A novel subtype of muscarinic receptor identified by homology screening

A new member of the protein superfamily of G-protein coupled receptors has been isolated by homology screening. By virtue of its homology with other muscarinic acetylcholine receptors and its ability to bind muscarinic specific antagonists, this muscarinic receptor subtype is designated M4. The M4 mRNA is preferentially expressed in certain brain regions. The existence of multiple receptor subtypes encoded by distinct genes in the brain has functional implications for the molecular mechanisms underlying information transmission in neuronal networks.     

Diverse pre- and post-synaptic expression of m1-m4 muscarinic receptor proteins in neurons and afferents in the rat neostriatum

We have utilized subtype specific antibodies to determine the cellular and subcellular distributions of the muscarinic acetylcholine receptor subtypes that are highly expressed in the rat striatum (m1-m4). Each receptor is expressed in distinct populations of striatal neurons in the relative proportions predicted by their mRNAs. They concentrate at post-synaptic sites and each of the four subtypes are also transported to pre-synaptic sites. m2 appears to be the only presynaptic autoreceptor in the striatum, but it is also localized in non-cholinergic terminals. These distinct pre- and post-synaptic localizations suggest that muscarinic receptor subtype diversity evolved to enable increasingly complex responses to acetylcholine release.     

Regulation of signal transduction at M2 muscarinic receptor

Muscarinic acetylcholine receptors mediate transmission of an extracellular signal represented by released acetylcholine to neuronal or effector cells. There are five subtypes of closely homologous muscarinic receptors which are coupled by means of heterotrimeric G-proteins to a variety of signaling pathways resulting in a multitude of target cell effects. Endogenous agonist acetylcholine does not discriminate among individual subtypes and due to the close homology of the orthosteric binding site the same holds true for most of exogenous agonists. In addition to the classical binding site muscarinic receptors have one or more allosteric binding sites at extracellular domains. Binding of allosteric modulators induces conformational changes in the receptor that result in subtype-specific changes in orthosteric binding site affinity for both muscarinic agonists and antagonists. This overview summarizes our recent experimental effort in investigating certain aspects of M2 muscarinic receptor functioning concerning i) the molecular determinants that contribute to the binding of allosteric modulators, ii) G-protein coupling specificity and subsequent cellular responses and iii) possible functional assays that exploit the unique properties of allosteric modulators for characterization of muscarinic receptor subtypes in intact tissue. A detailed knowledge of allosteric properties of muscarinic receptors is required to permit drug design that will modulate signal transmission strength of specific muscarinic receptor subtypes. Furthermore, allosteric modulation of signal transmission strength is determined by cooperativity rather than concentration of allosteric modulator and thus reduces the danger of overdose.     

Muscarinic receptor subtypes mediating central and peripheral antinociception studied with muscarinic receptor knockout mice: a review

To gain new insight into the physiological and pathophysiological roles of the muscarinic cholinergic system, we generated mutant mouse strains deficient in each of the five muscarinic acetylcholine receptor subtypes (M(1)-M(5)). In this chapter, we review a set of recent studies dealing with the identification of the muscarinic receptor subtypes mediating muscarinic agonist-dependent analgesic effects by central and peripheral mechanisms. Most of these studies were carried out with mutant mouse strains lacking M(2) or/and M(4) muscarinic receptors. It is well known that administration of centrally active muscarinic agonists induces pronounced analgesic effects. To identify the muscarinic receptors mediating this activity, wild-type and muscarinic receptor mutant mice were injected with the non-subtype-selective muscarinic agonist, oxotremorine (s.c., i.t., and i.c.v.), and analgesic effects were assessed in the tail-flick and hot-plate tests. These studies showed that M(2) receptors play a key role in mediating the analgesic effects of oxotremorine, both at the spinal and supraspinal level. However, studies with M(2)/M(4) receptor double KO mice indicated that M(4) receptors also contribute to this activity. Recent evidence suggests that activation of muscarinic receptors located in the skin can reduce the sensitivity of peripheral nociceptors. Electrophysiological and neurochemical studies with skin preparations from muscarinic receptor mutant mice indicated that muscarine-induced peripheral antinociception is mediated by M(2) receptors. Since acetylcholine is synthesized and released by different cell types of the skin, it is possible that non-neuronally released acetylcholine plays a role in modulating peripheral nociception. Our results highlight the usefulness of muscarinic receptor mutant mice to shed light on the functional roles of acetylcholine released from both neuronal and non-neuronal cells.     

