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A Network of Conserved Intramolecular Contacts Defines the Off-state of the Transmembrane Switch Mechanism in a Seven-transmembrane Receptor

2000; Elsevier BV; Volume: 275; Issue: 8 Linguagem: Inglês

10.1074/jbc.275.8.5682

1083-351X

Zhi-Liang Lu, Edward C. Hulme,

Nicotinic Acetylcholine Receptors Study

Activation of the rhodopsin-like 7-transmembrane (7-TM) receptors requires switching interhelical constraints that stabilize the inactive state to a new set of contacts in the activated state, which binds the cognate G-protein. The free energy to drive this is provided by agonist binding, which has higher affinity to the active than to the inactive conformation. We have sought specific interhelical constraint contacts, using the M1 muscarinic acetylcholine receptor as a model. Histidine substitutions of particular groups of amino acids, in transmembrane domains 3, 6, and 7, created high-affinity Zn2+ binding sites, demonstrating the close proximity of their side chains in the inactive state. Alanine point substitutions have shown the effect of weakening the individual intramolecular contacts. In each case, the acetylcholine affinity was increased, implying promotion of the activated state. These amino acids are highly conserved throughout the 7-TM receptor superfamily. We propose that they form an important part of a network of conserved interhelical contacts that defines the off-state of a general transmembrane switch mechanism. Activation of the rhodopsin-like 7-transmembrane (7-TM) receptors requires switching interhelical constraints that stabilize the inactive state to a new set of contacts in the activated state, which binds the cognate G-protein. The free energy to drive this is provided by agonist binding, which has higher affinity to the active than to the inactive conformation. We have sought specific interhelical constraint contacts, using the M1 muscarinic acetylcholine receptor as a model. Histidine substitutions of particular groups of amino acids, in transmembrane domains 3, 6, and 7, created high-affinity Zn2+ binding sites, demonstrating the close proximity of their side chains in the inactive state. Alanine point substitutions have shown the effect of weakening the individual intramolecular contacts. In each case, the acetylcholine affinity was increased, implying promotion of the activated state. These amino acids are highly conserved throughout the 7-TM receptor superfamily. We propose that they form an important part of a network of conserved interhelical contacts that defines the off-state of a general transmembrane switch mechanism. transmembrane domain muscarinic acetylcholine receptor acetylcholine (−)-N-methylscopolamine phosphoinositide Mutations which cause agonist-independent activation of 7-TM1 receptors have suggested the presence of structural constraints; for instance, a salt bridge between TM 3 and TM 7 may stabilize the ground-state conformation of rhodopsin (1.Robinson P.R. Cohen G.B. Zhukovsky E.A. Oprian D.D. Neuron. 1992; 9: 719-725Abstract Full Text PDF PubMed Scopus (442) Google Scholar) and the α1B receptor (2.Porter J.E. Hwa J. Perez D.M. J. Biol. Chem. 1996; 271: 28318-28323Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Recently, we identified a patch of amino acids in TM 3 of the M1 mAChR, including the highly conserved residues Leu116 and Ser120, whose mutation increased ACh affinity and caused constitutive activation (3.Hulme E.C. Lu Z.-L. J. Physiol. (Paris). 1998; 92: 269-274Crossref PubMed Scopus (13) Google Scholar, 4.Lu Z.