Affinity For Glycine Research Articles

Subunit-dependent inhibition of recombinant rodent N-methyl- d-aspartate receptors by a HIV-1 glycoprotein 120 derived peptide

Considerable evidence suggests that low (picomolar) concentrations of the HIV-1 envelope glycoprotein gp120 induce neuronal cell death by stimulating the release of microglial toxins, which in turn activate N-methyl- d-aspartate (NMDA) receptors. Conversely, high (micromolar) concentrations of gp120 have been reported to directly inhibit NMDA receptor-mediated currents and do not induce neurotoxicity. Here we show that micromolar concentrations of a synthetic peptide corresponding to the V3-loop of gp120 (V3-pep) inhibited agonist responses of recombinant heteromeric rodent NMDA receptors expressed in Xenopus laevis oocytes by decreasing their apparent glycine affinity. Different combinations of NMDA receptor subunits displayed differential sensitivities to inhibition by V3-pep, with a potency rank order of NR1/2B>NR1/2D>NR1/2C≥NR1/2A. Our observations may provide an explanation for the reduced neurotoxicity of high doses of gp120 in cell cultures and may be useful for the pharmacological discrimination of NMDA receptor subtypes.

Interaction of neutral and zwitterionic glycine with Zn2+ in gas phase:ab initio and SIBFA molecular mechanics calculations

The interaction of Zn2+ with glycine (Gly) in the gas phase is studied by a combination of ab initio and molecular mechanics techniques. The structures and energetics of the various isomers of the Gly–Zn2+ complex are first established via high-level ab initio calculations. Two low-energy isomers are characterized: one in which the metal ion interacts with the carboxylate end of zwitterionic glycine, and another in which it chelates the amino nitrogen and the carbonyl oygen of neutral glycine. These calculations lead to the first accurate value of the gas-phase affinity of glycine for Zn2+. Ab initio calculations were also used to evaluate the performance of various implementations of the SIBFA force field. To assess the extent of transferability of the distributed multipoles and polarizabilities used in the SIBFA computations, two approaches are followed. In the first, approach (a), these quantities are extracted from the ab initio Hartree–Fock wave functions of glycine or its zwitterion in its entirety, and for each individual Zn2+-binding conformation. In the second, approach (b), they are assembled from the appropriate constitutive fragments, namely methylamine and formic acid for neutral glycine, and protonated methylamine and formate for the zwitterion; they undergo the appropriate vector or matrix rotation to be assembled in the conformation studied. The values of the Zn2+–glycine interaction energies are compared to those resulting from ab initio SCF and MP2 computations using both the all-electron 6-311+G(2d,2p) basis set and an effective core potential together with the valence CEP 4-31G(2d) basis set. Approach (a) values closely reproduce the ab initio ones, both in terms of the total interaction energies and of the individual components. Approach (b) can provide a similar match to ab initio interaction energies as does approach (a), provided that the two constitutive Gly building blocks are considered as separate entities having mutual interactions that are computed simultaneously with those occurring with Zn2+. Thus, the supermolecule is treated as a three-body rather than a two-body system. These results indicate that the current implementation of the SIBFA force field should be adequate to undertake accurate studies on zinc metallopeptides. © 2000 John Wiley & Sons, Inc. J Comput Chem 21: 963–973, 2000

A mutation in the glycine binding pocket of the N-methyl- d-aspartate receptor NR1 subunit alters agonist efficacy

Alanine 714 of the NMDA receptor NR1 subunit resides in the glycine binding pocket. The Ala714Leu mutation substantially shifts glycine affinity, but here no effect on antagonism by DCK is detected. Ala714Leu is also found to limit the efficacy of a partial agonist without altering its apparent affinity. The differential sensitivity of Ala714Leu to glycine agonists suggests that alanine 714 may be an intermediary in transducing the ligand binding signal.

Molecular determinants of glycine receptor subunit assembly.

