Jan Jakubík

Constitutive activity of the M1–M4 subtypes of muscarinic receptors in transfected CHO cells and of muscarinic receptors in the heart cells revealed by negative antagonists

We investigated whether muscarinic receptors of the M1–M4 receptor subtypes are constitutively active. We have found that the synthesis of cyclic AMP was enhanced by the muscarinic antagonists atropine and N‐methylscopolamine (NMS) in Chinese hamster ovary (CHO) cells stably transfected with human m2 and m4 muscarinic receptor genes and in rat cardiomyocytes expressing the M2 receptor subtype, and that the production of inositol phosphates was inhibited by atropine and NMS in CHO cells stably transfected with human m1 and m3 and with rat m1 muscarinic receptor genes. The muscarinic antagonists quinuclidinyl benzilate and AF‐DX 116 had no effect in some cases and acted like atropine and NMS in others. We conclude that the M1–M4 subtypes of muscarinic receptors are constitutively active in the CHO cell lines expressing them and in cardiomyocytes and that atropine and NMS act as negative antagonists on these receptor subtypes by stabilizing them in the inactive conformation.

Differences in kinetics of xanomeline binding and selectivity of activation of G proteins at M1 and M2 muscarinic acetylcholine receptors

Xanomeline is a functionally selective M1/M4 muscarinic acetylcholine receptor agonist that nevertheless binds with high affinity to all five subtypes of muscarinic receptors. A novel mode of interaction of this ligand with the muscarinic M1 receptors characterized by persistent binding and receptor activation after extensive washout has been shown previously. In the present study, using human M1 and M2 receptors expressed in Chinese hamster ovary cells and [3H]N-methylscopolamine as a tracer, we show that persistent binding of xanomeline also occurs at the M2 receptor with similar affinity as at the M1 receptor (KI = 294 and 296 nM, respectively). However, kinetics of formation of xanomeline wash-resistant binding to M2 receptors was markedly slower than to M1 receptors. Xanomeline was a potent fast-acting full agonist in stimulating guanosine 5′-O-(3-[35S]thio)triphosphate binding at M1 receptors, whereas at M2 receptors it behaved as a potent partial agonist (40% of carbachol maximal response) only upon preincubation for 1 h. Development of xanomeline agonistic effects at the M2 receptor was slower than its ability to attenuate carbachol responses. We also demonstrate that xanomeline discriminates better between G protein subtypes at M1 than at M2 receptors. Our data support the notion that xanomeline interacts with multiple sites on the muscarinic receptor, resulting in divergent conformations that exhibit differential effects on ligand binding and receptor activation. These conformations are both time- and concentration-dependent and vary between the M1 and the M2 receptor.

Protection by Alcuronium of Muscarinic Receptors Against Chemical Inactivation and Location of the Allosteric Binding Site for Alcuronium

Abstract: We have found earlier that the neuromuscular blocker alcuronium binds to cardiac muscarinic receptors simultaneously with their specific antagonist [3H]methyl‐N‐scopolamine ([3H]NMS) and allosterically increases their affinity to this ligand. Nothing is known about the allosteric site with which alcuronium interacts. To gain an insight, we have now investigated how the binding of [3H]NMS is affected by agents known to modify specific residues in proteins and how their effects are altered by alcuronium. Reagents that covalently modify the tyrosyl residues (p‐nitrobenzenesulfonyl fluoride and 4‐chloro‐7‐nitrobenzofurazan) and the carboxyl groups of aspartate and glutamate [1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide, N,N′‐dicyclohexylcarbodiimide, and N‐ethyl‐5‐phenylisoxazolium‐3′‐sulfonate] blocked the binding of [3H]NMS to receptors in rat heart atria. Their action was probably due to the modification of tyrosyl and aspartyl residues directly in the muscarinic binding sites because it was antagonized by atropine and carbamoylcholine. Alcuronium and gallamine, another allosteric ligand, also protected the [3H]NMS binding sites against the inactivation by tyrosine‐ and carboxyl‐directed chemical modifiers just as well as by benzilylcholine mustard, known to attach covalently to the muscarinic binding sites. Protection by alcuronium has also been observed on cerebrocortical muscarinic receptors. The effect of alcuronium indicates that the drug interferes with the access of chemical modifiers to the muscarinic sites. In view of the unspecific nature of most of the modifiers used (with regard to muscarinic mechanisms), the protection by alcuronium appears to be best explained on the assumption that the drug binds in close vicinity of the “classical” muscarinic site and sterically blocks the access to this site.

Weak toxin WTX from Naja kaouthia cobra venom interacts with both nicotinic and muscarinic acetylcholine receptors.

