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Biophysical Journal Dec 2002Functional N-methyl-D-aspartate receptors (NMDARs) are heteromultimers formed by NR1 and NR2 subunits. The M3 segment, as contributed by NR1, forms the core of the...
Functional N-methyl-D-aspartate receptors (NMDARs) are heteromultimers formed by NR1 and NR2 subunits. The M3 segment, as contributed by NR1, forms the core of the extracellular vestibule, including binding sites for channel blockers, and represents a critical molecular link between ligand binding and channel opening. Taking advantage of the substituted cysteine accessibility method along with channel block and multivalent coordination, we studied the contribution of the M3 segment in NR2C to the extracellular vestibule. We find that the M3 segment in NR2C, like that in NR1, contributes to the core of the extracellular vestibule. However, the M3 segments from the two subunits are staggered relative to each other in the vertical axis of the channel. Compared to NR1, homologous positions in NR2C, including those in the highly conserved SYTANLAAF motif, are located about four amino acids more externally. The staggering of subunits may represent a key structural feature underlying the distinct functional properties of NMDARs.
Topics: Aminacrine; Amino Acid Sequence; Animals; Cations; Cell Membrane; Cells, Cultured; Cysteine; Extracellular Space; Female; Glutamic Acid; Ion Channel Gating; Ion Channels; Macromolecular Substances; Membrane Potentials; Mesylates; Molecular Sequence Data; Mutagenesis, Site-Directed; Oocytes; Protein Conformation; Protein Subunits; Receptors, N-Methyl-D-Aspartate; Sequence Homology, Amino Acid; Silver; Xenopus
PubMed: 12496098
DOI: 10.1016/S0006-3495(02)75331-9 -
Neuron Jan 2002Many N-methyl-D-aspartate receptor (NMDAR) channel blockers that have therapeutic potential can be trapped in the closed state. Using a combination of the substituted...
Many N-methyl-D-aspartate receptor (NMDAR) channel blockers that have therapeutic potential can be trapped in the closed state. Using a combination of the substituted cysteine accessibility method and open channel blockers, we found that the M3 segment forms the core of the extracellular vestibule, including a deep site for trapping blockers. The M3 segment, as well as more superficial parts of the extracellular vestibule, undergo extensive remodeling during channel closure, but do not define the activation gate, which is located deeper in the pore. Rather, the pore walls lining the extracellular vestibule constrict during channel closure. This movement is essential for coupling ligand binding to activation gate opening and accounts for the different mechanisms of open channel block, including trapping.
Topics: Aminacrine; Animals; Cell Membrane; Central Nervous System; Cysteine; Drug Interactions; Excitatory Amino Acid Antagonists; Extracellular Space; Female; Glutamic Acid; Ion Channel Gating; Membrane Potentials; Mutagens; Mutation; Oocytes; Protein Structure, Tertiary; Receptors, N-Methyl-D-Aspartate; Synaptic Transmission; Xenopus laevis
PubMed: 11779481
DOI: 10.1016/s0896-6273(01)00560-8 -
Biochimica Et Biophysica Acta Jan 2002We have previously shown that anacardic acid has an uncoupling effect on oxidative phosphorylation in rat liver mitochondria using succinate as a substrate (Life Sci. 66...