The inhibitory glycine receptor (GlyR) is a pentameric transmembrane protein composed of homologous alpha and beta subunits. Single expression of alpha subunits generates functional homo-oligomeric GlyRs, whereas the beta subunit requires a co-expressed alpha subunit to assemble into hetero-oligomeric channels of invariant stoichiometry (alpha(3)beta(2)). Here, we identified eight amino acid residues within the N-terminal region of the alpha1 subunit that are required for the formation of homo-oligomeric GlyR channels. We show that oligomerization and N-glycosylation of the alpha1 subunit are required for transit from the endoplasmic reticulum to the Golgi apparatus and later compartments, and that addition of simple carbohydrate side chains occurs prior to GlyR subunit assembly. Our data are consistent with both intersubunit surface and conformational differences determining the different assembly behaviour of GlyR alpha and beta subunits.

Amino Acid Volume and Hydropathy of a Transmembrane Site Determine Glycine and Anesthetic Sensitivity of Glycine Receptors

Two specific amino acid residues in transmembrane segments (TM) 2 and 3 are critical for the enhancement of glycine receptor (GlyR) function by volatile anesthetics. To determine which physicochemical characteristics of these sites determine their roles in anesthetic actions, an extensive series of single amino acid mutations at amino acid residue 288 (Ala-288) in TM3 of the alpha1 GlyR subunit was tested for modulation by volatile anesthetics. The mutations changed the apparent affinities of receptors for glycine; replacements with larger volumes and less hydropathy exhibited higher affinities for glycine. Potentiation by anesthetics was reduced by specific mutations at Ala-288. The molecular volume of the substituents was negatively correlated with the extent of potentiation by isoflurane, enflurane, and 1-chloro-1,2,2-trifluorocyclobutane, whereas there was no correlation between anesthetic enhancement and polarity, hydropathy, or hydrophilicity of substituents. In contrast to anesthetics, no correlation was found between the effects of the nonanesthetics 1,2-dichlorohexafluorocyclobutane or 2, 3-dichlorooctafluorobutane and any physicochemical property of the substituent. These results suggest that the molecular volume and hydropathy of the amino acid at position 288 in TM3 regulate glycine and anesthetic sensitivity of the GlyR and that this residue might represent one determinant of an anesthetic binding site.

Zn2+ differentially modulates glycine receptors versus GABA receptors in isolated carp retinal third-order neurons

It was previously reported that strychnine-sensitive glycine receptors and GABAA receptors co-existed on carp retinal third-order neurons (amacrine/ganglion cells) (Li, P. and Yang, X.-L., Strong synergism between GABAA and glycine receptors on isolated carp third-order neurons. NeuroReport, 9 (1998) 2785–2789.). In this study, the effects of the divalent cation Zn2+ on these two receptors were studied in amacrine/ganglion cells acutely isolated from carp, with the use of the whole-cell patch clamp recording technique. The glycine-induced currents were steadily potentiated by Zn2+ of lower concentrations (0.1–10 μM), while being dose-dependently inhibited by Zn2+ of higher concentrations (>100 μM). Both the effects involved changes in apparent glycine affinity of the glycine receptor. In contrast, Zn2+ consistently suppressed the GABAA mediated currents of these cells, and did not show similar dual effect. The differential modulation by Zn2+ of glycine receptors versus GABAA receptors provides a versatility for regulating inhibitory glycinergic and GABAergic inputs converging on most amacrine/ganglion cells.

α2-adrenoceptor-mediated enhancement of glycine response in rat sacral dorsal commissural neurons

