昆虫分子生物学的一些进展：神经递质和离子通道

昆虫分子生物学的一些进展：神经递质和离子通道翟启慧（中国科学院动物研究所北京１０００８０）１神经递质神经递质（ｎｅｕｒｏｔｒａｎ。ｍｉｔｔｅｒ）是在化学突触神经ｆＧ。１问传递信息的化学物质。神经递质有许多不同类型,如乙酸胆碱、丫一氨基丁酸、生物胺等。...     

Proton NMR and CD solution conformation determination and opioid receptor binding studies of a dynorphin A(1-17) model peptide

The opioid peptide dynorphin A(1-17) contains a peptide segment in residues 7-15 with the potential to form an amphiphilic beta-strand. This amphiphilic structure may, like the amphiphilic alpha-helices found in many other peptide hormones, be an important determinant of its interactions with membranes and receptors. In order to investigate and characterize these interactions, we have synthesized a 17-residue dynorphin analogue (YGGFLKKVKPKVKVKSS) that incorporates a peptide model of this amphiphilic secondary structure with minimized homology (25%) relative to the native sequence. This peptide exhibits the full biological potency of dynorphin in assays of kappa-opioid receptor binding, and is more selective for this type of opioid receptor than the natural peptide. The conformation of the model peptide in aqueous solution has been investigated in detail by NMR spectroscopy. The values of the NH-CH alpha coupling constants together with rotating frame NOEs indicate the presence of an amphiphilic structure together with some beta-strand structure in residues 7-15, and demonstrate that a peptide model that stabilizes this structure in aqueous solution and enhances kappa-opioid receptor selectivity can be successfully designed using using alternating lysine and valine residues.     

The biology of beta-adrenergic receptors: analysis in human epidermoid carcinoma A431 cells.

1. G-protein-linked transmembrane signaling has emerged as a major pathway for information transduction across the cell membrane. 2. In addition to photopigments that propagate the signal from light, cell-surface receptors for hormones, neurotransmitters, and autacoids propagate signals from ligand binding to membrane-bound effector units via G-proteins. 3. Biochemical and molecular features of one prominent member of these receptors, the beta-adrenergic receptor, will be highlighted in the present article. 4. The role of the human epidermoid carcinoma A431 cells as a model for the study of the structure and biology of beta-adrenergic receptors will be emphasized. 5. A model for receptor regulation, gleaned from recent advances in the biochemistry, cell and molecular biology of beta-adrenergic receptors, is discussed.     

Spectroscopic studies of PhTX facilitated cation movement across membranes.

Philanthotoxins, noncompetitive inhibitors of the nicotinic acetylcholine receptor and various glutamate receptors, were found to be capable of mediating cation transport across lipid bilayers. With respect to the relatively weak binding constants of these amphiphilic polyamines to neuronal receptor proteins, this finding implies that their interaction with cell membranes might have to be considered in addition to that with protein receptors to fully understand the molecular mechanism of these neurotoxins.     

Characterization of a Digitonin-Solubilized Bovine Brain H3 Histamine Receptor Coupled to a Guanine Nucleotide-Binding Protein

The H3 receptor is a high-affinity histamine receptor that inhibits release of several neurotransmitters, including histamine. We have characterized H3 receptor binding in bovine brain and developed conditions for its solubilization. Particulate [3H]histamine binding showed an apparently single class of sites (KD = 4.6 nM; Bmax = 78 fmol/mg of protein). Of the detergents tested, digitonin at a detergent/protein ratio of 1:1 (wt/wt) yielded the greatest amount of solubilized receptors, typically 15-30% of particulate binding. Neither equilibrium binding of [3H]histamine to receptors (KD = 6.1 nM; Bmax = 92 fmol/mg of protein) nor the inhibitor profile was substantially altered by digitonin solubilization. However, solubilization did increase the rate of [3H]histamine association with and dissociation from the receptor. Size-exclusion chromatography indicated an apparent molecular weight of 220,000 for the solubilized receptor, and peak binding from this column retained its guanine nucleotide sensitivity. These last two observations are consistent with the solubilized receptor occurring in complex with a guanine nucleotide-binding protein.     

