昆虫毒蕈碱型乙酰胆碱受体的研究进展

毒蕈碱型乙酰胆碱受体(Muscarinic Acetylcholine Receptors,mAChRs)是昆虫神经系统中一类重要的G蛋白偶联受体.昆虫mAChRs可以分为A、B、C型三大类,它们通过偶联不同的G蛋白激活不同的第二信使,完成信号转导过程,从而发挥其功能.mAChRs参与调控昆虫多种生理反应和行为过程,如...     

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.     

Structure-function analysis of muscarinic acetylcholine receptors

The structural basis underlying the G protein coupling selectivity of different muscarinic receptor subtypes was analyzed by using a combined molecular genetic/biochemical approach. These studies led to the identification of key residues on the receptors as well as the associated G proteins that are critically involved in determining proper receptor/G protein recognition. Mutational analysis of the m3 muscarinic receptor showed that most native cysteine residues are not required for productive receptor/G protein coupling. The putative extracellular disulfide bond was found to be essential for efficient trafficking of the receptor protein to the cell surface but not for receptor-mediated G protein activation.     

The conformational switch in muscarinic acetylcholine receptors

The recently-determined structure of rhodopsin has provided a suitable basis for modeling the three-dimensional structure of the M1 muscarinic acetylcholine receptor. Using this as a framework for interpreting mutagenesis studies, we have been able to suggest most of the contacts which the receptor makes with acetylcholine and many of the intramolecular contacts which are important for the ground-state structure of the receptor. It is possible to outline a mechanism of G-protein interaction.     

Antibodies to muscarinic acetylcholine receptors in myasthenia gravis

IgG obtained from patients with myasthenia gravis block the specific binding of the muscarinic antagonists (³H)-N-methyl-4-piperidyl benzilate (4NMPB) and (³H)-Quinuclidinyl benzilate to rat brain muscarinic acetylcholine receptors. IgG obtained from healthy controls have a much smaller effect. The inhibitory effect of the myasthenic IgG increases linearly with immunoglobulin concentration and has no effect on the affinity of the muscarinic receptors towards (³H)-4NMPB (K_D = 0.7 ± 0.1 nM). This implies that the inhibition is probably due to the blocking of some of the muscarinic receptors by the myasthenic IgG, and not due to alteration in affinity of all the receptors. it remains to be established whether the presence of antimuscarinic receptor activity in the serum of myasthenic patients is of importance in the pathophysiology and diagnosis of myasthenia gravis.     

Solubilization of muscarinic acetylcholine receptors of bovine brain

The effectiveness of several detergents and salts in solubilizing the muscarinic acetylcholine receptor (identified by its atropine-sensitive [³H]3-quinuclidinyl benzilate (QNB) binding) from bovine striatal membranes is reported. The highest density of receptor is obtained by extraction with 1% digitonin-0.1 mM EDTA. Although the total solubilized muscarinic receptors (sites/ml) are increased and the nonspecific binding is decreased when 1 M NaCl is included in this extraction medium, the receptor density (sites/mg protein) is lower. The solubilized receptors have the same specific QNB binding affinity, and sensitivity to a variety of drugs, as the membrane-bound muscarinic receptors.     

Purification of muscarinic acetylcholine receptors by affinity chromatography

Calf forebrain homogenates contain 2.8 pM muscarinic acetylcholine receptors per mg of protein. [3H]Antagonist saturation binding experiments under equilibrium conditions revealed a single class of sites with equilibrium dissociation constants of 0.82 nM for [3H]dexetimide and 0.095 nM for [3H]quinuclidinyl benzilate. Displacement binding studies with agonists revealed the presence of low and high affinity sites. Here we describe the solubilization of muscarinic acetylcholine receptors with digitonin and their purification by affinity chromatography using an affinity gel which consisted of dexetimide coupled to Affi-Gel 10 (i.e., carboxy N-hydroxysuccinimide esters linked via a 1 nm spacer arm to agarose beads). Purified proteins were obtained by specific elution with muscarinic drugs, i.e., the antagonist atropine and the irreversible ligand propylbenzilylcholine mustard. SDS-polyacrylamide gel electrophoresis of the radioiodinated purified preparations revealed a major 70-K protein.     

