Abstract
The binding of allosteric modulators to G protein-coupled receptors (GPCRs) is often described by an equilibrium allosteric ternary complex model (ATCM). This study evaluated the effects of three modulators on the binding of [3H]N-methylscopolamine ([3H]NMS) to the human M2 muscarinic acetylcholine receptor (mAChR). The binding of each modulator was more complex than predicted by the ATCM; the inhibitors heptane-1,7-bis-(dimethyl-3-phthalimidopropyl)-ammonium bromide and gallamine yielded biphasic curves that were described empirically by a two-site binding model, whereas the enhancer alcuronium yielded a bell-shaped curve. Radioligand dissociation assays revealed that the modulators retarded [3H]NMS kinetics such that the system never attained equilibrium. Subsequent application of a kinetic ATCM accommodated and quantified all experimental observations. Our findings confirm and extend previous studies on the use of a kinetic ATCM for mAChR allosteric enhancers, but also highlight how complex curves displayed by allosteric inhibitors can be misinterpreted in terms of multisite orthosteric binding. It is possible that similar behavior of other allosteric modulators at GPCRs may reflect nonequilibrium binding artifacts rather than deviation from an ATCM.
Footnotes
-
This work was supported by Project Grant 251538 of the National Health and Medical Research Council of Australia (NHMRC). A.C. and P.S. are Senior Research Fellows of the NHMRC. V.A. and L.M. are recipients of a Melbourne Research Scholarship.
-
DOI: 10.1124/jpet.103.059840.
-
ABBREVIATIONS: GPCR, G protein-coupled receptor; mAChR, muscarinic acetylcholine receptor; ATCM, allosteric ternary complex model; [3H]NMS, [3H]N-methylscopolamine; C7/3-phth, heptane, 1,7-bis-(dimethyl-3′-phthalimidopropyl)-ammonium bromide; CHO, Chinese hamster ovary; AIC, Akaike's Information Criterion; K-L, Kullback-Liebler.
- Received September 10, 2003.
- Accepted November 26, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|