Iodinated [125I] weak toxin from Naja kaouthia (WTX) cobra venom was injected into mice, and organ‐specific binding was monitored. Relatively high levels of [125I]WTX were detected in the adrenal glands. Rat adrenal membranes were therefore used for analysis of [125I]WTX‐binding sites. Specific [125I]WTX binding was partially inhibited by both α‐cobratoxin, a blocker of the α7 and muscle‐type nicotinic acetylcholine receptors (nAChRs), and by atropine, an antagonist of the muscarinic acetylcholine receptor (mAChR). Binding to rat adrenal nAChR had a Kd of 2.0 ± 0.8 μm and was inhibited by α‐cobratoxin but not by a short‐chain α‐neurotoxin antagonist of the muscle‐type nAChR, suggesting a specific interaction with the α7‐type nAChR. WTX binding was reduced not only by atropine but also by other muscarinic agents (oxotremorine and muscarinic toxins from Dendroaspis angusticeps), indicating an interaction with mAChR. This interaction was further characterized using individual subtypes of human mAChRs expressed in Chinese hamster ovary cells. WTX concentrations up to 30 μm did not inhibit binding of [3H]acetylcholine to any subtype of mAChR by more than 50%. Depending on receptor subtype, WTX either increased or had no effect on the binding of the muscarinic antagonist [3H]N‐methylscopolamine, which binds to the orthosteric site, a finding indicative of an allosteric interaction. Furthermore, WTX alone activated G‐protein coupling with all mAChR subtypes and reduced the efficacy of acetylcholine in activating G‐proteins with the M1, M4, and M5 subtypes. Our data demonstrate an orthosteric WTX interaction with nAChR and an allosteric interaction with mAChRs.

Two populations of muscarinic binding sites in the chick heart distinguished by affinities for ligands and selective inactivation

1 By measuring the binding of N‐[3H‐methyl]‐scopolamine ([3H]‐NMS) and of unlabelled subtype‐specific muscarinic antagonists, two populations of muscarinic binding sites can be distinguished in the membranes of cardiac ventricles taken from 1‐day‐old chicks. One of them, corresponding to approximately 80% of [3H]‐NMS binding sites, has higher affinities for AF‐DX116 (pKi = 6.42) and methoct‐ramine (pKi = 7.33); the rate of [3H]NMS dissociation from these sites is fast. The other population, corresponding to approximately 20% of [3H]‐NMS binding sites, has lower affinities for AF‐DX116 (pKi = 5.00) and methoctramine (pKi = 6.19); the rate of [3H]‐NMS dissociation from these sites is slow. Both populations have high affinities for pirenzepine, but the affinity of the former (major) population is lower (pKi = 7.99) than that of the latter (minor) population (pKi = 10.14). 2 Since it has been shown earlier that two mRNAs for muscarinic receptors are expressed in the chick heart, one of them close to the genetically defined m2 and the other to the m4 subtype, we propose that the major population of binding sites with high affinities for AF‐DX116 and methoctramine and the lower affinity for pirenzepine represents the M2‐like receptors, while the minor population represents the M4‐like receptors. 3 It proved possible to obtain isolated samples of either population by selectively protecting the M2‐like sites with AF‐DX116 and the M4‐like sites with pirenzepine, and by inactivating the unprotected sites with benzilylcholine mustard. The properties of the isolated populations corresponded to those derived from the analysis of [3H]‐NMS binding to the original mixed population. 4 Alcuronium exerted positive allosteric action on the binding of [3H]‐NMS both to the M2‐like and the M4‐like population and severely slowed down [3H]‐NMS dissociation from them; its affinity for the M2‐like sites was 3–10 times higher.

Positive allosteric interactions on cardiac muscarinic receptors: effects of chemical modifications of disulphide and carboxyl groups.

Changes in the allosteric effects of alcuronium on rat cardiac muscarinic receptors were investigated after chemical modifications of S-S bonds or free carboxyl groups. In membranes pretreated with dithiothreitol, alcuronium lost its positive action on the binding of [3H]methyl-N-scopolamine while its inhibitory effect on radioligand dissociation was preserved. In membranes pretreated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), known to modify free carboxyl groups in proteins, the ability to bind [3H]methyl-N-scopolamine was preserved if the pretreatment had been performed in the presence of alcuronium, methyl-N-scopolamine or carbachol, while the positive cooperative effect of alcuronium on [3H]methyl-N-scopolamine binding was only preserved in membranes that had been exposed to EDC in the presence of alcuronium. Methyl-N-scopolamine, carbachol and alcuronium differed in their ability to protect (against EDC) the action of alcuronium on the rate of [3H]methyl-N-scopolamine dissociation. The results suggest that the disulphide bridge connecting the first two extracellular loops of muscarinic receptors is important for the positive allosteric action of alcuronium and that three carboxyl groups (presumably aspartate residues) are involved in receptor interactions with alcuronium and methyl-N-scopolamine. The first group is important for the effect of alcuronium on the affinity for methyl-N-scopolamine, the second is critical for the effect of alcuronium on the rate of methyl-N-scopolamine dissociation, and the third is critical for methyl-N-scopolamine binding. Presumably, the two charged nitrogens of alcuronium associate with the first and the second of the three groups involved.