We have previously shown that anacardic acid has an uncoupling effect on oxidative phosphorylation in rat liver mitochondria using succinate as a substrate (Life Sci. 66 (2000) 229-234). In the present study, for clarification of the physicochemical characteristics of anacardic acid, we used a cyanine dye (DiS-C3(5)) and 9-aminoacridine (9-AA) to determine changes of membrane potential (DeltaPsi) and pH difference (DeltapH), respectively, in a liposome suspension in response to the addition of anacardic acid to the suspension. The anacardic acid quenched DiS-C3(5) fluorescence at concentrations higher than 300 nM, with the degree of quenching being dependent on the log concentration of the acid. Furthermore, the K(+) diffusion potential generated by the addition of valinomycin to the suspension decreased for each increase in anacardic acid concentration used over 300 nM, but the sum of the anacardic acid- and valinomycin-mediated quenching was additively increasing. This indicates that the anacardic acid-mediated quenching was not due simply to increments in the K(+) permeability of the membrane. Addition of anacardic acid in the micromolar range to the liposomes with DeltaPsi formed by valinomycin-K(+) did not significantly alter 9-AA fluorescence, but unexpectedly dissipated DeltaPsi. The DeltaPsi preformed by valinomycin-K(+) decreased gradually following the addition of increasing concentrations of anacardic acid. The DeltaPsi dissipation rate was dependent on the pre-existing magnitude of DeltaPsi, and was correlated with the logarithmic concentration of anacardic acid. Furthermore, the initial rate of DeltapH dissipation increased with logarithmic increases in anacardic acid concentration. These results provide the evidence for a unique function of anacardic acid, dissimilar to carbonylcyanide p-trifluoromethoxyphenylhydrazone or valinomycin, in that anacardic acid behaves as both an electrogenic (negative) charge carrier driven by DeltaPsi, and a 'proton carrier' that dissipates the transmembrane proton gradient formed.
Topics: Aminacrine; Anacardic Acids; Benzothiazoles; Carbocyanines; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Dose-Response Relationship, Drug; Fluorescent Dyes; Gramicidin; Hydrogen-Ion Concentration; Liposomes; Membrane Potentials; Models, Chemical; Molecular Structure; Salicylates; Spectrometry, Fluorescence; Valinomycin
PubMed: 11750264
DOI: 10.1016/s0005-2736(01)00422-9 -
Plant Physiology Sep 2000Electron transport and the electrochemical proton gradient across the thylakoid membrane are two fundamental parameters of photosynthesis. A combination of the electron...
Electron transport and the electrochemical proton gradient across the thylakoid membrane are two fundamental parameters of photosynthesis. A combination of the electron acceptor, ferricyanide and the DeltapH indicator, 9-aminoacridine, was used to measure simultaneously electron transport rates and DeltapH solely by changes in the fluorescence of 9-aminoacridine. This method yields values for the rate of electron transport that are comparable with those obtained by established methods. Using this method a relationship between the rate of electron transport and DeltapH at various uncoupler concentrations or light intensities was obtained. In addition, the method was used to study the effect of reducing the disulfide bridge in the gamma-subunit of the chloroplast ATP synthase on the relation of electron transport to DeltapH. When the ATP synthase is reduced and alkylated, the threshold DeltapH at which the ATP synthase becomes leaky to protons is lower compared with the oxidized enzyme. Proton flow through the enzyme at a lower DeltapH may be a key step in initiation of ATP synthesis in the reduced enzyme and may be the way by which reduction of the disulfide bridge in the gamma-subunit enables high rates of ATP synthesis at low DeltapH values.
Topics: Adenosine Triphosphate; Aminacrine; Electron Transport; Fluorescence; Fluorescent Dyes; Hydrogen-Ion Concentration; Indicators and Reagents; Proton-Translocating ATPases; Spinacia oleracea; Thylakoids
PubMed: 10982453
DOI: 10.1104/pp.124.1.407 -
The Journal of Neuroscience : the... Aug 1999NMDA receptor-mediated calcium transients play a critical role in synaptogenesis, synaptic plasticity, and excitotoxicity. NMDA receptors are heteromeric complexes of...