-L. Hulme E.C. J. Biol. Chem. 1999; 274: 7309-7315Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). We proposed that this follows the deletion of specific intramolecular contacts. Although the high-resolution structures of the 7-TM receptors are unknown, a general model of the α-carbon backbone has been published, based on sequence analysis in the context of a low-resolution electron crystallographic structure of rhodopsin (5.Unger V.M. Hargrave P.A. Baldwin J.M. Schertler G.F.X. Nature. 1997; 389: 203-206Crossref PubMed Scopus (482) Google Scholar, 6.Baldwin J.M. Schertler G.F.X. Unger V.M. J. Mol. Biol. 1997; 272: 144-164Crossref PubMed Scopus (635) Google Scholar). Interactions between highly conserved amino acids within the receptor core were proposed to mediate receptor conformational changes. In reference to the model, potential contacts for Leu116 are Phe374 (TM 6), Asn414 (TM 7), and Tyr418 (TM 7), while Ser120 may contact Tyr208 (TM 5). Mutations of Phe374 cause constitutive activation of the M5mAChR (7.Spalding T.A. Burstein E.S. Henderson S.C. Ducote K.R. Brann M.R. J. Biol. Chem. 1998; 273: 21563-21568Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), whereas Asn414 and Tyr418 have proved important for signal transduction in all of the 7-TM receptors in which they have been investigated (8.Barak L.S. Ménard L. Ferguson S.S.G. Colapietro A.-M. Caron M.G. Biochemistry. 1995; 34: 15407-15414Crossref PubMed Scopus (140) Google Scholar, 9.Gabilondo A.M. Krasel C. Lohse M.J. Eur. J. Pharmacol. 1996; 307: 243-250Crossref PubMed Scopus (40) Google Scholar, 10.Shyu J.-F. Inoue D. Baron R. Horne W.C. J. Biol. Chem. 1996; 271: 31127-31134Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 11.Wang J. Zheng J. Anderson J.L. Toews M.L. Mol. Pharmacol. 1997; 52: 306-313Crossref PubMed Scopus (34) Google Scholar, 12.Böhm S.K. Khitin L.M. Smeekens S.O. Grady E.F. Payan D.G. Bunnett N.W. J. Biol. Chem. 1997; 272: 2363-2372Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 13.Audoly L. Breyer R.M. Mol. Pharmacol. 1997; 51: 61-68Crossref PubMed Scopus (44) Google Scholar, 14.Vichi P. Whelchel A. Posada J. J. Biol. Chem. 1999; 274: 10331-10338Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). The locations of these residues are indicated in Fig. 1,a and b. We have used polymerase chain reaction mutagenesis to make combinatorial histidine substitutions of these residues, creating Zn2+ binding sites which have allowed us to explore this network of contacts in the M1 mAChR. Mutants were constructed by a polymerase chain reaction method and validated by di-deoxy sequencing. Mutant receptors were subcloned into the pCD expression vector and expressed transiently in COS-7 cells by electroporation as described (4.Lu Z.-L. Hulme E.C. J. Biol. Chem. 1999; 274: 7309-7315Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 15.Lu Z.-L. Curtis C.A.M. Jones P.G. Pavia J. Hulme E.C. Mol. Pharmacol. 1997; 51: 234-241Crossref PubMed Scopus (88) Google Scholar, 16.Page K.M. Curtis C.A.M. Jones P.G. Hulme E.C. Eur. J. Pharmacol. 1995; 289: 429-437Crossref PubMed Scopus (50) Google Scholar, 17.Jones P.G. Curtis C.A.M. Hulme E.C. Eur. J. Pharmacol. 1995; 288: 251-257Crossref PubMed Scopus (68) Google Scholar). Binding studies were conducted in 20 mm Na-Hepes buffer plus 100 mm NaCl, pH 7.5, for 2 h at 30 °C. (−)-N-[3H]methylscopolamine ([3H]NMS) binding curves were fitted to a one-site model to yield a total concentration of binding sites,Rt (expressed relative to a wild-type control in each transfection) and an affinity constant,K NMS. Expression of the wild-type M1mAChR varied from 0.7 to 1.2 pmol/mg of protein in different transfections. The affinity of [3H]NMS for the wild-type receptor was 1.0 ± 0.05 × 1010m−1. Inhibition curves for ACh and ZnCl2 were fitted to the Hill equation, and the binding constants, K ACh and K Zn, were corrected for the Cheng-Prusoff shift, as necessary (4.Lu Z.