Research ArticleGPCR SIGNALING

The mode of agonist binding to a G protein–coupled receptor switches the effect that voltage changes have on signaling

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Science Signaling  03 Nov 2015:
Vol. 8, Issue 401, pp. ra110
DOI: 10.1126/scisignal.aac7419
  • Fig. 1 The M1 receptor is sensitive to voltage changes.

    (A) Scheme of the FRET-based receptor biosensor M1-cam. Ligand binding induces a conformational change in the receptor that results in decreased FRET. (B) Activation of M1-cam in HEK 293 cells by carbachol (CCh) was detected as a decrease in FRET (F535/F480). Upon application of carbachol, M1-cam displayed robust activation at −90 mV and switched to an even more active state during a depolarization step to +60 mV (top panel, n = 6 cells). The biosensor was not activated by voltage alone (bottom panel, n = 5 cells). (C) Concentration-response curves for the carbachol-dependent activation of M1-cam in HEK 293 cells at −90 mV (black circles) and at +60 mV (open circles). Data are from paired experiments and are means ± SEM of 11 individual cells per data point. (D) FRET response of M1-cam in HEK 293 cells to 10 μM carbachol at different membrane potentials in the presence (black curve, solid circles) or absence (blue curve, open circles) of 500 μM intracellular GTPγS. The data were normalized to the holding potential (FRET/FRET−90 mV) and fitted to a Boltzmann equation. The calculated V0.5 values of M1-cam for control cells and GTPγS-treated cells are depicted. Data are from 11 to 14 cells per data point. The detailed experimental protocol is shown in fig. S1. All data are means ± SEM of the indicated number of individual cells.

  • Fig. 2 Signaling of the M1 receptor to Gq is sensitive to voltage changes.

    (A) Scheme of a FRET biosensor that monitors the Gq protein cycle. (B) In transfected HEK 293 cells expressing the M1 receptor and the Gq sensor, 50 nM carbachol induced Gq activation at about 25% of the maximal extent, which was induced by 10 μM carbachol. The trace represents mean ± SEM of five individual cells. (C) Average traces of Gq sensor activation in HEK 293 cells in response to carbachol (left) or pilocarpine (right) at −90 mV (black) or +60 mV (red). The traces are normalized to the maximal activation of the Gq sensor (induced by 10 μM carbachol). Data are average traces of four to six cells per membrane potential and per agonist. (D) Representative trace of a global Ca2+ transient of a HEK 293 cell transfected with plasmid encoding the Ca2+ indicator Green-GECO1.1. 50 nM carbachol induced a detectable Ca2+ transient in six of eight cells tested. (E) Representative Ca2+ transient of a cell that was exposed to 20 nM carbachol and then subjected to a voltage step from −90 mV to +60 mV. Four of 10 cells responded to the voltage switch with a Ca2+ signal. (F and G) No Ca2+ transients were detected in the presence of 20 nM carbachol at −90 mV (F; six cells tested) or at +60 mV in the absence of carbachol (G; five cells tested).

  • Fig. 3 The effect of depolarization on Gq-coupled muscarinic acetylcholine receptors is receptor subtype–specific.

    (A and B) The activity of wild-type (WT) M1 (A) and M3 (B) receptors was assessed with the Gq FRET biosensor. Application of carbachol resulted in activation of both receptors at −90 mV. During depolarization of the membrane to +60 mV, the M1 receptor induced increased activity of Gq (A; average trace of seven individual cells), whereas the activity of Gq during depolarization was reduced in cells expressing the M3 receptor (B; average trace of six individual cells). (C) Determination of M3 receptor sensitivity to carbachol at +60 mV and at −90 mV. Data are from five to seven cells per concentration tested. (D) Scheme of an arrestin FRET biosensor. Recruitment of arrestins by the active GPCR is detected as an increase in the FRET ratio. Tur, turquoise. (E) Activation of the M1 receptor by acetylcholine (ACh) led to an enhanced FRET signal at −90 mV that was facilitated by depolarization of the membrane (average trace of four individual cells). (F) Activation of the M3 receptor by acetylcholine resulted in a similar FRET signal at −90 mV, which was reduced during depolarization of the membrane to +60 mV (average trace of eight individual cells). All average traces are presented as means (solid lines) ± SEM (gray shading) of the indicated number of individual cells.

  • Fig. 4 Sensitivity of the M3 receptor to voltage changes is ligand-specific.

    (A) Average traces of Gq activation in HEK 293 cells expressing the M3 receptor and the Gq biosensor were measured in response to carbachol (left) and pilocarpine (right) at −90 mV (black) and +60 mV (red). (B) Representative trace of a single cell at −90 mV that was exposed to 100 nM carbachol or to 1 μM pilocarpine and subjected to depolarization of the membrane to +60 mV during the application of each agonist. Note that carbachol was removed completely before pilocarpine was applied (dotted line). (C and D) Calculated binding poses of carbachol and acetylcholine docked to the orthosteric binding pocket of the WT M3 receptor. The poses of carbachol (C) and acetylcholine (D) were very similar and showed an interaction with N6.52 of TM6. (E) Voltage-induced attenuation of receptor activation for 100 nM carbachol and 10 nM acetylcholine. The trace represents an average of four single transfected HEK cells expressing the M3 receptor and the Gq protein biosensor. Carbachol was removed completely before acetylcholine was applied, as indicated by the dotted line. (F) The pose of pilocarpine showed a different orientation toward D3.32 of TM3. The black dashed lines highlight ligand-receptor interactions with residues 3.32 or 6.52 of the orthosteric site. The black numbers indicate minimum distances (in angstrom) between the ligand and the interacting residue. See table S1 for details.