NMDA receptor-mediated calcium transients play a critical role in synaptogenesis, synaptic plasticity, and excitotoxicity. NMDA receptors are heteromeric complexes of NR1A combined with NR2A, NR2B, NR2C, and/or NR2D subunits. The NR2 subunits determine a variety of electrophysiological and pharmacological properties of the NMDA receptor complex. In this report, we provide evidence for the first time that there is also a significant difference in peak channel open probability (P(o)) between NMDA receptors composed of NR1A/NR2A and those of NR1A/NR2B subunits. First, whole-cell patch-clamp recordings from human embryonic kidney (HEK) 293 cells expressing NMDA receptors revealed that NR1A/NR2A-mediated peak current densities are approximately four times larger than those of NR1A/NR2B. We show that this fourfold difference is unlikely caused by differences in receptor surface expression, since these levels were similar for the two subtypes by Western blot analysis. To determine whether P(o) contributed to the difference in peak current densities, we used two different open channel antagonists, MK-801 and 9-aminoacridine, in a variety of experimental paradigms. Our results indicate that peak P(o) is significantly higher (twofold to fivefold) for NR1A/NR2A than NR1A/NR2B, with estimated values of approximately 0.35 and 0.07, respectively. These results suggest that a change in the relative expression levels of NR2A and NR2B can regulate peak amplitude of NMDA receptor-mediated excitatory postsynaptic potentials and therefore may play a role in mechanisms underlying synaptic plasticity.
Topics: Aminacrine; Cell Line; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Humans; Neuronal Plasticity; Patch-Clamp Techniques; Peptide Fragments; Probability; Receptors, N-Methyl-D-Aspartate; Recombinant Proteins; Transfection
PubMed: 10436042
DOI: 10.1523/JNEUROSCI.19-16-06844.1999 -
Biochimica Et Biophysica Acta Jan 1999NMDA receptor channel responses were recorded from acutely isolated rat hippocampal neurons, using whole-cell patch-clamp techniques. In the continuous presence of... (Comparative Study)
Comparative Study
NMDA receptor channel responses were recorded from acutely isolated rat hippocampal neurons, using whole-cell patch-clamp techniques. In the continuous presence of aspartate, tetraethylammonium, tetrabutylammonium, 1-amino-3-propyl-adamantane and 9-aminoacridine caused changes in the current through NMDA channels, which were described by two-exponential functions. It was established that depending on the behavior of the amplitude of the fast component for the recovery kinetics, the blocker action can be assigned to one of five types described by the simplest models. The effects of tetraethylammonium, tetrabutylammonium and 1-amino-3-propyl-adamantane were well described by these models. Using 9-aminoacridine as an example, it was shown that the simplest models cannot describe all possible types of the blocker-channel interaction. In such cases, the method of the simplest models combination can be used. The application of the simplest kinetic models analysis allowed to make the following conclusions: at least two molecules of 1-amino-3-propyl-adamantane or 9-aminoacridine can simultaneously bind to the open channel and block it; the occupation of 9-aminoacridine blocking sites in the channel can proceed in at least two different ways; the binding of tetrabutylammonium and 9-aminoacridine prevented the closure of the activation and/or desensitization gates of the channel, while that of tetraethylammonium did not.
Topics: Aminacrine; Animals; Cations; Kinetics; Patch-Clamp Techniques; Pyramidal Cells; Quaternary Ammonium Compounds; Rats; Receptors, N-Methyl-D-Aspartate; Tetraethylammonium
PubMed: 9889324
DOI: 10.1016/s0005-2736(98)00211-9 -
Nucleic Acids Research Aug 1997Four nitrogen mustards have been used in this study to examine protein-DNA interactions in intact human cells, specifically at the locus control region hypersensitive...
