Involvement of Ca<Superscript>2+</Superscript>/CaM in the signal transduction of acetylcholine regulating stomatal movement

It has been known that the neurotransmitter acetylcholine (ACh) also exists in plants and is able to regulate the movement of stomata.In another aspect,Ca^2 /CaM as the second messengers have a critical role of signal transduction in stomatal guard-cell,Here we showed that Ca^2 /CaM were also involved in the ACh regulated stomatal movement,In the medium containing Ca^2 ,the Ca^2 channel blockers (NIF and Ver)and CaM inhibitors (TFP and W7 ) could neutralize the ACh induced stomatal opening,however,they are ineffective in the medium containing K^ ,Those results indicated that Ca^2 /CaM were involved in the signal transduction pathway of ACh regulating stomatal movement.     

Localization of muscarinic acetylcholine receptor in plant guard cells

Acetylcholine (ACh), as an important neurotransmitter in animals, also plays a significant role in various kinds of physiological functions in plants. But relatively little is known about its receptors in plants. A green fluorescence BODIPY FL-labeled ABT, which is a high affinity ligand of muscarinic acetylcholine receptor (mAChR), was used to localize mAChR in plant guard cells. InVicia faba L. andPisum sativum L., mAChR was found both on the plasma membrane of guard cells. mAChR may also be distributed on guard cell chloroplast membrane ofVicia faba L. The evidence that mAChR localizes in the guard cells provides a new possible signal transduction pathway in ACh mediated stomata movement.     

Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist

The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.     

A single GTP-binding protein regulates K+-channels coupled with dopamine, histamine and acetylcholine receptors

Recently, a GTP-binding protein sensitive to islet activating protein (IAP) has been suggested to be important in producing K+-currents when the muscarinic receptor of the atrial muscle is activated by acetylcholine (ACh). Here we confirm the blocking effects of IAP and GTP gamma S (a nonhydrolysable analogue of GTP) on the ACh-induced K+-current recorded from the ganglion cells of the sea slug Aplysia and compare their effects on histamine (HA)-induced and dopamine (DA)-induced K+-currents. Intracellular injections of IAP irreversibly and selectively block the openings of K+-channels activated by either ACh, HA, or DA without affecting the resting potential or conductance states of the membranes. Intracellular application of GTP gamma S alone caused extremely slow, irreversible opening of K+-channels; however, repetitive receptor activations significantly increase the rate of the GTP gamma S effect. These results strongly suggest that a GTP-binding protein such as Gi regulates the opening of K+-channels coupled with these receptors.     

Cloning, sequencing and expression of complementary DNA encoding the muscarinic acetylcholine receptor

Cloning and sequence analysis of DNA complementary to porcine cerebral messenger RNA encoding the muscarinic acetylcholine receptor predict the complete amino-acid sequence of this protein. Expression of the complementary DNA produced functional muscarinic receptor in Xenopus oocytes. The muscarinic receptor is homologous with the beta-adrenergic receptor and rhodopsin in both amino-acid sequence and suggested transmembrane topography.     

乙酰胆碱对人胃粘膜上皮细胞和胃腺癌细胞内Ca2+、Mg2+浓度的影响

目的:探讨乙酰胆碱对人胃粘膜上皮细胞和胃腺癌细胞内Ca2 、Mg2 浓度的影响。方法:利用大型生化分析仪,检测了乙酰胆碱作用后培养的人胃粘膜上皮细胞和胃腺癌细胞内Ca2 、Mg2 的浓度。结果:与正常胃粘膜上皮细胞比较,胃腺癌细胞内Ca2 、Mg2 浓度偏低(P>0 01)。在乙酰胆碱作用下,胃粘膜上皮细胞内Ca2 浓度升高(P<0 01),Mg2 浓度降低(P<0 01);胃腺癌细胞仅有Ca2 浓度升高(P<0 05),Mg2 浓度无变化。阿托品能阻断乙酰胆碱的作用(P<0 05)。结论:乙酰胆碱对胃粘膜上皮细胞和胃腺癌细胞的作用是通过毒蕈碱受体实现的。     