The effect of noradrenaline on the glycine response was investigated in neurons acutely dissociated from the rat sacral dorsal commissural nucleus using nystatin perforated patch recording configuration under voltage-clamp conditions. Noradrenaline reversibly potentiated the 10 −5M glycine-induced Cl − current in a concentration-dependent manner. Single channel recordings in a cell-attached mode revealed that noradrenaline decreased the closing time of the glycine-activated channel activity. Noradrenaline neither changed the reversal potential of the glycine response nor affected the affinity of glycine to its receptor. Clonidine mimicked and yohimbine blocked the noradrenaline action on glycine response. N-[2(methylamino)ethyl]-5-misoquinoline sulfonamide dihydrochloride, protein kinase A inhibitor, mimicked the effect of noradrenaline on glycine response. Noradrenaline failed to affect the glycine response in the presence of these intracellular cyclic AMP and protein kinase A modulators. However, noradrenaline further enhanced the glycine response even in the presence of phorbol-12-myristate-13-acetate and chelerythrine, a protein kinase C inhibitor. Pertussis toxin treatment for 6–8 h blocked the noradrenaline facilitatory effect on the glycine response. In addition, noradrenaline potentiated the strychnine-sensitive postsynaptic currents evoked in a slice preparation of sacral dorsal commissural nucleus. These results suggest that the activation of α2-adrenoceptor by noradrenaline coupled with pertussis toxin-sensitive G-proteins reduces intracellular cyclic AMP formation through the inhibition of adenyl cyclase. The reduction of cyclic AMP decreases the protein kinase A activity, thus resulting in the potentiation of the glycinergic inputs to the sacral dorsal commissural neurons. It is thus feasible that the noradrenergic input to the sacral dorsal commissural nucleus modulates such nociceptive signals as pain by intracellular enhancing the glycine response.

Modulation of glycine-induced currents by zinc and other metal cations in neurons acutely dissociated from the dorsal motor nucleus of the vagus of the rat

Effects of Zn 2+ and other polyvalent cations on glycine-induced currents in the freshly dissociated rat dorsal motor nucleus of the vagus neurons were investigated under voltage-clamp conditions by the use of the nystatin-perforated patch recording configuration. Glycine evoked a Cl − current in a concentration-dependent manner with a half-maximum effective concentration (EC 50) of 3.3×10 −5 M. Strychnine inhibited the 3×10 −5 M glycine-induced current in a concentration-dependent manner with a half-maximal inhibitory concentration (IC 50) of 6.8×10 −7 M. At low concentrations (3×10 −8 M–3×10 −6 M), Zn 2+ potentiated the current elicited by 3×10 −5 M glycine. On the other hand, at concentrations higher than 10 −5 M, Zn 2+ inhibited the glycine response. The biphasic action of Zn 2+ was mimicked by Ni 2+, but La 3+ and Co 2+ had only potentiating effect. Zn 2+ shifted the concentration–response curve for the glycine-induced current without changing the maximum response, and the EC 50 values for the glycine response in the absence and presence of 10 −6 M and 10 −4 M Zn 2+ were 3.3×10 −5 M, 1.3×10 −5 M and 1.3×10 −4 M, respectively. These results suggest that the biphasic modulation of glycine response by Zn 2+ results from changes in apparent glycine affinity.

Zinc potentiation of the glycine receptor chloride channel is mediated by allosteric pathways.

Molecular mechanisms of zinc potentiation were investigated in recombinant human alpha1 glycine receptors (GlyRs) by whole-cell patch-clamp recording and [3H]strychnine binding assays. In the wild-type (WT) GlyR, 1 microM zinc enhanced the apparent binding affinity of the agonists glycine and taurine and reduced their concentrations required for half-maximal activation. Thus, in the WT GlyR, zinc potentiation apparently occurs by enhancing agonist binding. However, analysis of GlyRs incorporating mutations in the membrane-spanning domain M1-M2 and M2-M3 loops, which are both components of the agonist gating mechanism, indicates that most mutations uncoupled zinc potentiation from glycine-gated currents but preserved zinc potentiation of taurine-gated currents. One such mutation in the M2-M3 loop, L274A, abolished the ability of zinc to potentiate taurine binding but did not inhibit zinc potentiation of taurine-gated currents. In this same mutant where taurine acts as a partial agonist, zinc potentiated taurine-gated currents but did not potentiate taurine antagonism of glycine-gated currents, suggesting that zinc interacts selectively with the agonist transduction pathway. The intracellular M246A mutation, which is unlikely to bind zinc, also disrupted zinc potentiation of glycine currents. Thus, zinc potentiation of the GlyR is mediated via allosteric mechanisms that are independent of its effects on agonist binding.

