Channel-opening mechanism of a kainate-activated glutamate receptor: kinetic investigations using a laser-pulse photolysis technique

Kainate is an excitatory neurotransmitter that binds to the kainate and AMPA receptor subtypes of the glutamate receptor and triggers the formation of cation permeable transmembrane channels in these receptors. In the present report the channel-opening mechanism of the AMPA receptors by kainate has been determined in rat hippocampal neurons using two different kinetic methods, namely, the rapid-flow method (cell-flow) with a 10 ms time resolution and a laser-pulse photolysis technique with a approximately 65 microseconds time resolution. The whole-cell currents induced by kainate, using the cell-flow method, are nondesensitizing and inhibited significantly by CNQX and hence pertain to activation of the AMPA receptors and not the kainate receptors. The cell-flow measurements were used to evaluate the constants pertaining to the minimum mechanism that could account for the concentration of the receptor in the open-channel form over a 500-fold range of kainate concentration. These constants, namely, the intrinsic dissociation constant of kainate from the AMPA receptor and the channel-opening equilibrium constant, were determined to be 140 +/- 30 microM and 8 +/- 2, respectively. On the other hand, the kinetics of the steps leading to channel opening was evaluated using the laser-pulse photolysis techniques. In this technique whole-cell currents were obtained by releasing kainate in the submillisecond time scale near the cell by photolysis of N-(alpha-carboxy-2-nitrobenzyl) kainate. The concentration of the released kainate was calculated by comparing the whole-cell currents obtained from the laser-pulse photolysis experiments with the whole currents obtained with 100 microM kainate on the same cell using cell-flow measurements. The rate constants for channel opening and closing were then determined from the observed rate constants for the current rise obtained as a function of kainate concentration. These rates were 5000 +/- 2000 and 640 +/- 30 s-1, respectively. The rate and equilibrium constants obtained in the present report allow an evaluation of the fraction of the receptors in the open-channel form as a function of time and kainate concentration, hence providing insight into the role of kainate in neuronal signal transmission.     

Formation and properties of peroxynitrite as studied by laser flash photolysis, high-pressure stopped-flow technique, and pulse radiolysis

Flash photolysis of alkaline peroxynitrite solutions results in the formation of nitrogen monoxide and superoxide. From the rate of recombination it is concluded that the rate constant of the reaction of nitrogen monoxide with superoxide is (1.9 +/- 0.2) x 10(10) M-1 s-1. The pKa of hydrogen oxoperoxonitrate is dependent on the medium. With the stopped-flow technique a value of 6.5 is found at millimolar phosphate concentrations, while at 0.5 M phosphate the value is 7.5. The kinetics of decay do not follow first-order kinetics when the pH is larger than the pKa, combined with a total peroxynitrite and peroxynitrous acid concentration that exceeds 0.1 mM. An adduct between ONOO- and ONOOH is formed with a stability constant of (1.0 +/- 0.1) x 10(4) M. The kinetics of the decay of hydrogen oxoperoxonitrate are not very pressure-dependent: from stopped-flow experiments up to 152 MPa, an activation volume of 1.7 +/- 1.0 cm3 mol-1 was calculated. This small value is not compatible with homolysis of the O-O bond to yield free nitrogen dioxide and the hydroxyl radical. Pulse radiolysis of alkaline peroxynitrite solutions indicates that the hydroxyl radical reacts with ONOO- to form [(HO)ONOO].- with a rate constant of 5.8 x 10(9) M-1 s-1. This radical absorbs with a maximum at 420 nm (epsilon = 1.8 x 10(3) M-1 cm-1) and decays by second-order kinetics, k = 3.4 x 10(6) M-1 s-1. Improvements to the biomimetic synthesis of peroxynitrite with solid potassium superoxide and gaseous nitrogen monoxide result in higher peroxynitrite to nitrite yields than in most other syntheses.     

Relationship of a dystrophin-associated glycoprotein to junctional acetylcholine receptor clusters in rat skeletal muscle

The relationship of a member of the transmembrane dystrophin-associated glycoprotein (DAG) complex to acetylcholine receptors (AChRs) was investigated using immunofluorescence techniques at rat neuromuscular junctions (NMJs) viewed en face. These results were compared with those from a similar previous study of dystrophin and an autosomal homologue (utrophin or dystrophin-related protein, DRP) (Bewick et al. Neuro Report 1992; 3: 857-860). The region of highest 43 K DAG (43DAG) labelling projected beyond the AChRs by approximately 0.3 microns, as does that for dystrophin. By contrast DRP labelling precisely co-localizes with the AChRs. These results suggest that at the NMJ, the region of high 43DAG concentration encompasses the area of highest intensity labelling for both DRP and dystrophin.     