Mice lacking the M3 muscarinic acetylcholine receptor are hypophagic and lean

Members of the muscarinic acetylcholine receptor family (M1-M5) have central roles in the regulation of many fundamental physiological functions. Identifying the specific receptor subtype(s) that mediate the diverse muscarinic actions of acetylcholine is of considerable therapeutic interest, but has proved difficult primarily because of a lack of subtype-selective ligands. Here we show that mice deficient in the M3 muscarinic receptor (M3R-/- mice) display a significant decrease in food intake, reduced body weight and peripheral fat deposits, and very low levels of serum leptin and insulin. Paradoxically, hypothalamic messenger RNA levels of melanin-concentrating hormone (MCH), which are normally upregulated in fasted animals leading to an increase in food intake, are significantly reduced in M3R-/- mice. Intra-cerebroventricular injection studies show that an agouti-related peptide analogue lacked orexigenic (appetite-stimulating) activity in M3R-/- mice. However, M3R-/- mice remained responsive to the orexigenic effects of MCH. Our data indicate that there may be a cholinergic pathway that involves M3-receptor-mediated facilitation of food intake at a site downstream of the hypothalamic leptin/melanocortin system and upstream of the MCH system.     

Nitric oxide suppresses stomatal opening by inhibiting inward-rectifying K<Stack><Subscript>in</Subscript><Superscript>+</Superscript></Stack> channels in <Emphasis Type="Italic">Arabidopsis</Emphasis> guard cells

We explore nitric oxide （NO） effect on K^＋in, channels in Arabidopsis guard cells. We observed NO inhibited K^＋in, currents when Ca^2＋ chelator EGTA （Ethylene glycol-bis（2-aminoethylether）-N,N,N′,N;tetraacetic acid） was not added in the pipette solution; K^＋in currents were not sensitive to NO when cytosolic Ca^2＋ was chelated by EGTA. NO inhibited the Arabidopsis stomatal opening, but when EGTA was added in the bath solution, inhibition effect of NO on stomatal opening vanished. Thus, it implies that NO elevates cytosolic Ca^2＋ by activating plasma membrane Ca^2＋ channels firstly, then inactivates K^＋in, chartnels, resulting in stomatal opening suppressed subsequently.     

Rearrangements of microtubule cytoskeleton in stomatal closure of <Emphasis Type="Italic">Arabidopsis</Emphasis> induced by nitric oxide

NO （nitric oxide）, known as a key signal molecule in plant, plays important roles in regulation of stomatal movement. In this study, microtubule dynamics and its possible mechanism in the NO signal pathway were investigated. The results were as follows： （i） In vivo stomatsl aperture assays revealed that both vinblastine （microtubule-disrupUng drug） and SNP （exogenous NO donor） prevented stomatsl opening in the light, and vinblastine even could enhance the inhibitory effect of SNP, whereas tsxol （a microtubule-stsbilizing agent） was able to reduce this effect; （ii） microtubules in the opening Arabi-dopsis guard cells expressing GFP：a-tubulin-6 （AtGFP：a-tubulin-6） were organized in parallel, straight and dense bundles, radiating from the ventral side to the dorsal side, and most of them were localized perpendicularly to the ventral wall; （iii） under the same environmental conditions, treated with SNP for 30 min, the radial arrays of microtubules in guard cells began to break down, twisted partially and be- came oblique or exhibited a random pattern; （iv） furthermore, the involvement of cytosolic Ca^2＋ in this event was tested. Stomatal aperture assays revealed that BAPTA-AM （a chelator of Ca^2＋） greatly suppressed the effect of NO on stomatal closure; however, it did not show the same function on stomatal closure induced by vinblastine. When BAPTA-AM was added to the SNP-pretreated solution, the SNP-induced disordered microtubulue cytoskeleton in guard cells underwent rearrangement in a time-dependent manner. After 30 min of treatment with BAPTA-AM, the cortical microtubules resumed the original radial distribution, almost the same as the control. All this indicates that NO may promote rearrangement of microtubule cytoskeleton via elevation of [Ca^2＋]cyt （free Ca^2＋ concentration in the cytoplasm）, finally leading to stomatsl closure.     

A stepwise mechanism for acetylcholine receptor channel gating

Muscle contraction is triggered by the opening of acetylcholine receptors at the vertebrate nerve-muscle synapse. The M2 helix of this allosteric membrane protein lines the channel, and contains a 'gate' that regulates the flow of ions through the pore. We used single-molecule kinetic analysis to probe the transition state of the gating conformational change and estimate the relative timing of M2 motions in the alpha-subunit of the murine acetylcholine receptor. This analysis produces a 'Phi-value' for a given residue that reflects its open-like versus closed-like character at the transition state. Here we show that most of the residues throughout the length of M2 have a Phi-value of approximately 0.64 but that some near the middle have lower Phi-values of 0.52 or 0.31, suggesting that alphaM2 moves in three discrete steps. The core of the channel serves both as a gate that regulates ion flow and as a hub that directs the propagation of the gating isomerization through the membrane domain of the acetylcholine receptor.     

