FRET-Based Detection of M1 Muscarinic Acetylcholine Receptor Activation by Orthosteric and Allosteric Agonists

Muscarinic acetylcholine receptors (mAChRs) are 7-transmembrane, G protein-coupled receptors that regulate a variety of physiological processes and represent potentially important targets for therapeutic intervention. mAChRs can be stimulated by full and partial orthosteric and allosteric agonists, however the relative abilities of such ligands to induce conformational changes in the receptor remain unclear. To gain further insight into the actions of mAChR agonists, we have developed a fluorescently tagged M(1) mAChR that reports ligand-induced conformational changes in real-time by changes in Förster resonance energy transfer (FRET).Variants of CFP and YFP were inserted into the third intracellular loop and at the end of the C-terminus of the mouse M(1) mAChR, respectively. The optimized FRET receptor construct (M(1)-cam5) was expressed stably in HEK293 cells.The variant CFP/YFP-receptor chimera expressed predominantly at the plasma membrane of HEK293 cells and displayed ligand-binding affinities comparable with those of the wild-type receptor. It also retained an ability to interact with Gα(q/11) proteins and to stimulate phosphoinositide turnover, ERK1/2 phosphorylation and undergo agonist-dependent internalization. Addition of the full agonist methacholine caused a reversible decrease in M(1) FRET (F(EYFP)/F(ECFP)) that was prevented by atropine pre-addition and showed concentration-dependent amplitude and kinetics. Partial orthosteric agonists, arecoline and pilocarpine, as well as allosteric agonists, AC-42 and 77-LH-28-1, also caused atropine-sensitive decreases in the FRET signal, which were smaller in amplitude and significantly slower in onset compared to those evoked by methacholine.The M(1) FRET-based receptor chimera reports that allosteric and orthosteric agonists induce similar conformational changes in the third intracellular loop and/or C-terminus, and should prove to be a valuable molecular reagent for pharmacological and structural investigations of M(1) mAChR activation

Acceptor photobleaching of the three M₁ mAChR cameleons showing primarily plasma membrane localization.

<p>HEK293 cells were transiently transfected with M₁-cam1, M₁-cam2 or M₁-cam5 and imaged by confocal microscopy (458 nm excitation, 470–500 nm emission). Areas of the plasma membrane (delineated by red lines in each image) were bleached using repeated brief exposures to high intensity 514 nm illumination. The graphs show the signal at 458 nm excitation for 470–500 nm emission (ECFPc, cyan line) and >530 nm (EYFP^F46L emission, yellow line), with acceptor photobleach initiated at the arrow. The fluorescence signals from a non-photobleached region were also assessed as a control, which was comparable for all constructs, but only shown for M₁-cam1 (area outlined in cyan within the image and fluorescence within the graph for ECFPc (dark blue) and EYFP^46L (red)). These findings are representative of photobleaching experiments from at least 3 separate transfections for each construct.</p

Cellular localization of M₁-cameleons transiently expressed in HEK293 cells.

<p>HEK293 cells were transiently transfected with M₁-cam1 (<b>A</b>), M₁-cam2 (<b>B</b>), M₁-cam3 (<b>C</b>), M₁-cam4 (<b>D</b>) or M₁-cam5 (<b>E</b>). Images were acquired by confocal microscopy and show fluorescence emission at >530 nm following excitation at 514 nm. Scale bar, 15 µm.</p

Comparisons of maximal FRET changes and rate constants for a variety of orthosteric and allosteric ligands in HEK293-M₁-cam5 cells.

<p>Cells were stimulated with a maximally effective concentration of each agonist and FRET changes (<b>A</b>) and K<i>_obs</i> values (<b>B</b>) were determined as described above. Data are presented as means ± s.e.m. from at least three independent experiments. One-way AVOVA (*<i>p</i><0.05; **<i>p</i><0.005; ***<i>p</i><0.0001).</p

Internalization characteristics of the M₁-cam5 mAChR stably expressed in HEK293 cells.

<p>HEK293-M₁-cam5 cells were treated with various concentration of MCh for 45 min (to assess the concentration-dependency of receptor internalization), or with MCh (300 µM) for 0–60 min (to assess the time-dependency of receptor internalization). Cellular distributions of M₁-cam5 mAChR were monitored by confocal microscopy. For quantification of intracellular fluorescence at least 10 individual cells in five random fields of view were examined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029946#s2" target="_blank">Methods</a> section. Data represent means ± s.e.m. from three independent experiments.</p

Overview of M₁ AChR chimeric constructs.

<p>Primers used for introducing <i>AgeI</i> site into the i3 loop are shown where applicable.</p

MCh-induced changes in FRET in HEK293 M₁-cam5 cells.

<p>HEK293 cells stably expressing M₁-cam5 were observed using fluorescence imaging with single wavelength excitation (436 nm) and dual wavelength emission (436 nm to detect ECFPc and 535 nm to detect EYFP^46L. <b>A</b>. Representative images showing plasma membrane distribution of ECFPc (480 nm, left panel) and EYFP^46L (535 nm, middle panel), which overlap (right panel) as expected for signals from the same population of receptors. <b>B–D</b>, right panels: blue and yellow traces represent signals from ECFP and EYFP, respectively; left panels: red traces represent the FRET signal (ratio of F_EYFP/F_ECFP). Addition of MCh (100 µM) induced decreases in FRET, which remained constant throughout the application period (30–40 s; <b>B</b>); this effect was reversed on addition of atropine (1 µM; <b>C</b>); and the MCh-induced change in FRET ratio could be completely prevented by pre-addition of atropine (<b>D</b>). FRET data have been normalized so that the initial FRET signal is 100%. Emission traces are expressed as the change in fluorescence intensity from the basal fluorescence level (<i>F</i>/<i>F</i>₀). Representative traces of at least three independent experiments are shown.</p

Apparent binding affinities (expressed as −log <i>K</i>_B values) for various mAChR agonists and antagonists at the M₁-cam5 AChR, determined by [³H]NMS competition binding.

<p>Data are shown as means ± s.e.m. for duplicate determinations in the number of separate experiments (n) indicated.</p

Concentration-dependency and dynamics of MCh-induced FRET changes in HEK293-M₁-cam5 cells.

<p><b>A</b>. HEK293 cells stably expressing M₁-cam5 were stimulated with various concentration of MCh (0.3–300 µM) as above, and change in FRET ratio recorded. Data represent the means ± SEM from at least three independent experiments. <b>B</b>. Correlation between the rate constant (K<i>_obs</i>) and MCh concentration was analysed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029946#s2" target="_blank">Methods</a> section. K<i>_obs</i> values were obtained by fitting the FRET data to a single-phase exponential decay. Data represent the means ± s.e.m. from at least three independent experiments.</p