Modulation of the M2 Muscarinic Cholinergic Receptor by Cholesterol

M2 muscarinic receptor extracted from Sf9 cells in cholate-NaCl differs from that extracted from porcine sarcolemmal membranes. Whereas the latter has been shown to exhibit non-competitive effects in the binding of N-methylscopolamine (NMS) and quinuclidinylbenzilate (QNB), which can be explained in terms of cooperativity within a receptor that is at least tetravalent, binding to the former is essentially competitive. Levels of cholesterol in Sf9 membranes were only 5% of those in sarcolemmal membranes and were increased to about 100% by means of cholesterol-methyl-β-cyclodextrin. M2 receptors extracted from CHL-treated Sf9 membranes resembled those from heart; that is, cholesterol induced a pronounced heterogeneity detected in the binding of both radioligands, including a shortfall in the apparent capacity for [3H]NMS, and there were marked discrepancies in the apparent affinity of NMS as estimated directly and via the inhibition of [3H]QNB. The data can be described quantitatively in terms of cooperative effects among six or more interacting sites, apparently within an oligomer. Cholesterol also was found to increase the affinity of the receptor for NMS and QNB, and the effect was examined for its possible relationship to the known interconversion of cardiac muscarinic receptors between an agonist-specific (R*) and an antagonist-specific (R) state. Cholesterol and N-ethylmaleimide (NEM) were compared for their effect on the affinity of NMS, QNB and four muscarinic agonists, and the data were assessed in terms of an explicit mechanistic model for a receptor that interconverts spontaneously between two states. The data can be described equally well by an effect of cholesterol on either the distribution of receptors between R and R* or the affinity of all ligands for both states, with an accompanying effect of NEM on either the affinity or the distribution between states, respectively. Since NEM is known from other data to favor R* over R, cholesterol appears to increase affinity per se. Cholesterol therefore is a determinant of affinity and cooperativity in the binding of orthosteric ligands to the M2 receptor. Both effects are observed in solution and therefore appear to arise from a direct interaction between the lipid and the receptor.Ph

Localization of muscarinic receptors M1R, M2R and M3R in the human colon

BackgroundMuscarinic acetylcholine receptors (MR) are involved in multiple intestinal reflexes. The cellular localization of subtypes of MRs within enteric circuits mediating muscle and mucosal reflexes remains to be demonstrated. This study aimed to localize the three functionally significant subtypes of MRs in human colon.MethodsReverse transcriptase-PCR was used to determine expression levels of muscarinic receptor subtype (MRs) M1Rs, M2Rs and M3Rs in human colon. Indirect immunofluorescence and confocal microscopy was used to localize MRs in cryostat-cut sections of human colon. Sections were double labeled for multiple cellular and neurochemical markers. Western blotting was used to confirm specificity of the muscarinic antisera used.Key resultsAll three MR subtypes were expressed in human colon. Immunoreactivity (IR) for M2Rs and M3Rs was most abundant in circular and longitudinal muscle. M1R-IR was most abundant on myenteric and submucosal nerve cells, both cholinergic and nitrergic. M3R-IR was also present on populations on myenteric nerve cell bodies. Immunoreactivity for all three receptors was present on nerve fibers in the circular muscle.Conclusions & inferencesIn the human colon, subtypes of MRs were present on multiple cell types within the enteric circuits underlying motility, secretory and vasoactive reflexes. The cellular distribution for MRs found in this study agrees with data from functional studies, providing insight into the role MRs have in mediating enteric cholinergic neurotransmission.A. M. Harrington, C. J. Peck, L. Liu, E. Burcher, J. M. Hutson, & B. R. Southwel

Oligomeric Size of the M2 Muscarinic Receptor in Live Cells as Determined by Quantitative Fluorescence Resonance Energy Transfer*

Fluorescence resonance energy transfer (FRET), measured by fluorescence intensity-based microscopy and fluorescence lifetime imaging, has been used to estimate the size of oligomers formed by the M2 muscarinic cholinergic receptor. The approach is based on the relationship between the apparent FRET efficiency within an oligomer of specified size (n) and the pairwise FRET efficiency between a single donor and a single acceptor (E). The M2 receptor was fused at the N terminus to enhanced green or yellow fluorescent protein and expressed in Chinese hamster ovary cells. Emission spectra were analyzed by spectral deconvolution, and apparent efficiencies were estimated by donor-dequenching and acceptor-sensitized emission at different ratios of enhanced yellow fluorescent protein-M2 receptor to enhanced green fluorescent protein-M2 receptor. The data were interpreted in terms of a model that considers all combinations of donor and acceptor within a specified oligomer to obtain fitted values of E as follows: n = 2, 0.495 ± 0.019; n = 4, 0.202 ± 0.010; n = 6, 0.128 ± 0.006; n = 8, 0.093 ± 0.005. The pairwise FRET efficiency determined independently by fluorescence lifetime imaging was 0.20–0.24, identifying the M2 receptor as a tetramer. The strategy described here yields an explicit estimate of oligomeric size on the basis of fluorescence properties alone. Its broader application could resolve the general question of whether G protein-coupled receptors exist as dimers or larger oligomers. The size of an oligomer has functional implications, and such information can be expected to contribute to an understanding of the signaling process

Membrane cholesterol access into a G-protein-coupled receptor

Cholesterol is a key component of cell membranes with a proven modulatory role on the function and ligand-binding properties of G-protein-coupled receptors (GPCRs). Crystal structures of prototypical GPCRs such as the adenosine A2A receptor (A2AR) have confirmed that cholesterol finds stable binding sites at the receptor surface suggesting an allosteric role of this lipid. Here we combine experimental and computational approaches to show that cholesterol can spontaneously enter the A2AR-binding pocket from the membrane milieu using the same portal gate previously suggested for opsin ligands. We confirm the presence of cholesterol inside the receptor by chemical modification of the A2AR interior in a biotinylation assay. Overall, we show that cholesterol’s impact on A2AR-binding affinity goes beyond pure allosteric modulation and unveils a new interaction mode between cholesterol and the A2AR that could potentially apply to other GPCRs