Water-membrane partition thermodynamics of an amphiphilic lipopeptide: an enthalpy-driven hydrophobic effect

To shed light on the driving force for the hydrophobic effect that partitions amphiphilic lipoproteins between water and membrane, we carried out an atomically detailed thermodynamic analysis of a triply lipid modified H-ras heptapeptide anchor (ANCH) in water and in a DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) bilayer. Combining molecular mechanical and continuum solvent approaches with an improved technique for solute entropy calculation, we obtained an overall transfer free energy of ~−13 kcal mol⁻¹. This value is in qualitative agreement with free energy changes derived from a potential of mean force calculation and indirect experimental observations. Changes in free energies of solvation and ANCH conformational reorganization are unfavorable, whereas ANCH-DMPC interactions—especially van der Waals—favor insertion. These results are consistent with an enthalpy-driven hydrophobic effect, in accord with earlier calorimetric data on the membrane partition of other amphiphiles. Furthermore, structural and entropic analysis of molecular dynamics-generated ensembles suggests that conformational selection may play a hitherto unappreciated role in membrane insertion of lipid-modified peptides and proteins.     

Sphingolipid/cholesterol regulation of neurotransmitter receptor conformation and function

Like all other monomeric or multimeric transmembrane proteins, receptors for neurotransmitters are surrounded by a shell of lipids which form an interfacial boundary between the protein and the bulk membrane. Among these lipids, cholesterol and sphingolipids have attracted much attention because of their well-known propensity to segregate into ordered platform domains commonly referred to as lipid rafts. In this review we present a critical analysis of the molecular mechanisms involved in the interaction of cholesterol/sphingolipids with neurotransmitter receptors, in particular acetylcholine and serotonin receptors, chosen as representative members of ligand-gated ion channels and G protein-coupled receptors. Cholesterol and sphingolipids interact with these receptors through typical binding sites located in both the transmembrane helices and the extracellular loops. By altering the conformation of the receptors (“chaperone-like” effect), these lipids can regulate neurotransmitter binding, signal transducing functions, and, in the case of multimeric receptors, subunit assembly and subsequent receptor trafficking to the cell surface. Several sphingolipids (especially gangliosides) also exhibit low/moderate affinity for neurotransmitters. We suggest that such lipids could facilitate (i) the attachment of neurotransmitters to the post-synaptic membrane and in some cases (ii) their subsequent delivery to specific protein receptors. Overall, various experimental approaches provide converging evidence that the biological functions of neurotransmitters and their receptors are highly dependent upon sphingolipids and cholesterol, which are active partners of synaptic transmission. Several decades of research have been necessary to untangle the skein of a complex network of molecular interactions between neurotransmitters, their receptors, cholesterol and sphingolipids. This sophisticated crosstalk between all four distinctive partners may allow a fine biochemical tuning of synaptic transmission.     

A transferrin-binding protein of Trypanosoma brucei is encoded by one of the genes in the variant surface glycoprotein gene expression site.

A transferrin-binding protein (TFBP) with an apparent molecular weight of 42 kd was purified from detergent-soluble membrane proteins of bloodstream forms of Trypanosoma brucei. The protein is not expressed in the insect-borne stage of the parasite's life-cycle. Purified TFBP can be converted from an amphiphilic to a hydrophilic form by cleavage with T.brucei glycosylphosphatidylinositol (GPI)-specific phospholipase C, demonstrating that the C-terminus is modified by a GPI-membrane anchor. The TFBP is encoded by an expression-site-associated gene [ESAG 6 in the nomenclature of Pays et al. (1989) Cell, 57, 835-845] which is under the control of the promoter transcribing the expressed variant surface glycoprotein gene. The possible function of TFBP as a receptor for the uptake of transferrin in bloodstream forms is discussed.     

