Cardiac Muscarinic Receptors. Relationship between the G Protein and Multiple States of Affinity

Abstract

An expanded version of the mobile receptor model has been assessed in studies on the binding of N-[3H]methylscopolamine and [35S]GTPgammaS to cardiac muscarinic receptors and their attendant G proteins in ventricular membranes from hamster. The model comprises two pools of receptor, one of which lacks G proteins, and a heterogeneous population of G proteins that compete for the receptor within the G protein-containing pool. To guide the formulation of the model itself and to define the various parameters, data were combined from assays performed under various conditions with native membranes and following irreversible blockade of about 80% of the receptors with propylbenzilylcholine mustard. Multiple G proteins are indicated primarily by multiple states of affinity evident in the dose-dependent effect of guanyl nucleotides on the binding of carbachol; G protein-free receptors are indicated by sites of low affinity for carbachol that survive treatment with the mustard. The expanded model generally succeeds where more frugal schemes have been inadequate, but it nevertheless fails to yield a mechanistically consistent description of the data. Guanyl nucleotides and partial alkylation do not affect the inhibitory potency of carbachol in a manner consistent with their supposed effect on the equilibrium between uncoupled and G protein-coupled receptors. As inferred from the model, G proteins are lost upon alkylation of the receptor, and their numbers are regulated by guanyl nucleotides. Parameters estimated via N-[3H]methylscopolamine are wholly inconsistent with the same parameters estimated via [35S]GTPgammaS. The failure of the model suggests that multiple states of affinity may not arise from a ligand-regulated equilibrium between free receptors and G proteins on the one hand and one or more RG complexes on the other.

Oligomeric potential of the M2 muscarinic cholinergic receptor

“…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).…”

“…It has been suggested that the multiple states of affinity are induced by the G protein in an otherwise homogeneous population of mutually independent sites Gilman 1987;Birnbaumer et al 1990). This widely held view is problematic, however, and it is particularly difficult to sustain on quantitative grounds (Lee et al 1986;Sinkins and Wells 1993;Green et al 1997). An alternative view attributes the observed heterogeneity to nucleotide-sensitive cooperative effects between interacting sites (Chidiac and Wells 1992;Chidiac et al 1997).…”

Coupling of G Proteins to Reconstituted Monomers and Tetramers of the M2 Muscarinic Receptor

“…Two versions of the model were considered: a general case in which no restriction was placed on the affinity of the G protein for the receptor (Scheme 2) and the extreme case in which the values of K G and K G.N were set arbitrarily at 10 Ϫ15 M, approximating a stable RG complex (Scheme 3). Both schemes were applied with one class of G protein, as proposed originally (27), and with two classes (G 1 and G 2 ), the members of which compete for a single site on the receptor (33). The latter case of Scheme 2 is a specific example of ligandbiased signaling, in that the preference of the receptor for G 1 or G 2 will be determined by the relative affinity of the agonist for the free receptor on the one hand and each RG j complex on the other.…”

“…Scheme 2 is a variant of the ternary complex model (27) in which two different G proteins (G j , j ϭ 1 or 2) compete for a single site on the receptor (R) to form the spontaneously dissociating complex RG 1 or RG 2 (32,33 interact randomly with all receptors ([R] t ) within the relevant volume. The radioligand (P) and unlabeled ligands (A) bind to the receptor with equilibrium dissociation constants K P and K A , respectively, and a guanylyl nucleotide (N), such as GMP-PNP, binds to each G protein with the equilibrium dissociation constant K Nj .…”

“…The free concentrations of receptor, G proteins, and all ligands were calculated numerically from the total concentrations, as described previously (32,33). Parameters entered explicitly into the fitting procedure were…”