A specific multi-nutrient formulation enhances M1 muscarinic acetylcholine receptor responses in vitro

Recent evidence indicates that supplementation with a specific combination of nutrients may affect cell membrane synthesis and composition. To investigate whether such nutrients may also modify the physical properties of membranes, and affect membrane-bound processes involved in signal transduction pathways, we studied the effects of nutrient supplementation on G protein-coupled receptor activation in vitro. In particular, we investigated muscarinic receptors, which are important for the progression of memory deterioration and pathology of Alzheimer's disease. Nerve growth factor differentiated pheochromocytoma cells that were supplemented with specific combinations of nutrients showed enhanced responses to muscarinic receptor agonists in a membrane potential assay. The largest effects were obtained with a combination of nutrients known as Fortasyn? Connect, comprising docosahexaenoic acid, eicosapentaenoic acid, uridine monophosphate as a uridine source, choline, vitamin B6, vitamin B12, folic acid, phospholipids, vitamin C, vitamin E, and selenium. In subsequent experiments, it was shown that the effects of supplementation could not be attributed to single nutrients. In addition, it was shown that the agonist-induced response and the supplement-induced enhancement of the response were blocked with the muscarinic receptor antagonists atropine, telenzepine, and AF-DX 384. In order to determine whether the effects of Fortasyn? Connect supplementation were receptor subtype specific, we investigated binding properties and activation of human muscarinic M1, M2 and M4 receptors in stably transfected Chinese hamster ovary cells after supplementation. Multi-nutrient supplementation did not change M1 receptor density in plasma membranes. However, M1 receptor-mediated G protein activation was significantly enhanced. In contrast, supplementation of M2- or M4-expressing cells did not affect receptor signaling. Taken together, these results indicate that a specific combination of nutrients acts synergistically in enhancing muscarinic M1 receptor responses, probably by facilitating receptor-mediated G protein activation.     

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.     

Influence of MT7 toxin on the oligomerization state of the M1 muscarinic receptor1

Background information. The idea that GPCRs (G‐protein‐coupled receptors) may exist as homo‐ or hetero‐oligomers, although still controversial, is now widely accepted. Nevertheless, the functional roles of oligomerization are still unclear and gaining greater insight into the mechanisms underlying the dynamics of GPCR assembly and, in particular, assessing the effect of ligands on this process seems important. We chose to focus our present study on the effect of MT7 (muscarinic toxin 7), a highly selective allosteric peptide ligand, on the oligomerization state of the hM₁ (human M₁ muscarinic acetylcholine receptor subtype). Results. We analysed the hM₁ oligomerization state in membrane preparations or in live cells and observed the effect of MT7 via four complementary techniques: native‐PAGE electrophoresis analysed by both Western blotting and autoradiography on solubilized membrane preparations of CHO‐M1 cells (Chinese‐hamster ovary cells expressing muscarinic M₁ receptors); FRET (fluorescence resonance energy transfer) experiments on cells expressing differently tagged M₁ receptors using either an acceptor photobleaching approach or a novel fluorescence emission anisotropy technique; and, finally, by BRET (bioluminescence resonance energy transfer) assays. Our results reveal that MT7 seems to protect the M₁ receptor from the dissociating effect of the detergent and induces an increase in the FRET and BRET signals, highlighting its ability to affect the dimeric form of the receptor. Conclusions. Our results suggest that MT7 binds to a dimeric form of hM₁ receptor, favouring the stability of this receptor state at the cellular level, probably by inducing some conformational rearrangements of the pre‐existing muscarinic receptor homodimers.     

Alternative splicing of RGS8 gene changes the binding property to the M1 muscarinic receptor to confer receptor type-specific Gq regulation

RGS proteins constitute a large family that modulates heterotrimeric G-protein signaling. We previously showed that RGS8 suppressed Gq signaling in a receptor type-specific manner. To elucidate molecular mechanisms underlying receptor-specific attenuation by RGS8, we examined whether RGS8 can interact with certain G-protein-coupled receptors. By pull-down assay, we showed that RGS8 directly binds to the third intracellular (i3) loop of M1 and M3 muscarinic acetylcholine receptors (mAChRs). The binding of RGS8S, a splice variant with a different N-terminus, was weaker. RGS8 could bind specifically to the C-terminal part of M1i3 (containing amino acids of 304-353 of i3 of human M1-mAChR), but RGS8S could not. Moreover, deletion of the N-terminal 9 amino acids and substitution of both Arg-8 and Arg-9 of RGS8 with Ala resulted in reduced binding to M1i3. BRET experiments revealed that RGS8 actually interacts with M1-mAChR, but RGS8S does not interact in the living cells. The RGS8 mutant, which had less binding ability to M1i3, showed a reduced inhibitory function of Gq signaling through M1-mAChR. These results demonstrated that RGS8 can directly interact with M1-mAChR via its N-terminus and the i3 loop of the receptor, and this binding must play an essential role in receptor-specific suppression by RGS8.     