Surface viscosity, diffusion, and intermonolayer friction: simulating sheared amphiphilic bilayers

The flow properties of an amphiphilic bilayer are studied in molecular dynamics simulations, by exposing a coarse grained model bilayer to two shear flows directed along the bilayer surface. The first field, with a vorticity perpendicular to the bilayer, induces a regular shear deformation, allowing a direct calculation of the surface viscosity. In experiments this property is measured indirectly, by relating it to the diffusion coefficient of a tracer particle through the Saffman-Einstein expression. The current calculations provide an independent test of this relation. The second flow field, with a vorticity parallel to the bilayer, causes the two constituent monolayers to slide past one another, yielding the interlayer friction coefficient.     

Acetylcholinesterases of the nematode Steinernema carpocapsae. Characterization of two types of amphiphilic forms differing in their mode of membrane association.

We analyzed the molecular forms of acetylcholinesterase (AChE) in the nematode Steinernema carpocapsae. Two major AChEs are involved in acetylcholine hydrolysis. The first class of AChE is highly sensitive to eserine (IC50 = 0.05 microM). The corresponding molecular forms are: an amphiphilic 14S form converted into a hydrophilic 14.5S form by mild proteolysis and two hydrophilic 12S and 7S forms. Reduction of the amphiphilic 14S form with 10 mM dithiothreitol produces hydrophilic 7S and 4S forms, indicating that it is an oligomer of hydrophilic catalytic subunits linked by disulfide bond(s) to a hydrophobic structural element that confers the amphiphilicity to the complex. Sedimentation coefficients suggest that 4S, 7S, 12S forms correspond to hydrophilic monomer, dimer, tetramer and that the 14S form is also a tetramer linked to one structural element. The second class of AChE is less sensitive to eserine (IC50 = 0.1 mM). Corresponding molecular forms are hydrophilic and amphiphilic 4S forms (monomers) and a major amphiphilic 7S form converted into a hydrophilic dimer by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C. This amphiphilic 7S form thus possesses a glycolipid anchor. It appears that Steinernema (a very primitive invertebrate) presents AChEs with two types of membrane association that closely resemble those described for amphiphilic G2 and G4 forms of AChE in more evolved animals.     

Neurochemical and Molecular Pharmacological Aspects of the GABAB Receptor

Metabotropic gamma-aminobutyric acid (GABA)B receptors are known to modulate the synaptic release of various neurotransmitters in the nervous system. Activation of GABA(B) receptor induces the inhibition of adenylyl cyclase activity, while it does not stimulate the formation of inositol phosphates. Activation of a potassium conductance and suppression of a calcium conductance are also recognized, similarly to some of G protein-coupled receptors. Recent molecular cloning has revealed that GABA(B) receptor possesses a large extracellular domain including the binding site for GABA and seven transmembrane domains. Their molecular structures in the brain are unique and interesting because of heterodimerization consisting of two distinct genes: GABABR1 and GABABR2. Such assembled receptors can be classified as a novel type of the metabotropic receptor superfamily.     

Amphiphilic,glycophosphatidylinositol-specific phospholipase C (PI-PLC)-insensitive monomers and dimers of acetylcholinesterase

1. In a recent study, we distinguished two classes of amphiphilic AChE3 dimers in Torpedo tissues: class I corresponds to glycolipid-anchored dimers and class II molecules are characterized by their lack of sensitivity to PI-PLC and PI-PLD, relatively small shift in sedimentation with detergent, and absence of aggregation without detergent. 2. In the present report, we analyze the amphiphlic or nonamphiphilic properties of globular AChE forms in T28 murine neural cells, rabbit muscle, and chicken muscle. The molecular forms were identified by sucrose gradient sedimentation in the presence and absence of detergent and analyzed by nondenaturing charge-shift electrophoresis. Some amphiphilic forms showed an abnormal electrophoretic migration in the absence of detergent, because of the retention of detergent micelles. 3. We show that the amphiphilic monomers (G1a) from these tissues, as well as the amphiphilic dimers (G2a) from chicken muscle, resemble the class II dimers of Torpedo AChE. We cannot exclude that these molecules possess a glycolipidic anchor but suggest that their hydrophobic domain may be of a different nature. We discuss their relationship with other cholinesterase molecular forms.     

