3 at 37 degreesC) WDuo3 underwent the same loss of potency for the intera ction with either free or [H-3]NMS-liganded receptors. The loss of potency was quantified by a potency ratio (PR), i.e. the ratio between the concentr ations of the modulator leading to a half-maximal delay of [H-3]NMS associa tion or dissociation, respectively, in Mg,Tris,Cl,P-i compared with Na,K,P- i. For WDuo3 the ratios were PRass=22, respectively. For Duo3, the interact ion with free and [H-3]NMS-occupied receptors only slightly depended on the composition of the incubation medium: PRass=1.3, PRdiss=2.8. In contrast t o the other agents, the concentration-effect curves of which had slope fact ors nH not different from unity, the curves of Duo3 were steep (nH about -1 .6). For alcuronium the shift factors amounted to PRass=29 and PRdiss=25, f or gallamine to PRass=216 and PRdis=159. In conclusion, there was a wide va riation between the allosteric agents with regard to the respective buffer dependence of action. Yet, for a given allosteric agent, the interaction wi th either free or [H-3]NMS-occupied receptors was always characterized by t he same buffer-dependent shift. Thus, even the applied rod-shaped allosteri c agents do not appear to switch to the orthosteric site in free compared w ith orthoster-occupied M-2 receptors.