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

Abstract

Muscarinic M2 acetylcholine receptors contain an allosteric site that is probably located at the entrance of the ligand binding pocket above the orthosteric binding site. With the orthosteric area not occupied, allosteric agents might gain access to this site. The interaction of allosteric agents with orthoster-occupied receptors is known to depend on the buffer conditions in an alloster-specific fashion. Utilizing the buffer-dependent potency shift as an indicator, we aimed to find out for two rod-like shaped and flexible allosteric agents whether or not there is evidence for a switch in the site of attachment in free compared with [3H]N-methylscopolamine ([3H]NMS)-occupied porcine heart M2 receptors. These agents are the bispyridinium compounds WDuo3 (1,3-bis[4-(phthalimidomethoxyimino-methyl)-pyridinium-1-yl] propane dibromide) and Duo3 (4,4'-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1'-propane-1,3-diyl-bis-pyridinium dibromide). The prototype allosteric agents gallamine and alcuronium were included. Inhibition of [3H]NMS association was taken to reflect alloster interaction with free receptors, inhibition of [3H]NMS dissociation indicated binding to [3H]NMS-occupied receptors. In Na,K,Pi buffer (4 mM Na2HPO4, 1 mM KH2PO4, pH 7.4 at 23 degrees C) compared with Mg,Tris,Cl,Pi buffer (45 mM Tris-HCl, 2.6 mM MgHPO4, pH 7.3 at 37 degrees C) WDuo3 underwent the same loss of potency for the interaction with either free or [3H]NMS-liganded receptors. The loss of potency was quantified by a potency ratio (PR), i.e. the ratio between the concentrations of the modulator leading to a half-maximal delay of [3H]NMS association or dissociation, respectively, in Mg,Tris,Cl,Pi compared with Na,K,Pi. For WDuo3 the ratios were PRass=27 and PRdiss=22, respectively. For Duo3, the interaction with free and [3H]NMS-occupied receptors only slightly depended on the composition of the incubation medium: PRass=1.3, PRdiss=2.8. In contrast to the other agents, the concentration-effect curves of which had slope factors 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, for gallamine to PRass=216 and PRdiss=159. In conclusion, there was a wide variation between the allosteric agents with regard to the respective buffer dependence of action. Yet, for a given allosteric agent, the interaction with either free or [3H]NMS-occupied receptors was always characterized by the same buffer-dependent shift. Thus, even the applied rod-shaped allosteric agents do not appear to switch to the orthosteric site in free compared with orthoster-occupied M2 receptors.

Interactions of Orthosteric and Allosteric Ligands with [3H]Dimethyl-W84 at the Common Allosteric Site of Muscarinic M2 Receptors

“…However, tacrine and Duo3 also slow down the dissociation rate of [ 3 H]NMS with slope factors greater than 1 (Table 3, Fig. 7) (Potter et al, 1989;Trä nkle and Mohr, 1997;Schröter et al, 2000). In this experiment, the orthosteric site is occupied by [ 3 H]NMS; therefore, the effects causing the slope factors to be greater than 1 are allosteric in nature and independent of occupancy of the orthosteric site.…”

“…If AF-DX 384 were purely allosteric, as proposed for the analog AF-DX 116 (Lanzafame et al, 2001) Fig. 7) (Potter et al, 1989;Schröter et al, 2000). This might suggest simultaneous and positively cooperative binding of these ligands to both the orthosteric and allosteric sites.…”

“…These modulators may be distinguished from other allosteric modulators by their different interactions with the binding of radioligands to the orthosteric binding site (Potter et al, 1989;Trä nkle and Mohr, 1997;Schröter et al, 2000). In particular, their modulation of the binding of orthosteric ligands is characterized by slope factors greater than 1, which, at least in principle, is compatible with positive homotropic cooperativity.…”

Allosteric Site on Muscarinic Acetylcholine Receptors: A Single Amino Acid in Transmembrane Region 7 Is Critical to the Subtype Selectivities of Caracurine V Derivatives and Alkane-Bisammonium Ligands

“…On the other hand, affinities for the free receptor cannot be extracted from these data. There are, however, some indications that suggest that there is a common binding mode for allosteric ligands at the free and NMSliganded receptors (Ellis and Seidenberg, 2000;Schröter et al, 2000). Because [ 3 H]NMS dissociated much more rapidly from the M 2 subtype than from M 5 , one could argue that high-affinity binding of the allosteric ligand is associated with a fast dissociation and low-affinity binding with slow dissociation of [ 3 H]NMS.…”

Allosteric Binding Sites on Muscarinic Acetylcholine Receptors

“…One key observation supporting this concept is that certain indolocarbazole (Lazareno et al, 2000) and androstane (Lazareno et al, 2002) derivatives are predicted to bind to a nonorthosteric site different from that recognized by other allosteric muscarinic ligands such as gallamine or strychnine. Moreover, several allosteric muscarinic ligands, including the acetylcholinesterase inhibitor tacrine (Potter et al, 1989) and the bis-pyridinium derivative Duo3 (Trä nkle and Mohr, 1997;Schröter et al, 2000), have been identified that, in contrast to conventional allosteric muscarinic ligands, display concentration-effect curves with slope factors Ͼ1. The complex binding properties of Duo3 suggested that this ligand, similar to the above-mentioned indolocarbazole and androstane derivatives, may interact with a second allosteric binding site (Trä nkle and Mohr, TABLE 1 Major advantages associated with the potential therapeutic use of GPCR ligands acting on allosteric sites…”