  • Fig. 5 Analysis of ligand binding and the effects of voltage changes on signaling by the N6.52Q mutant M3 receptor.

    (A to C) Calculated binding poses for carbachol (A), acetylcholine (B), and pilocarpine (C) docked to the N6.52Q mutant M3 receptor. The side-chain mutation N6.52Q (green) induced a previously uncharacterized binding pose for carbachol (A) toward TM3, with fewer interactions with TM6. In contrast, the binding poses of acetylcholine (B) and pilocarpine (C) appeared to be similar to those for the WT receptor (compare to Fig. 4). The black lines highlight ligand-receptor interactions with residues 3.32 or 6.52 of the orthosteric site. The black numbers indicate minimum distances (in angstrom) between the ligand and the interacting residue. See table S1 for details. (D) The N6.52Q mutation reversed the directionality of the effect of a voltage change on the signaling of the mutant M3 receptor in response to carbachol (1 μM carbachol, average trace of five individual cells in red) compared to the effect of a voltage change on the WT receptor (50 nM carbachol, black average trace from Fig. 3B). The control N6.52A mutation did not change the effect of a voltage change on receptor signaling (1 μM carbachol, average trace of four individual cells, binding pose in fig. S4). (E) The N6.52Q mutation did not change the effect of voltage change on receptor signaling induced by 100 nM acetylcholine (average trace of five individual cells). Average traces are means (solid trace) or as means (solid trace) ± SEM (gray shading).

  • Fig. 6 Movement of residue 6.52 in TM6 during receptor activation in the WT and N6.52Q mutant M3 receptors.

    (A) Side view (left) and top view (right) of the receptor core highlighting TM5 and TM6. Left: The side-chain conformation of 6.52 in the structure of the inactive M2 receptor (3UON, light green) is very similar to the conformation of the inactive M3 receptor (4DAJ, dark green). The outer bilayer segment of helix 6 and N6.52 are closer to the receptor core in the structures of the active M2 receptor (4MQS in yellow and 4QMT in purple). Right: The different locations of TM6 for both activation states as viewed from the top. The arrows suggest the movement of N6.52 during receptor activation. (B) Superposition of the structures of inactive M3 receptor, its N6.52 mutant, and the active M2 receptor. The inactive state of the WT M3 receptor (green) is similar to the inactive state of the N6.52Q M3 mutant receptor, as shown in salmon. The active state of the M2 receptor is depicted in yellow. The elongated side chain of the N6.52Q mutant M3 receptor does not interfere with receptor activation.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/401/ra110/DC1

    Fig. S1. Analysis of the voltage sensitivity of the M1 receptor when uncoupled from Gq proteins.

    Fig. S2. Analysis of the voltage sensitivity of the M5 receptor.

    Fig. S3. Variations of the calculated ligand binding poses.

    Fig. S4. Analysis of the voltage sensitivity of the N6.52A and D3.32E mutant M3 receptors.

    Fig. S5. Analysis of the voltage sensitivity of the choline-activated M3 receptor.

    Fig. S6. Analysis of the voltage sensitivity of the N6.52Q mutant M1 receptor.

    Fig. S7. The N6.52Q mutation in the M3 receptor reduces its affinity for ligands.

    Table S1. Average distances between residues 6.52 of TM6 or 3.32 of TM3 and ligands in the wild-type and mutant M3 receptors.

  • Supplementary Materials for:

    The mode of agonist binding to a G protein–coupled receptor switches the effect that voltage changes have on signaling

    Andreas Rinne, Juan Carlos Mobarec, Martyn Mahaut-Smith, Peter Kolb, Moritz Bünemann*

    *Corresponding author. E-mail: moritz.buenemann{at}staff.uni-marburg.de

    This PDF file includes:

    • Fig. S1. Analysis of the voltage sensitivity of the M1 receptor when uncoupled from Gq proteins.
    • Fig. S2. Analysis of the voltage sensitivity of the M5 receptor.
    • Fig. S3. Variations of the calculated ligand binding poses.
    • Fig. S4. Analysis of the voltage sensitivity of the N6.52A and D3.32E mutant M3 receptors.
    • Fig. S5. Analysis of the voltage sensitivity of the choline-activated M3 receptor.
    • Fig. S6. Analysis of the voltage sensitivity of the N6.52Q mutant M1 receptor.
    • Fig. S7. The N6.52Q mutation in the M3 receptor reduces its affinity for ligands.
    • Table S1. Average distances between residues 6.52 of TM6 or 3.32 of TM3 and ligands in the wild-type and mutant M3 receptors.

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    Citation: A. Rinne, J. C. Mobarec, M. Mahaut-Smith, P. Kolb, M. Bünemann, The mode of agonist binding to a G protein–coupled receptor switches the effect that voltage changes have on signaling. Sci. Signal. 8, ra110 (2015).

    © 2015 American Association for the Advancement of Science

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