Ca^2＋ is involved in muscarine-acetylcholine-receptor-mediated acetylcholine signal transduction in guard cells of Vicia faba L.

Acetyicholine (ACh) is an important neuro-chemical transmitter in animals; it also exists in plants and plays a significant role in various kinds of physiological functions in plants. ACh has been known to induce the stomatal opening. By monitoring the changes of cytusolic Ca^2 with fluorescent probe Fiuo-3 AM under the confocal microscopy, we found that exogenous ACh increased cytosolic Ca^2 concentration of guard cells of Vicia faba L. Muscarlne, an agonist of muscarine acetyicholine receptor (mAChR), could do so as well. In contrast, atropine, the antagonist of mAChR abolished the ability of ACh to increase Ca^2 in guard cells. This mechanism is similar to mAChR in animals. When EGTA was used to chelate Ca^2 or ruthenium red to block Ca^2 released from vacuole respectively, the results showed that the increased cytosolic Ca^2 mainly come from intracellular Ca^2 store. The evidence supports that Ca^2 is involved in guard-cell response to ACh and that Ca^2 sigual is coupled to mAChRs in ACh signal transduction in guard cells.     

5-HT3 receptors mediate inhibition of acetylcholine release in cortical tissue

The release of cerebral acetylcholine from terminals in the cerebral cortex has been shown to be regulated by 5-hydroxytryptamine (5-HT) but it is not known which subtype of the 5-HT receptor is involved. 5-HT receptor agonists increase acetylcholine levels in vivo, indicating a reduced turnover, and reduce release of acetylcholine from striatal slices in vitro. Depleting 5-HT by inhibiting synthesis or by destroying the neurons containing 5-HT potentiates acetylcholine release, and increases acetylcholine turnover in the cerebral cortex and hippocampus. Selective antagonists for the 5-HT3 receptor subtypes which seem to have effects on mood and activity may exert their effect through the regulation of acetylcholine release in the cortex and limbic system. Radioligand binding studies show a high density of 5-HT3 receptors in the cholinergic-rich entorhinal cortex and we provide evidence that a reduction in cortical cholinergic function can be effected in vitro by 5-HT3 receptors.     

Isolation of a cDNA clone coding for a possible neural nicotinic acetylcholine receptor alpha-subunit

We have isolated a complementary DNA clone containing sequences homologous to those encoding the alpha-subunit of a mouse muscle nicotinic acetylcholine receptor. Based on the structural similarities between the encoded protein and the muscle acetylcholine receptor alpha-subunit, and the presence of hybridizing RNA species in the brain, we propose that this clone codes for a neural nicotinic acetylcholine receptor alpha-subunit.     

大鼠耳蜗血管纹胆碱能M受体的表达及意义

目的:研究胆碱能M受体在耳蜗血管纹的表达.方法:采用RT-PCR技术检测大鼠耳蜗血管纹上胆碱能M受体的基因表达.结果:扩增出标志M2、M3、M4和M5受体亚型基因表达的PCR产物,未扩增出标志M1受体亚型的产物.结论:胆碱能M2、M3、M4和M5受体亚型在耳蜗血管纹有表达,提示胆碱能M受体可能参与了血管纹功能的调节,为研究胆碱能M受体在血管纹上的功能提供分子生物学基础.     

Role of acetylcholine on plant root-shoot signal transduction

The role of accetylcholine (ACh) on plant root-shoot communication was investigated using the root-split system of Vicia faba L.In the experiments,slight osmotic stress caused the decrease of ACh content in root tips and the xylem sap transported up per time unit from root tip to the shool when the water potential of the shoot was kept unchanged .It also caused the decrease of ACh content in the abaxial epidermis,The decrease was highly correlative to the changes of transpiration rate,suggesting that the decrase of ACh content probably functions as a signal to regulate stomatal behavior,The effect of osmotic stress might be mainly through the inhibition of the ACh synthesis in root tip ;thus further influences the ACh content in root tip ,xylem sap and abaxial epidermis and resulting in the changes of stomata behavior,These results provide new evdence that plants transduce positive and negative signals among roots and shoot to coordinate stomatal behavior and adapt to variable environments.     