Activation of solubilized G-proteins by muscarinic acetylcholine receptors

Binding of GTP and its analogue, guanosine 5′-O-[γ-thio]triphosphate (GTP[S]) to G-proteins, and release of GTP[S] from G-proteins are stimulated by muscarinic acetylcholine (mACh) receptors in intact cardiac membranes. Upon solubilization of receptors and G-proteins by membrane extraction with the detergent, 3-[(cholamidopropyl)dimethylammonio]-1-propanesulphonate, followed by sucrose density gradient centrifugation, agonist-liganded mACh receptors stimulated binding of GTP[S] and hydrolysis of GTP by G-proteins with similar requirements as in intact membranes. One soluble agonist-activated mACh receptor induced binding of GTP[S] to several (about seven) soluble G-proteins. In contrast to intact membranes, however, agonist activation of mACh receptors did not induce release of GTP[S] from solubilized G-proteins. The data presented indicate that mACh receptors can interact with and efficiently activate G-proteins even in solution, whereas the possible interaction of receptors with GTP[S]-liganded G-proteins observed in intact membranes is lost upon solubilization of these components.     

Sphingosine kinase-mediated calcium signaling by muscarinic acetylcholine receptors

Based on the finding that G protein-coupled receptors (GPCRs) can induce Ca2+ mobilization, apparently independent of the phospholipase C (PLC)/inositol-1,4,5-trisphosphate (IP3) pathway, we investigated whether sphingosine kinase, which generates sphingosine-1-phosphate (SPP), is involved in calcium signaling by mAChR and other GPCRs. Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,/N-dimethylsphingosine markedly inhibited [Ca2+]i increases elicited by M2 and M3 mAChRs in HEK-293 cells without affecting PLC activation. Activation of M2 and M3 mAChR rapidly and transiently stimulated production of SPP. Furthermore, microinjection of SPP into HEK-293 cells induced rapid and transient Ca2+ mobilization. Pretreatment of HEK-293 cells with the calcium chelator BAPTA/AM fully blocked mAChR-induced SPP production. On the other hand, incubation of HEK-293 cells with calcium ionophores activated SPP production. Similar findings were obtained for formyl peptide and P2Y2 purinergic receptors in HL-60 cells. On the basis of these studies we propose, that following initial IP3 production by receptor-mediated PLC activation, a local discrete increase in [Ca2+]i induces sphingosine kinase stimulation, which ultimately leads to full calcium mobilization. Thus, sphingosine kinase activation most likely represents an amplification system for calcium signaling by mAChRs and other GPCRs.     

昆虫毒蕈碱型乙酰胆碱受体研究进展

毒蕈碱型乙酰胆碱受体（muscarinic acetylcholine receptor,mAchR）是昆虫神经系统中一种十分重要的神经递质受体,属于G蛋白偶联受体超家族,与配体结合后激活G蛋白并通过胞内第二信使产生生物学效应,参与昆虫多种神经生理功能,是农药开发的一个潜在靶标。昆虫毒蕈碱型乙酰胆碱受体的研究,有助于提高昆虫神经生理及行为调节机制等方面的认识,并为以此为靶标的新型杀虫剂研制提供新思路。本文综述了国内外对昆虫毒蕈碱型乙酰胆碱受体的研究概况,并对GenBank中已上传的昆虫mAchR序列进行了分子进化分析,最后对昆虫mAchR研究中存在的问题及前景进行了分析和展望。     