Molecular dynamics simulations of a lipovitellin-derived amphiphilic beta-sheet homologous to apoB-100 beta-sheets at a hydrophobic decane-water interface

Lipovitellin, an egg-yolk lipoprotein, transports lipids in a pocket surrounded by amphiphilic beta-sheets. Its X-ray structure provides possibilities to study interactions between lipophilic beta-sheets and lipids at the atomic level. Here, we studied a 67-residue-long amphiphilic beta-sheet of lipovitellin previously suggested a suitable working model for studies of the lipid-binding behaviour of amphiphilic beta-sheet regions in apolipoprotein B-100 (apoB-100). We performed four molecular dynamics simulations with different starting configurations to define characteristics of the amphiphilic beta-sheet model at a decane-water interface. In each simulation the model beta-sheet bound keenly to the decane layer via its hydrophobic surface. The structural profiles showed unchanged secondary structure of the beta-sheet during the attachment. Also, aromatic side chains, especially tryptophans and tyrosines, mediated the attachment to the hydrophobic layer and influenced the orientation of the decane molecules that are in contact with the beta-sheet. In conclusion, the present simulations reveal high affinity of a lipovitellin-derived amphiphilic beta-sheet to a hydrophobic decane layer. They lay thereby the basis for further studies of the interaction between amphiphilic beta-sheets and lipids in complex molecular systems, like LDL particles, in which the large apoB-100 is the main protein component.     

Artificial receptor-attached amphiphilic copolymer for barbiturate binding in aqueous media

A water-soluble self-associating amphiphilic copolymer was employed to provide a microenvironment for the solvation of a hydrogen-bonding barbiturate artificial receptor, to facilitate molecular recognition in water. The receptor-attached amphiphilic polymer (RP) was synthesized through random copolymerization of 3% (mol) barbiturate receptor-monomer, 70% (mol) 3-sulfopropyl methacrylate, and 27% (mol) n-dodecyl acrylate. Difference UV spectra of pH 6.5 aqueous solutions of phenobarbital and receptor-polymer (RP) gave peaks and valleys at 272 and 301 nm respectively, consistent with binding characteristics of monomeric barbiturate receptors in chloroform. Specific association between phenobarbital and the receptor-polymer was further indicated based on investigations of a receptor-free control polymer (CP) of similar polar/nonpolar monomer ratio. Micellar electrokinetic chromatography was applied for studying polymer-phenobarbital association, by capillary electrophoresis.     

Hippocampal astrocytes exhibit Ca2+-elevating muscarinic cholinergic and histaminergic receptors in situ

Recent findings suggest that astrocytes respond to neuronally released neurotransmitters with Ca2+ elevations. These Ca2+ elevations may trigger astrocytes to release glutamate, affecting neuronal activity. Neuronal activity is also affected by modulatory neurotransmitters that stimulate G protein-coupled receptors. These neurotransmitters, including acetylcholine and histamine, might affect neuronal activity by triggering Ca2+-dependent release of neurotransmitters from astrocytes. However, there is no physiological evidence for histaminergic or cholinergic receptors on astrocytes in situ. We asked whether astrocytes have these receptors by imaging Ca2+-sensitive dyes sequestered by astrocytes in hippocampal slices. Our results show that immunocytochemically identified astrocytes respond to carbachol and histamine with increases in intracellular free Ca2+ concentration. The H1 histamine receptor antagonist chlorpheniramine inhibited responses to histamine. Similarly, atropine and the M1-selective muscarinic receptor antagonist pirenzepine inhibited carbachol-elicited responses. Astrocyte responses to histamine and carbachol were compared with responses elicited by alpha1-adrenergic and metabotropic glutamate receptor agonists. Individual astrocytes responded to different subsets of receptor agonists. Ca2+ oscillations were the prevalent response pattern only with metabotropic glutamate receptor stimulation. Finally, functional alpha1-adrenergic receptors and muscarinic receptors were not detected before postnatal day 8. Our data show that astrocytes have acetylcholine and histamine receptors coupled to Ca2+. Given that Ca2+ elevations in astrocytes trigger neurotransmitter release, it is possible that these astrocyte receptors modulate neuronal activity.     