毒蕈乙酰胆碱受体亚型关系的研究

利用不同类型的生物学数据, 运用生物信息学方法和策略, 从分子进化、序列相似性、表达相关性以及蛋白互作4个层面对M受体亚型之间的关系进行了比较全面的研究。分析表明, 从分子进化和序列相似性角度,毒蕈乙酰胆碱受体5种亚型可分为2个亚类,分别为M1、M3、M5亚类(第一亚类)与M2、M4亚类(第二亚类),每一亚类内部亚型之间进化距离相对较近, 序列相似性较高。在表达层面发现第一亚类中受体亚型与第二亚类中受体亚型在某些组织中正表达相关, 呈现共表达趋势。在互作层面发现两亚类之间受体亚型存在着间接互作的关系, 呈现协同作用的现象。     

Distinct sequence elements control the specificity of G protein activation by muscarinic acetylcholine receptor subtypes.

Relatively little is understood concerning the mechanisms by which subtypes of receptors, G proteins and effector enzymes interact to transduce specific signals. Through expression of normal, hybrid and deletion mutant receptors in Xenopus oocytes, we determined the G protein coupling characteristics of the functionally distinct m2 and m3 muscarinic acetylcholine receptor (mAChR) subtypes and identified the critical receptor sequences responsible for G protein specificity. Activation of a pertussis toxin insensitive G protein pathway, leading to a rapid and transient release of intracellular Ca2+ characteristic of the m3 receptor, could be specified by the transfer of as few as nine amino acids from the m3 to the m2 receptor. In a reciprocal manner, transfer of no more than 21 residues from the m2 to the m3 receptor was sufficient to specify activation of a pertussis toxin sensitive G protein coupled to a slow and oscillatory Ca2+ release pathway typical of the m2 subtype. Notably, these critical residues occur within the same region of the third cytoplasmic domain of functionally distinct mAChR subtypes.     

Cloning of the rat M3, M4 and M5 muscarinic acetylcholine receptor genes by the polymerase chain reaction (PCR) and the pharmacological characterization of the expressed genes.

The coding sequence of the rat m3, m4 and m5 subtypes of muscarinic acetylcholine receptor (mAChR) genes was amplified by the polymerase chain reaction (PCR), cloned, and expressed in the murine fibroblast (B82) cell line. Sequencing of the cloned genes revealed some nucleotide differences when compared with the DNA sequence published in the literature. When the different sequence appeared in only one clone obtained by PCR, it was considered an error of the polymerase. The overall error frequency in the 25 cycles of PCR with either Taq polymerase or Replinase was 1 nucleotide in 1,692 base pairs. In order to evaluate the different nucleotide sequence from a PCR product as an error or as an allelic variant, at least three different clones were sequenced. The cloned genes were each stably expressed in a B82 cell line and pharmacologically evaluated. The affinity of the different antagonists to the muscarinic receptor subtypes was determined by [3H](-)MQNB/ligand inhibition experiments. In the m3, m4 and m5 transfected cells, carbachol appeared to stimulate [3H]inositol monophosphate (IP1) accumulation. Carbachol, at 3 microM, appeared to suppress the forskolin-stimulated cAMP formation in the m4 transfected cells. These findings suggest these mAChRs amplified by PCR, cloned, and expressed in the B82 cell lines exhibit the pharmacological characteristics of the muscarinic receptor subtypes.     

Affinity profiles of various muscarinic antagonists for cloned human muscarinic acetylcholine receptor (mAChR) subtypes and mAChRs in rat heart and submandibular gland.

A family of five subtypes of muscarinic acetylcholine receptors (mAChR) has been identified based on their molecular structures and second signal transduction pathways. In the present study, we examined the antagonist binding profiles of 9 muscarinic antagonists (atropine, 4-DAMP, pirenzepine, oxybutynin, tiquizium, timepidium, propiverine, darifenacin and zamifenacin) for human muscarinic acetylcholine receptor subtypes (m1, m2, m3, m4 and m5) produced by using a baculovirus infection system in Sf9 insect cells, and rat tissue membrane preparations (heart and submandibular gland). In a scopolamine methyl chloride [N-methyl-3H]- ([3H]NMS) binding assay, pirenzepine and timepidium displayed the highest affinities for the m1 and m2 subtypes, respectively, and both zamifenacin and darifenacin had the highest affinities for the m3 subtype, although the selectivities among the five subtypes were less than 10-fold. Propiverine showed a slightly higher affinity for the m5 subtype, whereas none of the drugs used in this study was uniquely selective for the m4 subtype. The binding affinities of muscarinic antagonists for rat heart and submandibular gland strong correlated with those for human cloned m2 and m3 subtypes, respectively. These data suggest that [3H]NMS binding studies using rat heart and submandibular gland might be useful methods which predict the affinities of test drugs for human muscarinic M2 and M3 receptor subtypes.     