-L. Hulme E.C. J. Biol. Chem. 1999; 274: 7309-7315Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 15.Lu Z.-L. Curtis C.A.M. Jones P.G. Pavia J. Hulme E.C. Mol. Pharmacol. 1997; 51: 234-241Crossref PubMed Scopus (88) Google Scholar).K ACh for the wild-type M1 mAChR was 1.1 ± 0.1 × 105m−1. Phosphoinositide (PI) dose-response curves to ACh were determined as described (4.Lu Z.-L. Hulme E.C. J. Biol. Chem. 1999; 274: 7309-7315Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 15.Lu Z.-L. Curtis C.A.M. Jones P.G. Pavia J. Hulme E.C. Mol. Pharmacol. 1997; 51: 234-241Crossref PubMed Scopus (88) Google Scholar) and fitted to a four-parameter logistic function, yielding an EC50 value and a maximum response (E max).E max values are expressed relative to wild-type control in each transfection. (−)-N-[3H]methylscopolamine (85 Ci/mmol), (−)-[3H]quinuclidinyl benzilate (50 Ci/mmol) andmyo-d-[3H]inositol (80 Ci/mmol) were obtained from Amersham Pharmacia Biotech. ZnCl2 was from Merck. With one exception (N414H-Y418H), the single, double, and triple His mutants were expressed at between 30 and 170% of the wild-type level in COS-7 cells, assayed by the specific binding of a high-affinity antagonist [3H]NMS (Table I). Most of the mutations caused less than a 3-fold change in [3H]NMS affinity. However, the F374H mutation caused a 7-fold reduction, which was partly reversed by the addition of a second His at position 116 or 414. The N414H-Y418H mutant gave 1% of wild-type expression, whereas its affinity was reduced by more than 10-fold. Both of these effects were substantially reversed by the incorporation of a third His at position 116 or 374.Table ILigand binding and functional activity of His-substituted M1 mAChRs[3H]NMS bindingACh bindingPI response to AChR tK NMSK AChpEC50E max% wild-typelog m −1logm −1log m −1% wild-typeWild-type(100)10.02 ± 0.045.04 ± 0.047.00 ± 0.03(100)L116H38 ± 810.28 ± 0.044.59 ± 0.055.98 ± 0.1090 ± 3F374H72 ± 79.14 ± 0.053.80 ± 0.045.16 ± 0.04101 ± 5N414H172 ± 210.23 ± 0.055.60 ± 0.010Y418H69 ± 510.44 ± 0.056.22 ± 0.086.63 ± 0.0853 ± 3S120H59 ± 59.95 ± 0.115.20 ± 0.015.64 ± 0.1451 ± 3Y208H91 ± 410.00 ± 0.054.53 ± 0.015.76 ± 0.1784 ± 3L116H-S120H39 ± 410.28 ± 0.154.58 ± 0.080L116H-Y208H38 ± 310.36 ± 0.074.58 ± 0.025.12 ± 0.0145 ± 6L116H-F374H52 ± 99.77 ± 0.014.04 ± 0.044.71 ± 0.0766 ± 5L116H-N414H56 ± 1510.06 ± 0.055.07 ± 0.080L116H-Y418H42 ± 410.52 ± 0.124.88 ± 0.015.36 ± 0.0831 ± 4S120H-Y208H72 ± 610.04 ± 0.034.98 ± 0.055.16 ± 0.0161 ± 3S120H-F374H71 ± 89.18 ± 0.064.12 ± 0.144.89 ± 0.1316 ± 3S120H-N414H85 ± 910.05 ± 0.085.88 ± 0.160S120H-Y418H53 ± 610.41 ± 0.016.07 ± 0.040F374H-N414H91 ± 99.78 ± 0.014.93 ± 0.180F374H-Y418H72 ± 119.43 ± 0.014.61 ± 0.044.54 ± 0.2118 ± 2L116H-S120H-Y208H37 ± 410.35 ± 0.074.62 ± 0.010L116H-S120H-F374H31 ± 59.85 ± 0.044.12 ± 0.130L116H-S120H-N414H38 ± 39.97 ± 0.074.98 ± 0.100L116H-S120H-Y418H30 ± 410.48 ± 0.055.26 ± 0.100L116H-F374H-N414H36 ± 59.44 ± 0.024.42 ± 0.040L116H-F734H-Y418H38 ± 29.83 ± 0.134.19 ± 0.044.65 ± 0.0713 ± 2N414H-Y418H1 ± 0.05<9.00NDaNon-detectable.0N414H-Y418H-L116H38 ± 79.89 ± 0.045.67 ± 0.030N414H-Y418H-F374H57 ± 49.38 ± 0.045.64 ± 0.040Ligand binding and PI assay were conducted as described under “Experimental Procedures.” Total concentration of receptor binding sites (R t) and the maximum PI response (E max) were expressed relative to a wild-type control in each transfection. K ACh values were calculated from IC50 values using the Cheng-Prusoff shift. Data are mean ± S.E. of three or more independent experiments.a Non-detectable. Open table in a new tab Ligand binding and PI assay were conducted as described under “Experimental Procedures.” Total concentration of receptor binding sites (R t) and the maximum PI response (E max) were expressed relative to a wild-type control in each transfection. K ACh values were calculated from IC50 values using the Cheng-Prusoff shift. Data are mean ± S.E. of three or more independent experiments. The mutation of F374H reduced ACh affinity by 17-fold (Table I), whereas mutation of Asn414 and Tyr418 to His (Table I) increased it by between 4- and 15-fold. The ACh affinities of the double and triple mutants were similar to the geometric means of the constituent single mutants. Signaling efficacies for the L116H and F374H mutants, calculated as described (3.Hulme E.C. Lu Z.