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Review
. 2013;89(6):226-56.
doi: 10.2183/pjab.89.226.

Molecular properties of muscarinic acetylcholine receptors

Tatsuya Haga  1
Affiliations
Review

Molecular properties of muscarinic acetylcholine receptors

Tatsuya Haga. Proc Jpn Acad Ser B Phys Biol Sci. 2013.

Abstract

Muscarinic acetylcholine receptors, which comprise five subtypes (M1-M5 receptors), are expressed in both the CNS and PNS (particularly the target organs of parasympathetic neurons). M1-M5 receptors are integral membrane proteins with seven transmembrane segments, bind with acetylcholine (ACh) in the extracellular phase, and thereafter interact with and activate GTP-binding regulatory proteins (G proteins) in the intracellular phase: M1, M3, and M5 receptors interact with Gq-type G proteins, and M2 and M4 receptors with Gi/Go-type G proteins. Activated G proteins initiate a number of intracellular signal transduction systems. Agonist-bound muscarinic receptors are phosphorylated by G protein-coupled receptor kinases, which initiate their desensitization through uncoupling from G proteins, receptor internalization, and receptor breakdown (down regulation). Recently the crystal structures of M2 and M3 receptors were determined and are expected to contribute to the development of drugs targeted to muscarinic receptors. This paper summarizes the molecular properties of muscarinic receptors with reference to the historical background and bias to studies performed in our laboratories.

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Figures

Figure 1.
Figure 1.
Affinity chromatography of muscarinic receptors.19) A digitonin extract of porcine cerebral membranes (100 ml) was applied to ABT-agarose gel (10 ml). Most proteins (filled circles) were eluted in the flow through fraction, and muscarinic receptors (open circles) were specifically eluted with a muscarinic ligand (arrow). Broken and dotted lines represent the amounts of proteins and muscarinic receptors in the original extract, respectively.
Figure 2.
Figure 2.
Topology models of muscarinic M1,26) M227) receptors, rhodopsin,37) and β adrenergic receptors.38) In the model of the muscarinic M1 receptor, ionic residues such as D, E, K, R, H are shown by white letters on the black background, and cysteine residues are numbered. In the models of muscarinic M2 receptors, rhodopsin and β adrenergic receptors, amino acid residues homologous to those in muscarinic M1 receptors are shown as black circles.
Figure 3.
Figure 3.
Interaction of muscarinic receptors with G protein Gi or Go.74) Muscarinic receptors purified from porcine cerebrum were reconstituted in lipid vesicles without or with G protein Gi or Go, which were purified from the same tissue, and were incubated with [3H]QNB in the presence of various concentrations of ACh or atropine (ATR) and in the presence or absence of GppNHp (GTP analogue). The interaction of muscarinic receptors with Gi or Go was demonstrated as GppNHp-sensitive, high-affinity agonist binding. The displacement curves by ATR of [3H]QNB binding were not affected by the presence or absence of Gi, Go or GppNHp. On the other hand, the displacement curves by ACh in the presence of Gi or Go, but not in their absence, were shifted to the right in the presence of GppNHp. Quantitative analysis indicated that approximately 50% of the muscarinic receptors showed a high affinity for ACh in the presence of Gi or Go and the absence of GppNHp. Interpretation of these results is given in the text.
Figure 4.
Figure 4.
Displacement by GDP of [35S]GTPγS binding to an M2-Giα fusion protein in the absence and presence of antagonists, partial agonists or agonists.79) Insect cell (Sf9) membranes expressing M2-Giα fusion proteins were incubated with 50 nM [35S]GTPγS and various concentrations of GDP in the presence of full agonists (ACh, carbmaylcholine), partial agonists (pilocarpine, McN-343), or antagonist (atropine), and [35S]GTPγS bound to the fusion proteins was trapped and counted. The displacement curves by GDP of [35S]GTPγS binding shifted to the right in the presence of partial agonists and full agonist, indicating that the affinity for GDP decreased in the presence of agonist, partial agonist, and antagonist in this order. In the model, M2-Giα fusion proteins (R-Gα) are assumed to take two conformations: one conformation with a high affinity for GDP is dominant in the absence of ligand or the presence of antagonist, the other conformation with a low affinity for GDP is dominant in the presence of full agonist, and the two conformations are assumed to be in equilibrium in the presence of partial agonists.
Figure 5.
Figure 5.
(a) Agonist-dependent phosphorylation of I3-GST in the presence of M2 or I3-deleted M2 receptors104) and (b) agonist-induced internalization and down-regulation of M2 and I3-deleted M2 receptors.106) (a) I3-GST was subjected to phosphorylation by GRK2 in the presence of M2 or I3-deleted M2 receptors with an agonist (carbachol) or antagonist (atropine), followed by SDS-PAGE of the reaction mixture and detection of [32P]labeled bands through autoradiography. (b) CHO cells expressing M2 or I3-deleted M2 receptors were incubated at 37oC with an agonist carbamylcholine for indicated time, and then subjected to [3H]NMS or [3H]QNB binding at 4℃. Agonist-induced internalization and down-regulation of M2 and I3-deleted M2 receptors were measured as the decrease in the [3H]NMS and [3H]QNB binding activity, which represent the amount of M2 receptors in the cell surface and in the whole cell, respectively.
Figure 6.
Figure 6.
A model of agonist-induced activation of G protein (“on” reaction) and agonist-induced phosphorylation and internalization of M2 receptors (“off” reaction).
Figure 7.
Figure 7.
Crystals and diffraction patterns of M2 receptors. I3-deleted M2 receptors were crystalized by the vapor-diffusion method. The resolution of diffraction points was 9 Å.
Figure 8.
Figure 8.
Crystal structures of rhodopsin,123) β adrenergic receptors124) and muscarinic M2 receptors.127) Rhodopin, β adrenergic receptors, and muscarinic M2 receptors bound with retinol, carazolol (β antagonist), and QNB, respectively, all of which are colored white and found at similar positions. The upper side is intracellular phase, and the bottom site is extracellular phase. Amino terminus is in the intracellular phase, and carboxy terminus in the extracellular phase. Transmembrane segments 1 to 7 in M2 receptors are colored with dark blue (TM1), blue (TM2), light blue (TM3), blue-green (TM4), green (TM5), orange (TM6) and red (TM7), respectively.
Figure 9.
Figure 9.
Interaction of muscarinic M2 receptors and QNB (orthosteric antagonist)127) (stereo view). QNB (green) is bound with Asn405 in TM6 through hydrogen bonds, interacts with Asp103 in TM3 electrostatically, and is bound with a lot of hydrophobic amino acids through van-del-Waals interaction. Hydrogen bonds and electrostatic interaction are demonstrated with dotted lines of yellow.
Figure 10.
Figure 10.
Gauche and trans forms of ACh in muscarinic M2 receptors.127,142) (a) A model for the conformational change of ACh in the transition of M2 receptors from the inactive, low affinity, free state to the active, high-affinity, Gi-bound state. (b) ACh with a gauche (pink) or trans (green) form of Cα–Cβ bond was inserted at the position of QNB in the M2-QNB crystal structure.
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