突触前α7烟碱受体对海马神经元兴奋性突触传递的调控

采用盲法膜片钳技术观察突触前烟碱受体(nicotinic acetylcholinel receptors,nAChRs)对海马脑片CAl区锥体神经元兴奋性突触传递的调控作用。结果显示,nAChRs激动剂碘化二甲基苯基哌嗪(dimethylphenyl—piperazinium iodide,DMPP)不能在CAl区锥体神经元上诱发出烟碱电流。DMPP对CAl区锥体神经元自发兴奋性突触后电流(spontaneous excitatory postsynaptic current,sEPSC)具有明显的增频和增幅作用,并呈现明显的浓度依赖关系。DMPP对微小兴奋性突触后电流(miniature excitatory postsynaptic current,mEPSC)具有增频作用,但不具有增幅作用。上述DMPP增强突触传递的作用不能被nAChRs拮抗剂美加明、六烃季铵和双氢-β-刺桐丁所阻断,但可被α-银环蛇毒素阻断。上述结果提示,海马脑片CAl区锥体神经元兴奋性突触前nAChRs含有对α-银环蛇毒素敏感的胡亚单位,其激活可增强海马CAl区锥体神经元突触前递质谷氨酸的释放,从而对兴奋性突触传递发挥调控作用。     

Temperature dependence of synaptic responses in guinea pig hippocampal CA1 neurons in vitro

1. Temperature-dependent properties of synaptic transmission were studied by recording orthodromic responses of the population spike and excitatory postsynaptic potential in CA1 pyramidal neurons of guinea pig hippocampal slices.2. Increasing the temperature of the perfusing medium from 30 to 43°C resulted in a decrease in the amplitude of the population spike (A-PS) and a reduced slope of the excitatory postsynaptic potential (S-EPSP). Bath application of the -aminobutyric acid receptor antagonist, picrotoxin, or a change in the calcium concentration of the perfusate did not affect the A-PS during heating.3. Increasing the strength of the synaptic input to that eliciting a PS with an amplitude 50, 75, or 100% of maximal at 30°C resulted in a significant increase in the A-PS during the middle phase of hyperthermia (35–39°C).4. The long-term potentiation (LTP) induced at either 30 or 37°C showed the same percentage increase in both the amplitude of the population spike and the S-EPSP after delivery of a tetanus (100 Hz, 100 pulses) to CA1 synapses.5. The results of the present study, therefore, indicate that the decrease in CA1 field potential was linearly related to the temperature of the slice preparation, while LTP was induced in these responses during heating from 30 to 37°C.     

From structure to clinic: Design of a muscarinic M1 receptor agonist with the potential to treat Alzheimer’s disease

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Peptide fragment of the m3 muscarinic acetylcholine receptor activates G(q) but not G(i2).

G(q), a heterotrimeric guanine nucleotide-binding protein, plays important roles such as the regulation of calcium mobilization and cell proliferation. This protein is considered as a promising drug target for the treatment of cardiac hypertrophy. Selective activation of G(q) would be quite useful for analyzing the role of G(q) in signaling pathways. We synthesized m3i3c-a peptide with 16 amino acid residues that corresponds to the junction between the C-terminus of the third intracellular loop and the sixth transmembrane helix (TM-VI) of human m3 muscarinic acetylcholine receptor, which couples to G(q) but not G(i2). At micromolar concentrations, this peptide was found to activate G(q) but not G(i2). This peptide is the first small compound that selectively activates G(q) but not G(i2). Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Rebuilding a macromolecular membrane complex at the atomic scale: Case of the Kir6.2 potassium channel coupled to the muscarinic acetylcholine receptor M2