Triazolam-induced modulation of muscarinic acetylcholine receptor in living brain slices as revealed by a new positron-based imaging technique

The effect of triazolam, a potent benzodiazepine (BZ) agonist, on muscarinic acetylcholinergic receptor (mAChR) binding was investigated in living brain slices by use of a novel positron-based imaging technique. Fresh rat brain slices were incubated with [11C]N-methyl-4-piperidylbenzilate ([11C]NMPB), a mAChR antagonist, in oxygenated Krebs-Ringer solution at 37 degrees C. During incubation, time-resolved imaging of [11C]NMPB binding in the slices was constructed on the storage phosphor screens. Addition of triazolam (1 microM) plus muscimol (30 microM), a GABA(A) receptor agonist, to the incubation mixture decreased the specific binding of [11C]NMPB. Ro15-1788, a BZ receptor antagonist, prevented this effect, indicating that the effect was exerted through the GABA(A)/BZ receptor complex. These results demonstrated that stimulation of the GABA(A)/BZ receptor lowers the affinity of the mAChR for its ligand, which may underlie the BZ-induced amnesia, a serious clinical side effect of BZ. No such effect in the P2-fraction instead implies that the integrity of the neuronal cells and/or their environment is prerequisite for the modulation of mAChR by GABA(A)/BZ stimulation.     

Potentiation and inhibition of nicotinic acetylcholine receptors by spermine in the TE671 human muscle cell line

Nicotinic acetylcholine receptors (nAChR) of the TE671 cell line were investigated using whole-cell and membrane patch recording techniques. At negative holding potentials (VH), pulses of acetylcholine (ACh) elicited whole-cell inward currents that rapidly desensitized. The EC50 value for ACh at VH = -60 mV was 7.8 microM. The ACh-induced current reversed at approximately 0 mV. Desensitization of nAChR by ACh was biphasic and reversible within approximately 20 sec. Spermine (1-100 microM) potentiated responses to ACh (10 microM - 1 mM) by reducing the rate of onset of desensitization; potentiation was inhibited by arcaine (10-100 microM). Spermine (1 mM) noncompetitively antagonized the AChinduced current. Antagonism by 1 to 5 mM spermine was voltage-dependent, increasing with negative VH. In 100 microM arcaine, this antagonism was shown to contain a voltage-independent component. Spermine (10 mM) increased the EC50 values for ACh, suggesting that at this concentration the polyamine is also a competitive antagonist. Single channel openings elicited during application of ACh to outside-out patches had a conductance of 47 pS at VH = -60 mV. At 10 and 100 microM, spermine increased channel open probability (po), but at 1 mM spermine, po was not significantly different from controls. The single channel conductance for ACh was unaffected by 10 and 100 microM spermine, but was decreased by 1 mM spermine. Spermine promoted the occurrence of approximately 27 pS openings. It is proposed that spermine acts at an excitatory modulatory site similar to that present on N-methyl-D-aspartate receptors and at least three inhibitory sites on nAChR of TE671 cells.     

Morphine-induced supersensitivity to acetylcholine in skeletal muscle and its mechanism of action

Studies conducted for over 20 years allowed the authors to accumulate a rich experience in the production and administration of an inactivated influenza vaccine applicable by nasal and oral route, prepared in the "Stefan S. Nicolau" Institute of Virology. Applied as a monovalent in a single dose of 1000 IU, the vaccine induced seroconversion of 70% of the vaccines and ensured a 2--4-fold decrease in influenza morbidity. Owing to these qualities the vaccine represents one of the most advantageous preparations used in influenza prophylaxis.     