Preparation and characterization of amphiphilic triblock terpolymer-based nanofibers as antifouling biomaterials

Antifouling surfaces are critical for the good performance of functional materials in various applications including water filtration, medical implants, and biosensors. In this study, we synthesized amphiphilic triblock terpolymers (tri-BCPs, coded as KB) and fabricated amphiphilic nanofibers by electrospinning of solutions prepared by mixing the KB with poly(lactic acid) (PLA) polymer. The resulting fibers with amphiphilic polymer groups exhibited superior antifouling performance to the fibers without such groups. The adsorption of bovine serum albumin (BSA) on the amphiphilic fibers was about 10-fold less than that on the control surfaces from PLA and PET fibers. With the increase of the KB content in the amphiphilic fibers, the resistance to adsorption of BSA was increased. BSA was released more easily from the surface of the amphiphilic fibers than from the surface of hydrophobic PLA or PET fibers. We have also investigated the structural conformation of KB in fibers before and after annealing by contact angle measurements, transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDX), and coarse-grained molecular dynamics (CGMD) simulation to probe the effect of amphiphilic chain conformation on antifouling. The results reveal that the amphiphilic KB was evenly distributed within as-spun hybrid fibers, while migrated toward the core from the fiber surface during thermal treatment, leading to the reduction in antifouling. This suggests that the antifouling effect of the amphiphilic fibers is greatly influenced by the arrangement of amphiphilic groups in the fibers.     

GABA is an endogenous ligand for synaptic glycine receptors

Experimental evidence refuting Dale's principle, the notion that each neuron synthesizes and releases only one neurotransmitter, has accumulated in the past four decades, and cotransmission by multiple neurotransmitters from the same axon terminal (and even from the same vesicle) now is well documented. Heretofore, in all examples of cotransmission, each released neurotransmitter acted on a different receptor. Lu, Rubio, and Trussell, however, demonstrate in this issue of Neuron the first instance of cotransmission in which two neurotransmitters act on the same postsynaptic receptor.     

Muscarinic acetylcholine receptors: structural basis of ligand binding and G protein coupling

Muscarinic acetylcholine receptors (m1-m5) were studied by a combined molecular genetic/pharmacologic approach to elucidate the molecular characteristics of the ligand binding site and of the receptor domains involved in G protein coupling. Site-directed mutagenesis studies of the rat m3 muscarinic receptor suggest that the acetylcholine binding domain is formed by a series of hydrophilic amino acids located in the "upper" half of transmembrane domains (TM) III, V, VI, and VII. Moreover, we showed that mutational modification of a TM VI Asn residue (Asn507 in the rat m3 receptor sequence) which is characteristic for the muscarinic receptor family has little effect on high-affinity acetylcholine binding and receptor activation, but results in dramatic reductions in binding affinities for certain subclasses of muscarinic antagonists. The N-terminal portion of the third intracellular loop (i3) of muscarinic and other G protein-coupled receptors has been shown to play a central role in determining the G protein coupling profile of a given receptor subtype. Insertion mutagenesis studies with the rat m3 muscarinic receptor suggest that this region forms an amphiphilic alpha-helix and that the hydrophobic side of this helix represents an important G protein recognition surface. Further mutational analysis of this receptor segment showed that Tyr254 located at the N-terminus of the i3 loop of the m3 muscarinic receptor plays a key role in muscarinic receptor-induced Gq activation. The studies described here, complemented by biochemical and biophysical approaches, should eventually lead to a detailed structural model of the ligand-receptor-G protein complex.