Cooperativity and Oligomeric Status of Cardiac Muscarinic Cholinergic Receptors

Abstract

Muscarinic cholinergic receptors can appear to be more numerous when labeled by [(3)H]quinuclidinylbenzilate (QNB) than by N-[(3)H]methylscopolamine (NMS). The nature of the implied heterogeneity has been studied with M(2) receptors in detergent-solubilized extracts of porcine atria. The relative capacity for [(3)H]NMS and [(3)H]QNB was about 1 in digitonin-cholate, 0.56 in cholate-NaCl, and 0.44 in Lubrol-PX. Adding digitonin to extracts in cholate-NaCl increased the absolute capacity for both radioligands, and the relative capacity increased to near 1. The latency cannot be attributed to a chemically impure radioligand, instability of the receptor, an irreversible effect of NMS, or a failure to reach equilibrium. Binding at near-saturating concentrations of [(3)H]QNB in cholate-NaCl or Lubrol-PX was blocked fully by unlabeled NMS, which therefore appeared to inhibit noncompetitively at sites inaccessible to radiolabeled NMS. Such an effect is inconsistent with the notion of functionally distinct, noninterconverting, and mutually independent sites. Both the noncompetitive effect of NMS on [(3)H]QNB and the shortfall in capacity for [(3)H]NMS can be described quantitatively in terms of cooperative interactions within a receptor that is at least tetravalent; no comparable agreement is possible with a receptor that is only di- or trivalent. The M(2) muscarinic receptor therefore appears to comprise at least four interacting sites, presumably within a tetramer or larger array, and ligands appear to bind in a cooperative manner under at least some conditions.

Oligomeric potential of the M2 muscarinic cholinergic receptor

“…A distinct and more pronounced heterogeneity has been observed more recently in the binding of antagonists to the M 2 muscarinic receptor and the D 2 dopamine receptor (Wreggett and Wells 1995;Armstrong and Strange 2001;Park et al 2002). In each case, a comparison of two radiolabeled antagonists revealed a difference in apparent capacity such that some receptors appeared to be of anomalously low affinity for one of the ligands.…”

“…Whereas the nucleotide-sensitive binding of agonists to myocardial membranes is inconsistent with the notion of a transient 1 : 1 complex between receptor and G protein (Lee et al 1986;Green et al 1997), the data can be described in terms of cooperativity within a receptor that is at least tetravalent . Cooperativity within a comparatively large aggregate also can account for otherwise paradoxical effects identified in the binding of radiolabeled antagonists to muscarinic receptor extracted from porcine atria in cholate-NaCl (Park et al 2002). A two-fold difference in the apparent capacity for N-[ 3 H]methylscopolamine and [ 3 H]quinuclidinylbenzilate and an accompanying noncompetitive effect of unlabeled N-methylscopolamine on the binding of [ 3 H]quinuclidinylbenzilate can be described in terms of cooperative effects among four or more interacting sites (Park et al 2002).…”

“…The analysis of cooperative effects in terms of explicit mechanistic models has suggested that the M 2 muscarinic receptor is at least tetrameric (Wreggett and Wells 1995;Chidiac et al 1997;Park et al 2002). Direct biochemical evidence for oligomers derives primarily from measurements of coimmunoprecipitation and energy transfer, which generally have not allowed dimers to be distinguished from larger aggregates.…”

Characterization of radioligand binding to a transmembrane receptor reconstituted into Lipobeads

“…Binding to proteoliposomes was measured essentially as described previously [11]. Aliquots of the liposomal preparation (3 lL) were added to buffer A (50 lL) containing [ 3 H]QNB and any unlabeled ligands, and the mixture was incubated at 30°C for 2 h. Routine estimates of capacity were performed at a saturating concentration of [ 3 H]QNB ($100 nM).…”

“…Further details regarding the analyses and related statistical procedures have been described elsewhere [11][12][13][14].…”