Association of muscle-specific kinase MuSK with the acetylcholine receptor in mammalian muscle

During synaptogenesis at the neuromuscular junction, a neurally released factor, agrin, causes the clustering of acetylcholine receptors (AChRs) in the muscle membrane beneath the nerve terminal. Agrin acts through a specific receptor which is thought to have a receptor tyrosine kinase, MuSK, as one of its components. In agrin-treated muscle cells, both MuSK and the AChR become tyrosine phosphorylated. To determine how the activation of MuSK leads to AChR clustering, we have investigated their interaction in cultured C2 myotubes. Immunoprecipitation experiments showed that MuSK is associated with the AChR and that this association is increased by agrin treatment. Agrin also caused a transient activation of the AChR-associated MuSK, as demonstrated by MuSK phosphorylation. In agrin-treated myotubes, MuSK phosphorylation increased with the same time course as phosphorylation of the beta subunit of the AChR, but declined more quickly. Although both herbimycin and staurosporine blocked agrin-induced AChR phosphorylation, only herbimycin inhibited the phosphorylation of MuSK. These results suggest that although agrin increases the amount of activated MuSK that is associated with the AChR, MuSK is not directly responsible for AChR phosphorylation but acts through other kinases.     

Photoaffinity labeling of Torpedo acetylcholine receptor by physostigmine

The plant alkaloid physostigmine, an established anti-cholinesterase agent of the carbamate type, has recently been shown to bind to the nicotinic acetylcholine receptor from Torpedo marmorata electrocytes [Okonjo, K. O., Kuhlmann, J. & Maelicke, A. (1991) Eur. J. Biochem. 200, 671-677]. Pharmacological studies of physostigmine-induced ion flux into nicotinic-acetylcholine-receptor-rich membrane vesicles, indicated distinct binding sites for physostigmine and acetylcholine. As shown in this study by photoaffinity labeling with [phenyl-(n)-3H](-)physostigmine, the physostigmine-binding site is located within the same subunit (alpha polypeptide) of the receptor as the acetylcholine-binding site. Using a variety of proteolytic cleavage conditions for the purified alpha polypeptide, several [3H]physostigmine-labeled peptides were isolated and sequenced. From the radioactivity released in the course of the Edman degradations of the labeled peptides, it was found that the label was associated in all cases with Lys125. These results identify a novel ligand-binding site for the Torpedo nicotinic acetylcholine receptor that is different in location from binding sites identified previously for acetylcholine, its established agonists and antagonists, and direct channel blockers.     

Authoritarian personality studied by a new variation of the sentence completion technique.

After taking a sentence completion test, the Ss are asked to indicate for each item whether the item, as completed by him, is true of himself or not. It is hypothesized that authoritarians will deny the self-reference of such responses more often than the equalitarians. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Mechanism of nicotinic acetylcholine receptor cluster formation by rapsyn

Rapsyn, a peripheral membrane protein of skeletal muscle, clusters nicotinic acetylcholine receptors (nAChRs) at high density in the postsynaptic membrane. The mechanism of nAChR clustering by rapsyn was analyzed by expressing nAChRs in HEK293T cells with various fragments of mouse rapsyn fused to green fluorescent protein. Membrane targeting of rapsyn is conferred solely by its acylated N terminus, as the myristoylated N-terminal 15 amino acids of rapsyn are sufficient to target green fluorescent protein to the plasma membrane. However, neither N-terminal myristoylation nor the conserved N-terminal amino acid sequence is essential. Membrane targeting, self-association, and nAChR clustering are preserved when the first 10 amino acids of rapsyn were replaced by those of src, which also contains a consensus sequence for N-myristoylation, or by those of GAP43, which contains a palmitoylation sequence. Rapsyn1-90, containing two tetratrichopeptide repeats is sufficient for self-association. Rapsyn1-360, lacking the cysteine rich domain, clusters nAChRs, while rapsyn1-287, containing seven tetratrichopeptide repeats, does not cluster nAChRs. We identified rapsyn298-331 as a potential coiled-coil domain, and established that mutations disrupting coiled-coil propensity prevent nAChR clustering. Thus the structural domains of rapsyn necessary for membrane targeting, self-association, and nAChR clustering are distinct, with nAChR-rapsyn interaction mediated by a previously unrecognized coiled-coil motif.     

Endurance training increases acetylcholine receptor quantity at neuromuscular junctions of adult rat skeletal muscle

The aim of the study was to test the hypothesis that a 16 week endurance training program would alter the abundance of endplate-associated nicotinic acetylcholine receptors (nAChR) in various rat skeletal muscles. We found a 20% increase in endplate-specific [125I]alpha-bungarotoxin binding in several muscles of trained rats, accompanied by equal susceptibility of toxin binding to the inhibitory effect of D-tubocurarine in sedentary and trained muscles. We conclude that the neuromuscular junction adaptations that occur with increased chronic activation include an increase in nAChR number. Results of experiments designed to determine nAChR turnover also suggest that this effect is mediated by an alteration in the receptor's metabolic state. The potential implications and mechanisms of this adaptation are discussed.     