-L. J. Physiol. (Paris). 1998; 92: 269-274Crossref PubMed Scopus (13) Google Scholar, 4.Lu Z.-L. Hulme E.C. J. Biol. Chem. 1999; 274: 7309-7315Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), were close to wild-type. Decreases were found for Y208H (4-fold), S120H (25-fold), and Y418H (35-fold), whereas the N414H mutant had, essentially, zero efficacy. These reductions mirror the decreased maximum PI responses for these mutants. The L116H-F374H, L116H-Y418H, F374H-Y418H, and L116H-F374H-Y418H mutants gave detectable PI responses to ACh, but multiple mutants in which Asn414 was altered were inactive (Table I). None of the His mutants showed raised basal PI signaling. The binding and functional data (Table I) suggested that, with the exception of N414H-Y418H, the His mutations caused only minor perturbations of the ground-state structure of the M1mAChR. Thus, probing the substituted structures with Zn2+ions, and determination of the chelate effect, should give information about the spatial proximities of the parent residues. Zn2+ binding was assayed by inhibition of the binding of [3H]NMS (2 × K dconcentration). The apparent K d of Zn2+for the wild-type receptor was 0.72 mm. The results are exemplified in Fig. 2 a and summarized in Fig. 3 a, which are expressed relative to the wild-type receptor.Figure 3Zn2+ binding to His-substitution mutants. a, changes in Zn2+ binding affinity, relative to wild-type. b, cooperativity of Zn2+ binding by substituted histidines. The values were calculated by the ratio ofK Zn/(K Zn1 +K Zn2 + ...) minus 1, which corresponded to the contribution to the free energy (ΔΔG ochelate) arising from the chelate effect, ΔΔG ochelate = −RTln(K Zn/(K Zn1 +K Zn2 + ...)) (23.Suh S.-S. Haymore B.L. Arnold F.H. Protein Eng. 1991; 4: 301-305Crossref PubMed Scopus (74) Google Scholar), whereK Zn is the association constant for Zn2+ binding to a double or triple histidine mutant,K Zn1,2, are the association constants for Zn2+ binding to the individual single histidine mutants,R is the gas constant (8.314 J/K/mol), and T= 298.15 K. Values are mean ± standard error of three or more independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Single histidine substitutions of Ser120, Tyr208, Phe374, Asn414, and Tyr418 gave less than 6-fold increases in the Zn2+ affinity. However, the mutation of Leu116increased the affinity by 14-fold. The double histidine mutant L116H-S120H, with a favorable iand i + 4 helical spacing, displayed a 43-fold increase in Zn2+ affinity (Fig. 2 a). The contribution to the free energy arising from cooperativity of the two histidines, calculated from the chelate effect (ΔΔG ochelate), was 2.6 kJ/mol (legend to Fig. 3). L116H-F374H and L116H-N414H yielded 71- and 32-fold increases in affinity, respectively, also giving positive chelate effects. However, L116H-Y208H, L116H-Y418H, as well as the combinations of S120H with Y208H, F374H, N414H, and Y418H did not show increased Zn2+ affinity relative to the single mutants and gave no chelate effect. The double mutations F374H-N414H and F374H-Y418H increased the affinity of Zn2+ by 8–14-fold (Fig.3 a). The triple histidine mutant generated by the addition of a third His at position 374 to L116H-S120H gave a further increase in Zn2+affinity of 