Ion channel‐coupled receptors (ICCR) are artificial proteins built from a G protein‐coupled receptor and an ion channel. Their use as molecular biosensors is promising in diagnosis and high‐throughput drug screening. The concept of ICCR was initially validated with the combination of the muscarinic receptor M2 with the inwardly rectifying potassium channel Kir6.2. A long protein engineering phase has led to the biochemical characterization of the M2‐Kir6.2 construct. However, its molecular mechanism remains to be elucidated. In particular, it is important to determine how the activation of M2 by its agonist acetylcholine triggers the modulation of the Kir6.2 channel via the M2‐Kir6.2 linkage. In the present study, we have developed and validated a computational approach to rebuild models of the M2‐Kir6.2 chimera from the molecular structure of M2 and Kir6.2. The protocol was first validated on the known protein complexes of the μ‐opioid Receptor, the CXCR4 receptor and the Kv1.2 potassium channel. When applied to M2‐Kir6.2, our protocol produced two possible models corresponding to two different orientations of M2. Both models highlights the role of the M2 helices I and VIII in the interaction with Kir6.2, as well as the role of the Kir6.2 N‐terminus in the channel opening. Those two hypotheses will be explored in a future experimental study of the M2‐Kir6.2 construct. Proteins 2014; 82:1694–1707. © 2014 Wiley Periodicals, Inc.     

Arrestin‐dependent but G‐protein coupled receptor kinase‐independent uncoupling of D2‐dopamine receptors

We reconstituted D2 like dopamine receptor (D2R) and the delta opioid receptor (DOR) coupling to G‐protein gated inwardly rectifying potassium channels (K_ir3) and directly compared the effects of co‐expression of G‐protein coupled receptor kinase (GRK) and arrestin on agonist‐dependent desensitization of the receptor response. We found, as described previously, that co‐expression of a GRK and an arrestin synergistically increased the rate of agonist‐dependent desensitization of DOR. In contrast, only arrestin expression was required to produce desensitization of D2R responses. Furthermore, arrestin‐dependent GRK‐independent desensitization of D2R‐K_ir3 coupling could be transferred to DOR by substituting the third cytoplasmic loop of DOR with that of D2R. The arrestin‐dependent GRK‐independent desensitization of D2R desensitization was inhibited by staurosporine treatment, and blocked by alanine substitution of putative protein kinase C phosphorylation sites in the third cytoplasmic loop of D2R. Finally, the D2R construct in which putative protein kinase C phosphorylation sites were mutated did not undergo significant agonist‐dependent desensitization even after GRK co‐expression, suggesting that GRK phosphorylation of D2R does not play an important role in uncoupling of the receptor.

     

Activation of D1 dopamine receptors increases surface expression of AMPA receptors and facilitates their synaptic incorporation in cultured hippocampal neurons

Considerable evidence indicates that neuroadaptations leading to addiction involve the same cellular processes that enable learning and memory, such as long-term potentiation (LTP), and that psychostimulants influence LTP through dopamine (DA)-dependent mechanisms. In hippocampal CA1 pyramidal neurons, LTP involves insertion of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors into excitatory synapses. We used dissociated cultures to test the hypothesis that D1 family DA receptors influence synaptic plasticity in hippocampal neurons by modulating AMPA receptor trafficking. Brief exposure (5 min) to a D1 agonist increased surface expression of glutamate receptor (GluR)1-containing AMPA receptors by increasing their rate of externalization at extrasynaptic sites. This required the secretory pathway but not protein synthesis, and was mediated mainly by protein kinase A (PKA) with a smaller contribution from Ca2+-calmodulin-dependent protein kinase II (CaMKII). Prior D1 receptor stimulation facilitated synaptic insertion of GluR1 in response to subsequent stimulation of synaptic NMDA receptors with glycine. Our results support a model for synaptic GluR1 incorporation in which PKA is required for initial insertion into the extrasynaptic membrane whereas CaMKII mediates translocation into the synapse. By increasing the size of the extrasynaptic GluR1 pool, D1 receptors may promote LTP. Psychostimulants may usurp this mechanism, leading to inappropriate plasticity that contributes to addiction-related behaviors.     

Novel contribution of cell surface and intracellular M1‐muscarinic acetylcholine receptors to synaptic plasticity in hippocampus

Muscarinic acetylcholine receptors (mAChRs) are well known to transmit extracellular cholinergic signals into the cytoplasm from their position on the cell surface. However, we show here that M1‐mAChRs are also highly expressed on intracellular membranes in neurons of the telencephalon and activate signaling cascades distinct from those of cell surface receptors, contributing uniquely to synaptic plasticity. Radioligand‐binding experiments with cell‐permeable and ‐impermeable ligands and immunohistochemical observations revealed intracellular and surface distributions of M1‐mAChRs in the hippocampus and cortex of rats, mice, and humans, in contrast to the selective occurrence on the cell surface in other tissues. All intracellular muscarinic‐binding sites were abolished in M1‐mAChR‐gene‐knockout mice. Activation of cell surface M1‐mAChRs in rat hippocampal neurons evoked phosphatidylinositol hydrolysis and network oscillations at theta rhythm, and transiently enhanced long‐term potentiation. On the other hand, activation of intracellular M1‐mAChRs phosphorylated extracellular‐regulated kinase 1/2 and gradually enhanced long‐term potentiation. Our data thus demonstrate that M1‐mAChRs function at both surface and intracellular sites in telencephalon neurons including the hippocampus, suggesting a new mode of cholinergic transmission in the central nervous system.     