Four nitrogen mustards have been used in this study to examine protein-DNA interactions in intact human cells, specifically at the locus control region hypersensitive site-2 (LCR HS-2) of the human beta-globin locus. Three of these nitrogen mustards are DNA-targeted by attachment of an acridine or amsacrine intercalating chromophore, while the fourth (chlorambucil) is a non-targeted mustard. The ligation-mediated PCR technique was used to determine the sites of damage at base pair resolution on DNA sequencing gels. A densitometric comparison was made between DNA damaged in intact erythroid K562 cells and in purified DNA. The intensity of DNA damage sites in the LCR HS-2 were found to differ significantly between intact K562 cells and purified DNA. At the NF-E2/AP-1 motif, pronounced damage protection was observed in DNA derived from drug treated cells. The nuclear factor- erythroid 2 (NF-E2) protein factor is thought to bind at this NF-E2/AP-1 motif in K562 cells. Other sites of protection and enhancement that corresponded to known transcription factor binding sites were also detected. These nitrogen mustards are therefore very effective compounds for detection of transcription factor binding to DNA in intact cells and are superior to other commonly used agents. The sequence selectivity of the compounds was determined using plasmid DNA and compared to that found in intact cells. The acridine-based nitrogen mustard had a preference for forming adducts at guanine bases, while the two amsacrine-based nitrogen mustards and chlorambucil formed adducts at both guanine and adenine bases.
Topics: Aminacrine; Amsacrine; Cell Line; Chlorambucil; Chromatin; DNA Damage; DNA-Binding Proteins; Gene Expression Regulation; Globins; Humans; Mechlorethamine; Promoter Regions, Genetic
PubMed: 9241238
DOI: 10.1093/nar/25.16.3255 -
Bioscience, Biotechnology, and... Jul 1997Some derivatives of 9-aminoacridine (1) were synthesized, and their frameshift mutagenicity and DNA binding affinity were studied. The introduction of a methyl group...
Some derivatives of 9-aminoacridine (1) were synthesized, and their frameshift mutagenicity and DNA binding affinity were studied. The introduction of a methyl group into the acridine ring of 1 reduced the mutagenic activity and the intercalative DNA binding affinity, while the introduction of chlorine increased them. Halogenated derivatives of 1 showed higher toxicity against Salmonella typhimurium TA1537.
Topics: Aminacrine; Animals; DNA; Frameshift Mutation; Halogens; Liver; Methylation; Mutagenicity Tests; Mutagens; Rats; Salmonella typhimurium; Spectrum Analysis; Structure-Activity Relationship
PubMed: 9255975
DOI: 10.1271/bbb.61.1121 -
Environmental Health Perspectives Feb 1997
Topics: Aminacrine; Animals; Anti-Infective Agents, Local; Birth Weight; Body Weight; Dose-Response Relationship, Drug; Female; Kidney; Liver; Male; Mice; Organ Size; Pregnancy; Reproduction
PubMed: 9114319
DOI: No ID Found -
Biochimica Et Biophysica Acta Jul 1995We analyze the adsorption of the fluorescent monoamine 9-aminoacridine to the membrane phase of photosynthetic chromatophores, in the physiological interval of pH values...
We analyze the adsorption of the fluorescent monoamine 9-aminoacridine to the membrane phase of photosynthetic chromatophores, in the physiological interval of pH values ranging from 5.5 to 8.5 and at ionic strengths of 0.005 and 0.150 M. The interaction of the probe with the membrane phase is described with S-shaped isotherms of the Hill type and is modulated by electrostatic effects as modelled with the Gouy-Chapman-Boltzman theory. This description is consistent with different values of the surface change density of the chromatophore membranes decreasing from about 1.3 x 10(-3) to about 0.5 x 10(-3) e-/A2, on changing the pH from 8.5/7.5 to 6.5/5.5, respectively. Furthermore we show that, when the free concentrations of the probe in the inner and outer vesicle compartments are computed from the adsorbing isotherms at the proper pH values, the model considering the equilibrium distribution of the neutral monoamine following the onset of a delta pH is sufficient to describe the dependence of the artificially induced transmembrane delta pH values on the observed quenching of the probe fluorescence.
Topics: Aminacrine; Cell Membrane; Chromatophores; Fluorescent Dyes; Hydrogen-Ion Concentration; Microdialysis
PubMed: 7619838
DOI: 10.1016/0005-2736(95)00075-e