5.7-fold, to 245-fold the wild-type value (ΔΔG ochelate = 6.3 kJ/mol) (Fig.2 a). In contrast, adding a third His at positions 208, 414, or 418 had little or no further effect. Combining L116H-F374H with N414H gave a 7-fold additional increase, to 504-fold the wild-type value (ΔΔG ochelate = 7.9 kJ/mol). Although it was impossible to measure the Zn2+affinity of the poorly expressed double mutant N414H-Y418H, the triple mutant F374H-N414H-Y418H showed a 75-fold increase relative to the wild-type receptor. This was 6- and 16-fold greater than the F374H-N414H or F374H-Y418H mutants, corresponding to a ΔΔG ochelate of 5.0 kJ/mol (Fig.3, a and b). An interaction between these residues is also supported by the rescue of receptor expression and affinity in the triple mutant relative to the N414H-Y418H mutant (TableI). Zn2+ inhibition of binding of the tertiary antagonist [3H]quinuclidinyl benzilate was also tested for selected mutants, giving results similar to those measured by [3H]NMS. 2Z.-L. Lu, unpublished observations. Increasing the concentration of [3H]NMS from 2K d to 6 K d or 18K d shifted the inhibition curves to higher concentrations of Zn2+(Fig. 2 b) but did not reduce the maximum inhibition, thus showing high negative cooperativity of inhibition. None of these mutants increased the affinity of Ni2+ more than 8-fold.2 The inhibition of [3H]NMS binding to the histidine mutants by Zn2+ at the IC50 concentration was reversed by subsequent addition of 1 mm excess of EDTA to chelate the Zn2+ ions. This had less effect on the nonspecific binding of Zn2+ to the wild-type receptor (Fig. 2 c). These results indicated that relatively specific Zn2+binding sites were created and that the Zn2+ binding to the specific sites was reversible. Zn2+(100–300 μm) inhibited the PI response evoked by ACh at the L116H-F374H mutant (70% inhibition of the effect of 10−4m ACh at 300 μmZn2+) while having little effect on the wild-type receptor (20% inhibition of the effect of 10−5m ACh at 300 μm Zn2+) or the F374H and L116H mutants (less than 20% inhibition of the effect of 10−4m ACh at 300 μm Zn2+). However, Zn2+(10–100 μm) also doubled the basal PI signal in untransfected COS-7 cells, indistinguishably from cells transfected with the inactive L116H-N414H, F374H-N414H, and L116H-F374H-N414H mutants. The occurrence of this background of nonspecific stimulation made it impossible to quantitate the effects of Zn2+ on the ACh-induced PI response, and these experiments were not pursued further. Three of the triple His mutants showed strong positive cooperativity of Zn2+ binding by histidine (Fig.3 b), corresponding to a ΔΔG ochelate in the range of 5.0–7.9 kJ/mol. The K d values for Zn2+of these triads (L116H-S120H-F374H, 2.9 μm; L116H-F374H-N414H, 1.4 μm; F374H-N414H-Y418H, 9.5 μm) are comparable with those reported for triple-His mutants in the NK-1 (18.Elling C.E. Nielsen S.M. Schwartz T.W. Nature. 1995; 374: 74-77Crossref PubMed Scopus (161) Google Scholar) and κ-opioid receptors (19.Thirstrup K. Elling C.E. Hjorth S.A. Schwartz T.W. J. Biol. Chem. 1996; 271: 7875-7878Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) and suggest the successful creation of high-affinity Zn2+ binding sites. The Hill coefficients of Zn2+ inhibition of [3H]NMS binding to these triple mutants were close to 1.0, consistent with the ligation of a single Zn2+ ion. The inhibition mechanism appeared near competitive, over the concentration ranges studied. It is likely that the introduction of a positively charged Zn2+ ion into the central cleft of the receptor strongly disfavors the binding of the positively charged radiolabeled antagonist, even though the altered residues do not overlap with the primary ligand binding residues (Fig. 1 a). The α-carbons of the residues composing the