Expression of muscarinic acetylcholine receptors and choline acetyltransferase enzyme in cultured antennal sensory neurons and non‐neural cells of the developing moth Manduca sexta

Antennal sensory neurons of Manduca sexta emerge from epidermal cells that also give rise to sheath cells surrounding the peripheral parts of the neurons and to glial cells that enwrap the sensory axons in the antennal nerve. Reciprocal interactions between sensory neurons and glial cells are believed to aid in axon growth and guidance, but the exact nature of these interactions is not known. We investigated the possibility of cholinergic interactions in this process by locating muscarinic acetylcholine receptors (mAChRs) and choline acetyltransferase (ChAT) enzyme in cultured antennal sensory neurons and non‐neural cells. ChAT and mAChRs were present in the sensory neurons from the first day in culture. Therefore, the sensory neurons are probably cholinergic, as previously suggested, but they may also be controlled by ACh. In 7‐day‐old cultures a subgroup of small non‐neural cells with processes expressed ChAT activity, and in 14‐day‐old cultures non‐neural cells that formed lamellipodia and scaffoldlike structures on the culture substrate were labeled with ChAT antibody. mAChR activity was detected in similar non‐neural cells but only in areas surrounding the nuclei. In addition, mAChRs were found in flat lamellipodia and filopodia forming cells that were present in 1‐day‐old cultures and grew in size during the 2 week investigation period. These findings suggest muscarinic cholinergic interactions between the neural and non‐neural cells during the development of Manduca antenna. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

Differential regulation of mTOR-dependent S6 phosphorylation by muscarinic acetylcholine receptor subtypes

Muscarinic receptors subserve many functions in both peripheral and central nervous systems. Some of these processes depend on increases in protein synthesis, which may be achieved by activation of mammalian target of rapamycin (mTOR), a kinase that regulates protein translation capacity. Here, we examined the regulation of mTOR-dependent signaling pathways by muscarinic receptors in SK-N-SH human neuroblastoma cells, and in human embryonic kidney (HEK) cell lines transfected with individual muscarinic receptor subtypes. In SK-N-SH cells, the acetylcholine analog carbachol stimulated phosphorylation of the ribosomal S6 protein, a downstream target of mTOR. The sensitivity of the response to subtype-selective muscarinic receptor antagonists indicated that it was mediated by M3 receptors. Carbachol-evoked S6 phosphorylation was blocked by the mTOR inhibitor rapamycin, but was independent of phosphoinositide 3-kinase activation. The response was significantly reduced by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also inhibited carbachol-evoked S6 phosphorylation in HEK cells expressing M2 receptors, but was ineffective in M3 receptor-expressing HEK cells, although carbachol activated MAPK in both transfected lines. The p90 ribosomal S6 kinase has been implicated in mTOR regulation by phorbol esters, but was not activated by carbachol in any of the cell lines tested. The protein kinase C inhibitor bisindolylmaleimide I reduced carbachol-stimulated S6 phosphorylation in SK-N-SH cells, and in HEK cells expressing M3 receptors, but not in HEK cells expressing M2 receptors. The results demonstrate that multiple muscarinic receptor subtypes regulate mTOR, and that both MAPK-dependent and -independent mechanisms may mediate the response in a cell context-specific manner.     

Identification of multiple allosteric sites on the M1 muscarinic acetylcholine receptor

Staurosporine and four staurosporine derivatives were docked on the rhodopsin-based homology model of the M1 muscarinic acetylcholine receptor in order to localize the possible allosteric sites of this receptor. It was found that there were three major allosteric sites, two of which are located at the extracellular face of the receptor, and one in the intracellular domain of the receptor. In the present study, the localization of these binding sites is described for the first time. The present study confirms the existence of multiple allosteric sites on the M1 muscarinic receptor, and lays the ground for further experimental and computational analysis to better understand how muscarinic receptors are modulated via their allosteric sites. These findings will also help to design and develop novel drugs acting as allosteric modulators of the M1 receptor, which can be used in the treatment of the Alzheimer's disease.     

