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 approximately 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.

FIGURE 1.

Generation and functionality of the muscarinic M₂ receptor sensor. A, schematic structure of the sensor. CFP was fused to the C terminus of the FLAG-tagged human muscarinic M₂ receptor via a 5-amino acid linker GSGEG, and the FlAsH motif CCPGCC was introduced into the shortened ICL3 with the indicated linkers. B, transfected HEK-TsA201 cells were labeled with FlAsH and analyzed by laser scanning microscopy. Confocal pictures show a distinct membrane staining in both the CFP and the FlAsH channels. C, radioligand binding studies with CHO cell membranes expressing wild-type M₂ receptor or M₂ receptor sensor are shown. Competitive [³H]NMS displacement curves for carbachol and gallamine are presented (data are shown as means ± S.E., n = 3). The IC₅₀ values were 7.3 ± 2.2 and 10.0 ± 1.7 μm for carbachol, 11.5 ± 1.4 and 11.5 ± 3.9 μm for gallamine, for wild-type receptor, and M₂ receptor sensor, respectively. D, GIRK current measurements in HEK-TsA201 cells transfected with the M₂ receptor sensor and GIRK1/4 and activated with different concentrations of acetylcholine show the signaling of the receptor sensor to the GIRK channels with EC₅₀ values of 100 ± 7 nm (mean ± S.E., n = 5). Cells without transfected receptor constructs showed no effects of acetylcholine on GIRK currents (data not shown). E, inhibition of forskolin-induced cAMP signals by the wild-type M₂ receptor and the M₂ receptor sensor was measured as described under “Experimental Procedures.” Concentration-response curves for carbachol in absence or presence of 100 μm gallamine are presented. The EC₅₀ values were 1.6 ± 0.3 and 2.8 ± 0.5 μm for carbachol, 31.4 ± 0.3 and 36.4 ± 0.4 μm for carbachol in presence of gallamine, for wild-type receptor, and M₂ receptor sensor, respectively (n = 4–5). Error bars, S.E.

FIGURE 2.

Effects of agonists on the FRET response of the M₂ receptor sensor. Fluorescence emission was measured at 480 nm (F₄₈₀) and at 535 nm (F₅₃₅) from cells expressing the M₂ receptor sensor and superfused for the indicated period of time with agonist. A, individual recording from a single cell shows that acetylcholine induced a rapid increase in F₄₈₀ and a corresponding decrease in F₅₃₅, resulting in a reduced ratio F₅₃₅/F₄₈₀, representing FRET (bottom trace). Upon washout of acetylcholine, all traces reverted to their original values. B, concentration-response curves for the FRET changes evoked by acetylcholine and carbachol revealed EC₅₀ values of 1.5 ± 0.3 μm and 5.1 ± 0.4 μm, respectively (means ± S.E. (error bars), n = 6–8).

FIGURE 3.

Reversal of agonist-induced FRET signals of the M₂ receptor sensor by antagonists. Shown are responses to the agonists acetylcholine (A) or carbachol (B). Top panels, F₅₃₅/F₄₈₀ traces (normalized to the starting values) as in Fig. 2, representative of at least six independent experiments. Washout of the agonist as well as addition of atropine in the continued presence of the respective agonist resulted in a fast reversal of the FRET signal. Bottom panels, time constants for the respective experiments, in which statistical significance was assessed using a t test. Error bars, S.E. **, p < 0.01.

FIGURE 4.

Effects of gallamine on the FRET signals of the M₂ receptor sensor. A–C, F₅₃₅/F₄₈₀ traces (normalized to the starting values) as in Fig. 2, representative for 10–15 independent experiments. A, gallamine (10 μm) caused no changes in FRET when superfused alone. Gallamine 10 μm reverted the signal induced by 100 μm acetylcholine (B, left) or 100 μm carbachol (C, left). B and C, right, average effects of the indicated concentrations of gallamine, expressed as percent reversal of the maximal agonist-induced FRET signal (means ± S.E. (error bars), n = 6–8).

FIGURE 5.

Effects of dimethyl-W84 on the FRET signals of the M₂ receptor sensor. A–C, F₅₃₅/F₄₈₀ traces (normalized to the starting values) as in Fig. 2, representative for 10–15 independent experiments. A, dimethyl-W84 (W84, 10 μm) caused no changes in FRET when superfused alone. Dimethyl-W84 (10 μm) reverted the signal induced by 100 μm acetylcholine (B, left) or 100 μm carbachol (C, left). B and C, right, average effects of the indicated concentrations of dimethyl-W84, expressed as percent reversal of the maximal agonist-induced FRET signal (means ± S.E. (error bars), n = 6–8).

FIGURE 6.

Kinetics of the reversal of agonist-induced FRET signals. Data are derived from experiments as in Figs. 4 and 5. A and B, average time constants (τ) of the effects of atropine (10 μm), methoctramine (100 μm), dimethyl-W84 (1 mm), and gallamine (1 mm) in reversing the FRET signals induced by 100 μm acetylcholine (A) or 100 μm carbachol (B); (means ± S.E. (error bars), n = 6–8). Statistical significance of the differences between the kinetics of atropine and allosteric ligands was assessed by analysis of variance. ***, p < 0,001. C–E, representative individual FRET traces for atropine, dimethyl-W84, and gallamine applied after 100 μm acetylcholine and fitted with a monoexponential function to estimate the τ values shown in A. F, reversal of the FRET signal in the continued presence of 100 μm acetylcholine induced by addition of gallamine (1 mm) or atropine (10 μm) to the superfusion buffer; shown is the percent reversal of the acetylcholine-induced FRET signal. Traces are representative of six to eight independent experiments.

FIGURE 7.

Saturation of time and rate constants for atropine, gallamine, and methoctramine in reverting 100 μm acetylcholine-induced effects on the M₂ receptor sensor. A, time constants (τ) for the three ligands as a function of the ligand concentration. B, rate constants (k_obs) for atropine, methoctramine, and gallamine at 1 mm. Data are means ± S.E. (error bars), n = 6–8. ***, p < 0.001 compared with atropine and methoctramine (analysis of variance).