红色赤潮藻对球形棕囊藻的化感作用研究

红色赤潮藻(Akashiwo sanguinea)和球形棕囊藻(Phaeocystis globosa)都是近年来在我国近岸海域频繁引发赤潮的藻类。为了探索红色赤潮藻和球形棕囊藻之间是否存在化感作用及作用方式,本研究采用不同起始密度培养红色赤潮藻与球形棕囊藻,并将前者的完整细胞培养液、无细胞滤液、藻液超声破碎液分别与球形棕囊藻进行混合培养,分析红色赤潮藻对球形棕囊藻细胞生长的影响。结果显示,在红色赤潮藻细胞起始密度保持5×10⁵ cells/L的混合培养条件下,起始密度分别为5×10⁵、1×10⁶、2×10⁶、1×10⁷ cells/L的球形棕囊藻生长均受到明显的抑制;无论是含有红色赤潮藻完整细胞的培养液,还是其无细胞滤液或藻液超声破碎液,均对球形棕囊藻的生长具有显著抑制作用(P<0.01),抑制强度由强至弱依次为完整细胞培养液、无细胞滤液和藻液超声破碎液;相反,球形棕囊藻的存在对红色赤潮藻细胞的生长无显著影响(P>0.05)。因此,红色赤潮藻具有抑制球形棕囊藻生长...     

发酵木糖高产乙醇树干毕赤酵母菌株的Co60诱变选育

【目的】选育能够利用木糖高产乙醇的酵母菌株。【方法】采用Co60诱变树干毕赤酵母(Pichia stipitis),筛选乙醇产量高的突变菌株,并对原始菌株、突变菌株的生长发酵特性和两个菌株对高浓度木糖、乙醇的耐受性进行比较。【结果】在YPX培养基上筛选获得1株能够高效发酵木糖的突变菌株1K-9。该菌株在50mL 15%FM发酵84h,发酵液乙醇含量最高达(51.034±0.112)g/L,比原始菌株提高10.05%;在500mL 15%FM发酵96h,乙醇含量最高达(51.390±0.119)g/L;在500mL 20%FM发酵156h,乙醇含量最高达(52.496±0.513)g/L。菌株1K-9在HSM培养基或含4%~5%乙醇的YPX培养基中生长良好,在含6%~7%乙醇的YPX培养基中生长缓慢。【结论】Co60诱变对于树干毕赤酵母(Pichia stipitis)菌株是有效的,能选育出木糖高产乙醇酵母菌株1K-9。     

Mapping the conformational wave of acetylcholine receptor channel gating

Allosteric transitions allow fast regulation of protein function in living systems. Even though the end points of such conformational changes are known for many proteins, the characteristics of the paths connecting these states remain largely unexplored. Rate-equilibrium linear free-energy relationships (LFERs) provide information about such pathways by relating changes in the free energy of the transition state to those of the ground states upon systematic perturbation of the system. Here we present an LFER analysis of the gating reaction pathway of the muscle acetylcholine receptor. We studied the closed <==> open conformational change at the single-molecule level following perturbation by series of single-site mutations, agonists and membrane voltages. This method provided a snapshot of several regions of the receptor at the transition state in terms of their approximate positions along the reaction coordinate, on a scale from 0 (closed-like) to 1 (open-like). The resulting map reveals a spatial gradient of positional values, which suggests that the conformational change proceeds in a wave-like manner, with the low-to-high affinity change at the transmitter-binding sites preceding the complete opening of the pore.     

Phosphorylation of the nicotinic acetylcholine receptor regulates its rate of desensitization

Recent studies have provided evidence for a role of protein phosphorylation in the regulation of the function of various potassium and calcium channels (for reviews, see refs 1, 2). As these ion channels have not yet been isolated and characterized, it has not been possible to determine whether phosphorylation of the ion channels themselves alters their properties or whether some indirect mechanism is involved. In contrast, the nicotinic acetylcholine receptor, a neurotransmitter-dependent ion channel, has been extensively characterized biochemically and has been shown to be directly phosphorylated. The phosphorylation of this receptor is catalysed by at least three different protein kinases (cyclic AMP-dependent protein kinase, protein kinase C and a tyrosine-specific protein kinase) on seven different phosphorylation sites. However, the functional significance of phosphorylation of the receptor has been unclear. We have now examined the functional effects of phosphorylation of the nicotinic acetylcholine receptor by cAMP-dependent protein kinase. We investigated the ion transport properties of the purified and reconstituted acetylcholine receptor before and after phosphorylation. We report here that phosphorylation of the nicotinic acetylcholine receptor on the gamma- and delta-subunits by cAMP-dependent protein kinase increases the rate of the rapid desensitization of the receptor, a process by which the receptor is inactivated in the presence of acetylcholine (ACh). These results provide the first direct evidence that phosphorylation of an ion channel protein modulates its function and suggest that phosphorylation of postsynaptic receptors in general may play an important role in synaptic plasticity.