high-affinity triads must be separated by less than 13 Å to allow the corresponding imidazole side chains to coordinate a metal ion (20.Higaki F.N. Flettterick R.J. Craik C.S. Trends Biochem. Sci. 1992; 17: 100-104Abstract Full Text PDF PubMed Scopus (100) Google Scholar). The results suggest that, in the inactive state of the M1 mAChR, a network of interactions exists between amino acid side chains, centered on Leu116 (TM 3)-Phe374 (TM 6)-Asn414(TM 7) and supported by Ser120 (TM 3) and Tyr418 (TM 7), which turn away from Leu116 and Asn414 by 40 degrees of arc (Fig.4). In contrast, it seems unlikely that Tyr208 (TM 5) is close enough to Ser120 (TM 3) to form a hydrogen bond; in the TSH receptor, the homologous tyrosine has been proposed to make a hydrogen bond with a carbonyl oxygen in the peptide backbone of TM6 (21.Biebermann H. Schöneberg T. Schulz A. Krause G. Grüters A. Schultz G. Gudermann T. FASEB J. 1998; 12: 1461-1471Crossref PubMed Scopus (83) Google Scholar). Ala substitution mutagenesis has suggested functions for some of the side chains of the amino acids in this network in activation of the M1 mAChR. Ala-substitutions of Leu116 and Ser120 simultaneously increased ACh affinity, raised basal activity, and enhanced signaling efficacy, suggesting that the intramolecular contacts made by these highly conserved residues help to stabilize the inactive ground state of the M1 mAChR (3.Hulme E.C. Lu Z.-L. J. Physiol. (Paris). 1998; 92: 269-274Crossref PubMed Scopus (13) Google Scholar, 4.Lu Z.-L. Hulme E.C. J. Biol. Chem. 1999; 274: 7309-7315Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). A similar role has been proposed for Phe374 in TM 6 (7.Spalding T.A. Burstein E.S. Henderson S.C. Ducote K.R. Brann M.R. J. Biol. Chem. 1998; 273: 21563-21568Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar,22.Baranski T.J. Herzmark P. Lichtarge O. Gerber B.O. Trueheart J. Meng E.C. Iiri T. Sheikh S.P. Bourne H.R. J. Biol. Chem. 1999; 274: 15757-15765Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Mutation of Ser120 to histidine caused little change in agonist and antagonist binding. In contrast with mutation of S120A and S120C,2 S120H strongly reduced signaling efficacy and maximum PI response. Double mutation of L116H-S120H and S120H-Y418H was inactive, but S120H-Y208H and S120H-F374H gave detectable PI response to ACh (Table I). Substitution of the small side chain of Ser120 by histidine may affect transition from the inactive to the active state, in which relative rotation and translation of helices are necessary, but without perturbation of the ground-state structure. His substitution of the TM 7 residues Asn414 and Tyr418 (Table I) increased ACh affinity but essentially abolished Gq-mediated phosphoinositide signaling. These findings were confirmed by Ala-substitution. In the case of N414A, as reported for L116A, there was also a large reduction in receptor expression. 3Z.-L. Lu and E. C. Hulme, manuscript in preparation. Asn414 and Tyr418 may resemble Leu116, Ser120, and Phe374 in making intramolecular contacts that stabilize the inactive ground state of the receptor. However, the loss of signal implies that they are also important for the formation of the agonist-receptor-Gqprotein signaling complex. Thus, in contrast to residues which act as pure constraints, they may have an additional role. In summary, several of the specific, conserved, interhelical contacts between TM 3, 6, and 7 of the rhodopsin-like 7-TM receptors which have been proposed from mutagenesis and modeling studies have been directly supported by the engineering of Zn2+ binding sites in the M1 mAChR. These contacts may be important in stabilizing the off-state of the receptor switch mechanism and be broken or rearranged during receptor activation.