Pharmacology of the nicotinic acetylcholine receptor from fetal rat muscle expressed in Xenopus oocytes

BACKGROUND: Firefly luciferase is a 62 kDa protein that catalyzes the production of light. In the presence of MgATP and molecular oxygen, the enzyme oxidizes its substrate, firefly luciferin, emitting yellow-green light. The reaction proceeds through activation of the substrate to form an adenylate intermediate. Firefly luciferase shows extensive sequence homology with a number of enzymes that utilize ATP in adenylation reactions. RESULTS: We have determined the crystal structure of firefly luciferase at 2.0 A resolution. The protein is folded into two compact domains. The large N-terminal domain consists of a beta-barrel and two beta-sheets. The sheets are flanked by alpha-helices to form an alphabetaalphabetaalpha five-layered structure. The C-terminal portion of the molecule forms a distinct domain, which is separated from the N-terminal domain by a wide cleft. CONCLUSIONS: Firefly luciferase is the first member of a superfamily of homologous enzymes, which includes acyl-coenzyme A ligases and peptide synthetases, to have its structure characterized. The residues conserved within the superfamily are located on the surfaces of the two domains on either side of the cleft, but are too far apart to interact simultaneously with the substrates. This suggests that the two domains will close in the course of the reaction. Firefly luciferase has a novel structural framework for catalyzing adenylate-forming reactions.     

Afferent mechanism of cortical myoclonus studied by proprioception-related SEPs

This questionnaire study examined perceived sources of stress and satisfaction at work among 121 mental health staff members. Five factors were derived from principal component analysis of sources of work stress items (stress from: role, poor support, clients, future, and overload), and accounted for 70% of the total variance. Four factors were derived from the items related to sources of job satisfaction (satisfaction from: career, working with people, management, and money), accounting for 68% of the variance. The associations of these factors with sociodemographic and job characteristics were examined, and they were entered as explanatory variables into regression models predicting mental health, burnout, and job satisfaction. Stress from "overload" was associated with being based outside an in-patient ward, and with emotional exhaustion and worse mental health. Stress related to the "future" was associated with not being white. Stress from "clients" was associated with the "depersonalization" component of burnout. Higher job satisfaction was associated with "management" and "working with people" as sources of satisfaction, whereas emotional exhaustion and poorer mental health were associated with less "career" satisfaction.     

Propidium as a probe of acetylcholine receptor binding sites

The peptidoglycan of Bifidobacterium globosum contains ornithine and lysine alternately in the same position of the peptide subunit. The uridine diphospho-N-acetylmuramyl-alanyl-D-glutamic acid: diamino acid ligase of this organism was purified 700-fold. Since the activities for the incorporation of ornithine and lysine into uridine diphospho-N-acetylmuramyl-tripeptide did not separate during purification and since the incorporation of ornithine is competitively inhibited by lysine and vice versa, both ornithine and lysine are assumed to be incorporated by one single enzyme. Studies on the specificity of the ligase toward analogs of ornithine have shown that the enzyme requires a diamino, monocarboxylic acid with 4-6 carbon atoms. Methylation of the epsilon-amino group or hydroxylation of the delta-carbon atom of lysine decreases the competitive properties of the analog, whereas the substitution of the gamma-methylen group by sulfur (S-2-aminoethyl cysteine) results in a highly competitive compound.     

Developmental change in the modulation of acetylcholine receptor channel by protein kinase C activation in Xenopus embryonic muscle cells

Protein phosphorylation is important in synaptic transmission and plasticity. We report here that phorbol 12-myristate 13-acetate (TPA), a protein kinase C (PKC) activator, enhances the postsynaptic response at developing neuromuscular junctions by increasing the open time of embryonic acetylcholine (ACh) channels at earlier stages of cultured myocytes. Compared with day-1 cultures, the effects of TPA declined or disappeared on day-3 cultures. Adenosine 5'-triphosphate (ATP) which is co-stored and co-released with ACh at motor nerve terminals and is reported to enhance spontaneous synaptic currents by the activation of PKC, also shows similar developmental changes in the modulation of embryonic ACh channels in Xenopus embryonic myocytes.