Allosteric Modulation of Muscarinic Acetylcholine Receptors.

An allosteric modulator is a ligand that binds to an allosteric site on the receptor and changes receptor conformation to produce increase (positive cooperativity) or decrease (negative cooperativity) in the binding or action of an orthosteric agonist (e.g., acetylcholine). Since the identification of gallamine as the first allosteric modulator of muscarinic receptors in 1976, this unique mode of receptor modulation has been intensively studied by many groups. This review summarizes over 30 years of research on the molecular mechanisms of allosteric interactions of drugs with the receptor and for new allosteric modulators of muscarinic receptors with potential therapeutic use. Identification of positive modulators of acetylcholine binding and function that enhance neurotransmission and the discovery of highly selective allosteric modulators are mile-stones on the way to novel therapeutic agents for the treatment of schizophrenia, Alzheimer’s disease and other disorders involving impaired cognitive function.

Subtype‐selective inhibition of [methyl‐3H]‐N‐methylscopolamine binding to muscarinic receptors by α‐truxillic acid esters

Seven esters of α‐truxillic acid have been synthesized: bis‐3‐piperidylpropyl ester and its quaternary bis‐N‐ethyl derivative, bis‐N‐diethylaminopropyl ester and its quaternary bis‐N‐methyl derivative, and bis‐4‐piperidylbutyl ester and its quaternary bis‐N‐methyl and bis‐N‐ethyl derivatives. All esters inhibited the specific binding of muscarinic receptor antagonist [methyl‐3H]‐N‐methylscopolamine ([3H]‐NMS) to muscarinic receptors in membranes of CHO cell lines stably expressing the human gene for the M1, M2, M3 or M4 subtype of muscarinic receptors. All esters displayed the highest potency at the M2 and the lowest potency at the M3 receptor subtype. In experiments performed on the M2 muscarinic receptor subtype, the affinity between the receptors and the esters was greatly increased when the concentration of ions was diminished. The highest affinities were found for the tertiary bis‐3‐piperidylpropyl and bis‐4‐piperidylbutyl aminoesters (equilibrium dissociation constants of 52 and 179 pM, respectively, in the low ionic strength medium). All investigated esters slowed down the dissociation of [3H]‐NMS from the M2 muscarinic receptor subtype. [3H]‐NMS dissociation from the M1, M3 and M4 muscarinic receptor subtypes was investigated in experiments with the bis‐4‐piperidylbutyl aminoester and also found to be decelerated. It is concluded that the esters of α‐truxillic acid act as M2‐selective allosteric modulators of muscarinic receptors and that, by their potency, the tertiary bis‐3‐piperidylpropyl and bis‐4‐piperidylbutyl aminoesters surpass the other known allosteric modulators of these receptors.

Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy

BACKGROUND AND PURPOSE Conventional determination of agonist efficacy at G‐protein coupled receptors is measured by stimulation of guanosine‐5′‐γ−thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M2 muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy.

Wash-Resistantly Bound Xanomeline Inhibits Acetylcholine Release by Persistent Activation of Presynaptic M2 and M4 Muscarinic Receptors in Rat Brain

We studied the effects of 3-[3-hexyloxy-1,2,5-thiadiazo-4-yl]-1,2,5,6-tetrahydro-1-methylpyridine (xanomeline) wash-resistant binding on presynaptic muscarinic regulation of electrically evoked [3H]acetylcholine (ACh) release from rat brain slices. In both cortical and striatal tissues that possess M2 and M4 autoreceptors, respectively, immediate application of 10 μM xanomeline had no effect on evoked [3H]ACh release or its inhibition by 10 μM carbachol. In contrast, preincubation with 1, 10, or 100 μM xanomeline for 15 min decreased evoked release of ACh measured after 53 min of washing in xanomeline-free medium in a concentration-dependent manner. The maximal inhibitory effect equaled the immediate effect of the muscarinic full agonist carbachol, and it was completely (at 1 and 10 μM xanomeline) or partially (at 100 μM xanomeline) blocked by 1 μM N-methylscopolamine. Neither presence of N-methylscopolamine during 100 μM xanomeline treatment nor previous irreversible inactivation of the classical receptor binding site using propylbenzylcholine mustard in cortical slices prevented the inhibitory effect of wash-resistantly bound xanomeline. Treatment of cortical slices with xanomeline slightly decreased the number of muscarinic binding sites, and it markedly decreased affinity for N-methylscopolamine. When applied as in acetylcholine release experiments, xanomeline did not impair presynaptic α2-adrenoceptor-mediated regulation of noradrenaline release. The functional studies in brain tissue reported in this work demonstrate that xanomeline can function as a wash-resistant agonist of native presynaptic muscarinic M2 and M4 receptors with both competitive and allosteric components of action.