Characterization of muscarinic acetylcholine receptors in the avian salt gland

Electrolyte and fluid secretion by the avian salt gland is regulated by activation of muscarinic acetylcholine receptors (R). In this study, these receptors were characterized and quantitated in homogenates of salt gland from domestic ducks adapted to conditions of low (freshwater, FW) and high (saltwater, SW) salt stress using the cholinergic antagonist [3H]-quinuclidinyl benzilate (QNB). Specific binding of the antagonist to receptors in both FW- and SW-adapted glands reveals a single population of high affinity binding sites (KdFW = 40.1 +/- 3.0 pM; KdSW = 35.1 +/- 2.1 pM). Binding is saturable; RLmaxFW = 1.73 +/- 0.10 fmol/micrograms DNA; RLmaxSW = 4.16 +/- 0.31 fmol/micrograms DNA (where L is [3H]QNB and RL the high affinity complex). Calculated average cellular receptor populations of 5,800 sites/cell in FW-adapted glands and 14,100 sites/cell in SW-adapted glands demonstrate that upward regulation of acetylcholine receptors in the secretory epithelium follows chronic salt stress. The receptor exhibits typical pharmacological specificities for muscarinic cholinergic antagonists (QNB, atropine, scopolamine) and agonists (oxotremorine, methacholine, carbachol). In addition, the loop diuretic furosemide, which interferes with ion transport processes in the salt gland, competitively inhibits [3H]QNB binding. Preliminary studies of furosemide effects on [3H]QNB binding to rat exorbital lacrimal gland membranes showed a similar inhibition, although the diuretic had no effect on antagonist binding to rat brain or atrial receptors.     

Direct and energy-transfer photolabelling of brain muscarinic acetylcholine receptors

Efficient photolabelling of muscarinic acetylcholine receptors was obtained using either two aryldiazonium salts or an azido derivative. These probes did not discriminate between muscarinic binding subtypes or affinity states and became irreversibly bound to the receptor sites, in an entirely atropine-protectable manner, upon ultraviolet irradiation. The extent of labelling was dependent both on probe concentration and on time of irradiation and reached up to 80% of the receptor population, under optimal alkylating conditions. In contrast to the azido derivative, both diazonium salts behave as potent irreversible labels of muscarinic receptors, provided energy-transfer photolabelling conditions were followed. Such an indirect activation of diazonium ligands, through an energy transfer from photoexcited tryptophan residues, has been previously found to increase the site-specificity and the rate of labelling of other acetylcholine binding proteins. Analogies in the photolabelling process of acetylcholinesterase or of nicotinic and muscarinic receptors by the two diazonium salts are discussed. Altogether, these findings suggest that these new probes may be promising tools to investigate the location and the topography of the agonist-antagonist binding domain on purified muscarinic receptors, through amino acid and/or sequence analyses of radioactive, photolabelled residues.     

Interactions of brain muscarinic acetylcholine receptors with plant lectins

We previously reported that muscarinic acetylcholine receptors (mAChRs) from porcine brains are glycoproteins. When porcine brain membranes were solubilized with digitonin or 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), approximately 20% of the receptors were solubilized, most (90% or more) of which bound to Sepharose 4B conjugated with wheat germ agglutinin (WGA). In contrast, when membranes were solubilized with Lubrol PX, a much larger fraction (approximately 60%) of the receptors were solubilized. However, about a third of this solubilized receptor population remained unbound to WGA-Sepharose even in the presence of an excess amount of the lectin-Sepharose. These results suggested a structural heterogeneity of the mAChR in terms of its carbohydrate moiety. The effects of lectins on the ligand binding properties of mAChRs were also studied. WGA or concanavalin A (ConA) was found to cause a 2- to 3-fold increase in the affinity of membrane-bound receptors to an antagonist [3H]quinuclidinyl benzylate [( 3H]QNB) without affecting the maximum number of sites, whereas the lectins had no significant effects on the binding of the agonist [3H]cis-methyldioxolane. When the membranes were dissolved with detergents, lectin did not increase the [3H]QNB affinity: These lectins caused an approximately 2 fold decrease in the affinity of digitonin-solubilized receptors for [3H]QNB. Thus the lectins exert differential effects on agonist and antagonist binding to the brain membrane mAChRs, most likely by modulating some intermolecular interactions.     

