A Fluorescence Resonance Energy Transfer-based M2 Muscarinic Receptor Sensor Reveals Rapid Kinetics of Allosteric Modulation

Abstract

Allosteric modulators have been identified for several G protein-coupled receptors, most notably muscarinic receptors. To study their mechanism of action, we made use of a recently developed technique to generate fluorescence resonance energy transfer (FRET)-based sensors to monitor G protein-coupled receptor activation. Cyan fluorescent protein was fused to the C terminus of the M 2 muscarinic receptor, and a specific binding sequence for the small fluorescent compound fluorescein arsenical hairpin binder, FlAsH, was inserted into the third intracellular loop; the latter site was labeled in intact cells by incubation with FlAsH. We then measured FRET between the donor cyan fluorescent protein and the acceptor FlAsH in intact cells and monitored its changes in real time. Agonists such as acetylcholine and carbachol induced rapid changes in FRET, indicative of agonist-induced conformational changes. Removal of the agonists or addition of an antagonist caused a reversal of this signal with rate constants between 400 and 1100 ms. The allosteric ligands gallamine and dimethyl-W84 caused no changes in FRET when given alone, but increased FRET when given in the presence of an agonist, compatible with an inactivation of the receptors. The kinetics of these effects were very rapid, with rate constants of 80 -100 ms and ≈200 ms for saturating concentrations of gallamine and dimethyl-W84, respectively. Because these speeds are significantly faster than the responses to antagonists, these data indicate that gallamine and dimethyl-W84 are allosteric ligands and actively induce a conformation of the M 2 receptor with a reduced affinity for its agonists.

Nonequilibrium Activation of a G-Protein-Coupled Receptor

“…FRET experiments were performed with whole cells as described previously (Vilardaga et al, 2003Hoffmann et al, 2005;Nikolaev et al, 2006;Zü rn et al, 2009;Maier-Peuschel et al, 2010;Reiner et al, 2010). In brief, transfected cells grown on coverslips were washed with HBSS and maintained in buffer A (140 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , and 10 mM HEPES, pH 7.3) at room temperature.…”

Fluorescence/Bioluminescence Resonance Energy Transfer Techniques to Study G-Protein-Coupled Receptor Activation and Signaling

“…A large number of similar GPCR activation sensors has been developed since then, including the ␤ 1 -and ␤ 2 -adrenergic, A 2A -adenosine, M 1 -, M 2 -, M 3 -, and M 5 -muscarinic, and B 2 -bradykinin receptors (Vilardaga et al, 2003Hoffmann et al, 2005;Chachisvilis et al, 2006;Rochais et al, 2007;Jensen et al, 2009;Maier-Peuschel et al, 2010;Reiner et al, 2010;Ziegler et al, 2011;see Table 2). The attachment of two relatively large fluorescent proteins is surprisingly well tolerated by many GPCRs, as evidenced by cell surface expression, maintained ligand binding, and often even signaling properties.…”

“…There is no systematic comparison of FlAsH-versus YFP-labeled FRET sensors to provide a clear explanation for this increase; a plausible cause is the more rigid attachment of the FlAsH label via the four cysteines to its binding sequence, which may add an orientational component to the FRET signal. After this initial description, a large number of CFP/ FlAsH-based GPCR sensors have been reported (Nikolaev et al, 2006c;Zü rn et al, 2009;Maier-Peuschel et al, 2010;Reiner et al, 2010;Ziegler et al, 2011).…”

Comparison of the Activation Kinetics of the M3Acetylcholine Receptor and a Constitutively Active Mutant Receptor in Living Cells

“…Over the past few years, we have reported the generation of conformational sensors based on fluorescence resonance energy transfer (FRET) for different GPCRs (Vilardaga et al, 2003;Hoffmann et al, 2005;Zü rn et al, 2009;Maier-Peuschel et al, 2010;Alvarez-Curto et al, 2011;Ziegler et al, 2011). Such conformational sensors are usually modified with CFP at the C terminus and a second fluorophore (YFP or a tetracysteine-motif capable of binding a small soluble fluorophore called FlAsH) within the third intracellular loop of the receptor (Hoffmann et al, 2008;Vilardaga et al, 2009).…”