Single channel studies of inward rectifier potassium channel regulation by muscarinic acetylcholine receptors

Negative regulation of the heartbeat rate involves the activation of an inwardly rectifying potassium current (I(KACh)) by G protein-coupled receptors such as the m2 muscarinic acetylcholine receptor. Recent studies have shown that this process involves the direct binding of G(betagamma) subunits to the NH(2)- and COOH-terminal cytoplasmic domains of the proteins termed GIRK1 and GIRK4 (Kir3.1 and Kir3.4/CIR), which mediate I(KACh). Because of the very low basal activity of native I(KACh), it has been difficult to determine the single channel effect of G(betagamma) subunit binding on I(KACh) activity. Through analysis of a novel G protein-activated chimeric inward rectifier channel that displays increased basal activity relative to I(KACh), we find that single channel activation can be explained by a G protein-dependent shift in the equilibrium of open channel transitions in favor of a bursting state of channel activity over a long-lived closed state.     

Subtype-specific regulation of the expression and function of muscarinic acetylcholine receptors in embryonic chicken retinal cells

We examined the effect of long-term agonist exposure on muscarinic acetylcholine receptor expression and function in embryonic chicken retinal cells. Long-term carbachol exposure induced a time- and concentration-dependent decrease in M2, M3 and M4 muscarinic receptor numbers. Kinetic analyses revealed a first-order process with similar rate constants for all three subtypes. Both the maximal decrease and the agonist potency for regulation of M3 were significantly higher than those for M2 and M4. Upon agonist removal, M2 and M4 numbers returned to control values, but M3 recovery after 24 h was no higher than 40%. Agonist treatment did not alter the levels of receptor mRNAs. Receptor inactivation with a covalent alkylating antagonist demonstrated that the partial M3 protein recovery was not due to a decreased intrinsic basal rate of synthesis, suggesting that it is induced by agonist treatment. Prolonged carbachol exposure induced concomitant decreases in muscarinic-mediated inhibition of cyclic AMP accumulation which were completely reversed after agonist removal. Sustained receptor activation also promoted significant decreases in muscarinic receptor-stimulated phosphoinositide turnover, which were only partially reversed after agonist removal. These data demonstrate subtype-specific regulation of the expression and function of muscarinic receptors in the retina.     

Differential regulation of metabotropic glutamate receptor 5-mediated phosphoinositide hydrolysis and extracellular signal-regulated kinase responses by protein kinase C in cultured astrocytes

The metabotropic glutamate receptor 5 (mGluR5) exhibits a rapid loss of receptor responsiveness to prolonged or repeated agonist exposure. This receptor desensitization has been seen in a variety of native and recombinant systems, and is thought to result from receptor-mediated, protein kinase C (PKC)-dependent phosphorylation of the receptor, uncoupling it from the G protein in a negative feedback regulation. We have investigated the rapid PKC-mediated desensitization of mGluR5 in cortical cultured astrocytes by measuring downstream signals from activation of mGluR5. These include activation of phosphoinositide (PI) hydrolysis, intracellular calcium transients, and extracellular signal-regulated kinase 2 (ERK2) phosphorylation. We present evidence that PKC plays an important role in rapid desensitization of PI hydrolysis and calcium signaling, but not in ERK2 phosphorylation. This differential regulation of mGluR5-mediated responses suggests divergent signaling and regulatory pathways which may be important mechanisms for dynamic integration of signal cascades.     

Constitutive phosphorylation by protein kinase C regulates D1 dopamine receptor signaling

The D(1) dopamine receptor (D(1) DAR) is robustly phosphorylated by multiple protein kinases, yet the phosphorylation sites and functional consequences of these modifications are not fully understood. Here, we report that the D(1) DAR is phosphorylated by protein kinase C (PKC) in the absence of agonist stimulation. Phosphorylation of the D(1) DAR by PKC is constitutive in nature, can be induced by phorbol ester treatment or through activation of Gq-mediated signal transduction pathways, and is abolished by PKC inhibitors. We demonstrate that most, but not all, isoforms of PKC are capable of phosphorylating the receptor. To directly assess the functional role of PKC phosphorylation of the D(1) DAR, a site-directed mutagenesis approach was used to identify the PKC sites within the receptor. Five serine residues were found to mediate the PKC phosphorylation. Replacement of these residues had no effect on D(1) DAR expression or agonist-induced desensitization; however, G protein coupling and cAMP accumulation were significantly enhanced in PKC-null D(1) DAR. Thus, constitutive or heterologous PKC phosphorylation of the D(1) DAR dampens dopamine activation of the receptor, most likely occurring in a context-specific manner, mediated by the repertoire of PKC isozymes within the cell.