Trafficking of M(2) muscarinic acetylcholine receptors

Internalization is an important mechanism regulating the agonist-dependent responses of G-protein-coupled receptors. The internalization of the M(2) muscarinic cholinergic receptors (mAChR) in HEK293 cells has been demonstrated to occur by an unknown mechanism that is independent of arrestins and dynamin. In this study we examined various aspects of the trafficking of the M(2) mAChR in HEK293 cells to characterize this unknown pathway of internalization. Internalization of the M(2) mAChR was rapid and extensive, but prolonged incubation with agonist did not lead to appreciable down-regulation (a decrease in total receptor number) of the receptors. Recovery of M(2) mAChRs to the cell surface following agonist-mediated internalization was a very slow process that contained protein synthesis-dependent and -independent components. The protein synthesis-dependent component of the recovery of receptors to the cell surface did not appear to reflect a requirement for synthesis of new receptors, as no changes in total receptor number were observed either in the presence or absence of cycloheximide. Phosphorylation of the M(2) mAChR did not appear to influence the rate or extent of the recovery of receptors to the cell surface, as the recovery of a phosphorylation-deficient mutant M(2) mAChR, the N,C(Ala-8) mutant, was similar to the recovery of the wild type M(2) mAChR. Finally, the constitutive, nonagonist-dependent internalization and recycling of the M(2) mAChR was very slow and also contained protein synthesis-dependent and -independent components, suggesting that a similar pathway controls the recovery from agonist-dependent and -independent internalization. Overall, these data demonstrated a variety of previously unappreciated facets involved in the regulation of M(2) mAChRs.     

Functional analysis of muscarinic acetylcholine receptors using knockout mice

Because of the low selectivity of available ligands, pharmacological approaches to elucidate the functional difference among muscarinic acetylcholine receptor (mAChR) subtypes have been problematic. As an alternative approach, we have established a series of mutant mouse lines deficient in each mAChR subtype (mAChR KO mice). The systematic analyses of these mice have been useful in revealing the functional difference among mAChR subtypes. Here, we review our prior research on these mutant mice and also some notable findings reported by other research groups.     

Structural determinants for the interactions between muscarinic toxin 7 and muscarinic acetylcholine receptors

Muscarinic acetylcholine receptors (mAChRs) have five subtypes and play crucial roles in various physiological functions and pathophysiological processes. Poor subtype specificity of mAChR modulators has been an obstacle to discover new therapeutic agents. Muscarinic toxin 7 (MT7) is a natural peptide toxin with high selectivity for the M1 receptor. With three to five residues substituted, M3, M4, and M5 receptor mutants could bind to MT7 at nanomolar concentration as the M1 receptor. However, the structural mechanisms explaining MT7–mAChRs binding are still largely unknown. In this study, we constructed 10 complex models of MT7 and each mAChR subtype or its mutant, performed molecular dynamics simulations, and calculated the binding energies to investigate the mechanisms. Our results suggested that the structural determinants for the interactions on mAChRs were composed of some critical residues located separately in the extracellular loops of mAChRs, such as Glu4.56, Leu4.60, Glu/Gln4.63, Tyr4.65, Glu/Asp6.67, and Trp7.35. The subtype specificity of MT7 was attributed to the non‐conserved residues at positions 4.56 and 6.67. These structural mechanisms could facilitate the discovery of novel mAChR modulators with high subtype specificity and enhance the understanding of the interactions between ligands and G‐protein‐coupled receptors. Copyright © 2015 John Wiley & Sons, Ltd.     

Expression of muscarinic and nicotinic acetylcholine receptors in the mouse urothelium

Acetylcholine (ACh) and its receptors play a crucial role in bladder physiology. Here, we investigated the presence of muscarinic receptor subtypes (MR) and nicotinic acetylcholine receptor (nAChR) alpha-subunits in the mouse urothelium by RT-PCR and immunohistochemistry. With RT-PCR, we detected mRNAs coding for all of the five different MR subtypes and for the nicotinic receptor subunits alpha2, alpha4, alpha5, alpha6, alpha7, alpha9 and alpha10, whereas the alpha3-subunit was not expressed. Using immunohistochemistry, we localised a panel of acetylcholine receptors in the different layers of the murine bladder urothelium, with predominant appearance in the basal plasma membrane of the basal cell layer and in the apical membrane of the umbrella cells. M2R and subunit alpha9 were observed exclusively in the umbrella cells, whereas the MR subtypes 3-5 and the nAChR subunits alpha4, alpha7 and alpha10 were also detected in the intermediate and basal cell layers. The subunit alpha5 was localised only in the basal cell layer. In conclusion, the murine urothelium expresses multiple cholinergic receptors, including several subtypes of both MR and nAChR, which are differentially distributed among the urothelial cell types. Since these receptors have different electrophysiological and pharmacological properties, and therefore are considered to be responsible for different cellular responses to ACh, this differential distribution is expected to confer cell type-specificity of cholinergic regulation in the bladder urothelium.