Evidence for Multiple Muscarinic Receptor Subtypes in Human Brain

Pirenzepine, a compound with selective antimuscarinic activity, was used to distinguish muscarinic acetylcholine receptor subtypes in normal human brain. Hill coefficients and IC50 values derived from the inhibition of specific [3H]L-quinuclidinyl benzilate receptor binding suggest the presence of two muscarinic binding sites, differing both in affinity for pirenzepine and in tissue distribution.     

Cloning and Expression of a G Protein-Linked Acetylcholine Receptor from Caenorhabditis elegans

Abstract : We have isolated a cDNA clone from the nematode Caenorhabditis elegans that encodes a protein of greatest sequence similarity to muscarinic acetylcholine receptors. This gene codes for a polypeptide of 682 amino acids containing seven putative transmembrane domains. The amino acid identities, excluding a highly variable middle portion of the third intracellular loop, to the human m1-m5 receptors are 28-34%. When this cloned receptor was coexpressed with a G protein-gated inwardly rectifying K⁺ channel (GIRK1) in Xenopus oocyte, acetylcholine was able to elicit the GIRK current. This acetylcholine-induced current was substantially inhibited by the muscarinic antagonist atropine in a reversible manner. However, another muscarinic agonist oxotremorine and antagonists scopolamine and pirenzepine had little or negligible effects on this receptor. Taken together, these results suggest that the cloned gene encodes a G protein-linked acetylcholine receptor that is most similar to but pharmacologically distinct from muscarinic acetylcholine receptors.     

Identification of subtypes of muscarinic receptors that regulate Ca2+ and K+ channel activity in sympathetic neurons

Many different G protein-coupled receptors modulate the activity of Ca2+ and K+ channels in a variety of neuronal types. There are five known subtypes (M1-M5) of muscarinic acetylcholine receptors. Knockout mice lacking the M1, M2, or M4 subtypes are studied to determine which receptors mediate modulation of voltage-gated Ca2+ channels in mouse sympathetic neurons. In these cells, muscarinic agonists modulate N- and L-type Ca2+ channels and the M-type K+ channel through two distinct, G-protein mediated pathways. The fast and voltage-dependent pathway is lacking in the M2 receptor knockout mice. The slow and voltage-independent pathway is absent in the M1 receptor knockout mice. Neither pathway is affected in the M4 receptor knockout mice. Muscarinic modulation of the M current is absent in the M1 receptor knockout mice, and can be reconstituted in a heterologous expression system using cloned channels and M1 receptors. Our results using knockout mice are compared with pharmacological data in the rat.     

Cholinergic receptor alterations in the cerebral cortex of spinal cord injured rat

Many areas of the cerebral cortex process sensory information or coordinate motor output necessary for control of movement. Disturbances in cortical cholinergic system can affect locomotor coordination. Spinal cord injury causes severe motor impairment and disturbances in cholinergic signalling can aggravate the situation. Considering the impact of cortical cholinergic firing in locomotion, we focussed the study in understanding the cholinergic alterations in cerebral cortex during spinal cord injury. The gene expression of key enzymes in cholinergic pathway - acetylcholine esterase and choline acetyl transferase showed significant upregulation in the cerebral cortex of spinal cord injured group compared to control with the fold increase in expression of acetylcholine esterase prominently higher than cholineacetyl transferase. The decreased muscarinic receptor density and reduced immunostaining of muscarinic receptor subtypes along with down regulated gene expression of muscarinic M1 and M3 receptor subtypes accounts for dysfunction of metabotropic acetylcholine receptors in spinal cord injury group. Ionotropic acetylcholine receptor alterations were evident from the decreased gene expression of alpha 7 nicotinic receptors and reduced immunostaining of alpha 7 nicotinic receptors in confocal imaging. Our data pin points the disturbances in cortical cholinergic function due to spinal cord injury; which can augment the locomotor deficits. This can be taken into account while devising a proper therapeutic approach to manage spinal cord injury.     

