(A) Gels were loaded with samples of FLAG- and c-Myc-tagged receptor extracted from co-infected Sf9 cells (lane 1, 20–30 fmol of receptor per lane) or from the precipitate obtained upon treatment of …
Panel B–Monomeric status of the purified M2 receptor after chemical cross-linking.
Panel C–Parametric values for the effect of gallamine on the rate of dissociation of [3H]QNB. Panel D–Parametric values for the effect of gallamine on the binding of [3H]NMS at equilibrium. Panels E–G–Parametric values for the effect of strychnine on the binding of [3H]NMS to oligomers and monomers.
Figure 1–figure supplement 1–Parametric values for the effect of strychnine on the binding of [3H]NMS and [3H]QNB to membrane-bound M2 receptor.
Figure 1-figure supplement 2–Rate constants for the simulated binding of strychnine and [3H]NMS according to Figure 6.
The binding of [3H]NMS (A) or [3H]QNB (B) to M2 receptor in membranes from CHO cells was measured at the concentrations of strychnine shown on the x-axes. Strychnine and the radioligand were added …
Total specific binding of the orthosteric ligand (RL + ARL) at graded concentrations of strychnine was simulated according to Figure 6 (Equations 13–17) with the parametric values listed in Figure …
(A) A computed molecular model of the M2 receptor (red) fused at the N-terminus to mCherry (yellow) and labeled with FlAsH (green) at a tetracysteine motif inserted in ECL2 between Val166 and Gly167.…
Panel A–Time-resolved fluorescence and fluorescence anistoropy of eGFP and eGFP-tagged M2 receptors.
Panel E–Levels of significance for ligand-dependent changes in the FRET efficiency of FlAsH-reacted mCh-M2-FCM.
CHO cells were transfected with plasmids coding for mCh-M2-FCM (A), mCh-M2(D103A)-FCM (B), M2-FCM (C), eGFP-M2 (D), mCh-M2 (E), and eGFP-M2-mCh (F). Images were recorded on a confocal microscope, as …
Membranes were prepared from CHO cells expressing M2 receptor bearing mCherry at the N-terminus and the FlAsH-reactive sequence FCM in the second extracellular loop. The binding of [3H]NMS was …
CHO cells expressing the eGFP- and mCherry-tagged M2 receptor were excited at 473 nm at a power of 0.37 µW. The emission spectrum was recorded from the region of the plasma membrane and analyzed to …
The sensor (mCh-M2-FCM) (A), a mutant thereof that does not bind NMS [mCh-M2(D103A)-FCM] (B), and the mutant plus the wild-type M2 receptor (C) were expressed or co-expressed in CHO cells and …
Panel D–Levels of significance for ligand-dependent changes in the FRET efficiency of FlAsH-reacted mCh-M2-FCM and mCh-M2(D103A)-FCM
(A) CHO cells were cotransfected with the plasmids coding for eGFP-M2 and mCh-M2(D103A)-FCM. The cells were treated with FlAsh, and those expressing both receptors were identified by the presence of …
Models of the liganded receptor were simulated by molecular dynamics as described in Materials and Methods. The region of the ligand-binding sites is shown in the figure, with NMS and strychnine (A, …
The ligands shown are: NMS, N-methylscopolamine; QNB, quinuclidinylbenzilate; Str, strychnine; and Gal, gallamine. Different liganded states of the receptor are identified as X_R_Y, where X and Y …
(A) The three residues of the aromatic cap (i.e., Tyr104, Tyr403, and Tyr426) and Trp422 are shown in an overlay of the results of three simulations: a vacant receptor (yellow), a QNB-bound receptor …
Figure 5–figure supplement 2–Distance between the α-carbon atoms of Tyr177 and Asn419 in crystal structures of the M2 receptor
Figure 5–figure supplement 3–Mean distances (Å) between the α-carbon atoms of Tyr177 and Asn419 in the M2 receptor with different combinations of allosteric and orthosteric ligands
Strychnine adopts a similar pose at the vacant receptor and in the presence of either NMS (A) or QNB at the orthosteric site. The position of gallamine at a vacant receptor (B) is consistent …
Changes in the distance between the α-carbon atoms of Tyr177 and Asn419 were taken as a measure of the effect of an orthosteric ligand on the conformation of the allosteric site. The structures of …
The distances from all frames of the molecular dynamics simulations over the production period of 30 ns are shown in the figure for a receptor with NMS (A) or QNB (B) at the orthosteric site. In …
Each ligand can bind separately to form AR or RL, but the ternary complex is accessible only via RL. The orthosteric site of the M2 receptor is located within the cluster of helical domains, with …
Correlates of electrostatic repulsion between orthosteric and allosteric ligands. The inter-cationic distance was calculated as that between the cationic nitrogen atom of the orthosteric ligand and …
Allosteric–orthosteric pair | ||||
---|---|---|---|---|
Str_R_NMSa | Str_R_QNB | Gal_R_NMS | Gal_R_QNBa | |
Inter-cationic distance (Å) | 13.7 | 15.7 | 16.5 | 16.8 |
Difference in electrostatic potential (kcal/mol) | 0.0085 | 0.063 | 0.096 | 0.41 |
a The distances are shown in Figures 4A and B.
Analysis of binding.
Calculation of FRET by spectral unmixing.
Simulation of the capped allosteric ternary complex model.