Muscarinic toxins: tools for the study of the pharmacological and functional properties of muscarinic receptors

Muscarinic receptors mediate metabotropic actions of acetylcholine in the CNS and PNS and autocrine functions of acetylcholine in non-neuronal systems. Because of the lack of highly selective muscarinic ligands, the precise location, functional role, and roles in various diseases of the five muscarinic receptor subtypes remain unclear. Muscarinic toxins isolated from the venom of Dendroaspis snakes have a natural high affinity and selectivity, associated with roles as competitive antagonists, allosteric modulators, and potential agonists. These toxins may therefore be invaluable tools for studying muscarinic receptors. We review data on the structural and pharmacological characterization of the muscarinic toxins, focusing on recent structure–function studies on toxin–receptor interactions. We discuss the potential benefits of using these toxins for investigating muscarinic function in vivo .     

Generation and pharmacological analysis of M2 and M4 muscarinic receptor knockout mice

Muscarinic acetylcholine receptors (M1-M5) play important roles in the modulation of many key functions of the central and peripheral nervous system. To explore the physiological roles of the two Gi-coupled muscarinic receptors, we disrupted the M2 and M4 receptor genes in mice by using a gene targeting strategy. Pharmacological and behavioral analysis of the resulting mutant mice showed that the M2 receptor subtype is critically involved in mediating three of the most striking central muscarinic effects, tremor, hypothermia, and analgesia. These studies also indicated that M4 receptors are not critically involved in these central muscarinic responses. However, M4 receptor-deficient mice showed an increase in basal locomotor activity and greatly enhanced locomotor responses following drug-induced activation of D1 dopamine receptors. This observation is consistent with the concept that M4 receptors exert inhibitory control over D1 receptor-mediated locomotor stimulation, probably at the level of striatal projection neurons where the two receptors are known to be coexpressed. These findings emphasize the usefulness of gene targeting approaches to shed light on the physiological and pathophysiological roles of the individual muscarinic receptor subtypes.     

Structure-activity relationships of methoctramine-related polyamines as muscarinic antagonist: effect of replacing the inner polymethylene chain with cyclic moieties

The aim of the present paper was to investigate the role of the octamethylene spacer of methoctramine (1) on the biological profile. Thus, this spacer was incorporated into a dianiline or dipiperidine moiety to determine whether flexibility and the basicity of the inner nitrogen atoms are important determinants of potency with respect to muscarinic receptors. The most potent compound was 4, which displayed, in the functional assays, a comparable potency at muscarinic M(2) receptors with respect to 1, and, in the binding assays, a loss of potency and selectivity toward muscarinic M(1) and M(3) receptor subtypes. Both compounds were endowed with antinociceptive activity. Furthermore, in microdialysis tests in rat parietal cortex, they enhanced acetylcholine release, most likely by antagonizing presynaptic muscarinic receptor subtypes.     

Constitutive activity of muscarinic acetylcholine receptors

We review the literature describing constitutive activity of the five muscarinic acetylcholine receptors in native and recombinant systems and discuss the effect of constitutive activity on muscarinic pharmacology in the context of modern models of receptor activation. We include a summary of mutations found to cause constitutive activity and discuss the implications of these data for the structure, function, and activation mechanism of muscarinic receptors. Finally, we discuss the possible physiological significance of constitutive activity of muscarinic receptors, incorporating information provided by targeted deletion of each of the muscarinic subtypes.     

MUSCARINIC ACETYLCHOLINE RECEPTOR SUBTYPE mRNAs IN THE HUMAN AND RAT VESTIBULAR PERIPHERY

The expression of the five muscarinic acetylcholine receptor (mAChR) subtypes (m1–m5) in the vestibular end-organs and in the primary afferent vestibular ganglia of the human and rat was studied using RT-PCR from the two tissue populations from both species. In the human, although all five mAChR subtypes were expressed in brain, only the m1, m2, and m5 mAChR subtypes were amplified from both the vestibular ganglia and the vestibular end-organs, while in the rat, all five mAChR subtypes were expressed. These data suggest that the efferent cholinergic axo-dendritic and axo-somatic synapses have a muscarinic component and that there are pharmacologic implications for patients with vestibular dysfunction.     

Constitutive Activity of Muscarinic Acetylcholine Receptors

We review the literature describing constitutive activity of the five muscarinic acetylcholine receptors in native and recombinant systems and discuss the effect of constitutive activity on muscarinic pharmacology in the context of modern models of receptor activation. We include a summary of mutations found to cause constitutive activity and discuss the implications of these data for the structure, function, and activation mechanism of muscarinic receptors. Finally, we discuss the possible physiological significance of constitutive activity of muscarinic receptors, incorporating information provided by targeted deletion of each of the muscarinic subtypes.