Salinity stress responses in the plant growth promoting rhizobacteria,Azospirillum spp

In order to adapt to the fluctuations in soil salinity/osmolarity the bacteria of the genusAzospirillum accumulate compatible solutes such as glutamate, proline, glycine betaine, trehalose, etc. Proline seems to play a major role in osmoadaptation. With increase in osmotic stress the dominant osmolyte inA. brasilense shifts from glutamate to proline. Accumulation of proline inA. brasilense occurs by both uptake and synthesis. At higher osmolarityA. brasilense Sp7 accumulates high intracellular concentration of glycine betaine which is taken up via a high affinity glycine betaine transport system. A salinity stress induced, periplasmically located, glycine betaine binding protein (GBBP) of ca. 32 kDa size is involved in glycine betaine uptake inA. brasilense Sp7. Although a similar protein is also present inA. brasilense Cd it does not help in osmoprotection. It is not known ifA. brasilense Cd can also accumulate glycine betaine under salinity stress and if the GBBP-like protein plays any role in glycine betaine uptake. This strain, under salt stress, seems to have inadequate levels of ATP to support growth and glycine betaine uptake simultaneously. ExceptA. halopraeferens, all other species ofAzospirillum lack the ability to convert choline into glycine betaine. Mobilization of thebet ABT genes ofE. coli intoA. brasilense enables it to use choline for osmoprotection. Recently, aproU-like locus fromA. lipoferum showing physical homology to theproU gene region ofE. coli has been cloned. Replacement of this locus, after inactivation by the insertion of kanamycin resistance gene cassette, inA. lipoferum genome results in the recovery of mutants which fail to use glycine betaine as osmoprotectant.

Characterization of desensitization in recombinant N-methyl- d-aspartate receptors: comparison with native receptors in cultured hippocampal neurons

In the present study we have characterized the effect of Ca 2+, glycine, and agonist concentration on inactivation and desensitization in native and recombinant N-methyl- d-aspartate (NMDA) receptors. In agreement with earlier studies on neurons, we found that in the presence of saturating glycine concentrations, lowering [Ca 2+] o, will decrease inactivation of NMDA receptors in cultured hippocampal neurons. However, unlike native NMDA receptors under the same recording conditions, recombinant receptors did not exhibit Ca 2+-dependent inactivation. We also show that the glycine-insensitive desensitization observed in the recombinant receptors is subunit dependent, as NR1a2A and NR1a2B receptors significantly desensitized while the NR1a2C combination did not. Furthermore, we show this form of desensitization in NR1a2A receptors is due to classic agonist-induced desensitization. In addition, we demonstrate the presence of glycine-dependent desensitization in recombinant receptors. The ability of glycine to inhibit desensitization correlates to the rank order of glycine's affinity for potentiating the peak response for each subtype. Finally, using ifenprodil in the presence of high and low glycine concentrations, we present evidence that both 2A-like and 2B-like subtypes of receptors can independently coexist in single neurons.

Developmental Changes in NMDA Receptor Glycine Affinity and Ifenprodil Sensitivity Reveal Three Distinct Populations of NMDA Receptors in Individual Rat Cortical Neurons

Previous work with recombinant receptors has shown that the identity of the NMDA NR2 subunit influences receptor affinity for both glutamate and glycine. We have investigated the developmental change in NMDA receptor affinity for both glutamate and glycine in acutely dissociated parietal cortex neurons of the rat, together with the expression during ontogeny of NR2A and NR2B mRNA and protein. Whereas there is little change in NMDA receptor glutamate affinity with age, a population of NMDA receptors emerges in 14- and 28-d-old animals with a markedly reduced affinity for glycine (mKD = approximately 800 nM) and a reduced sensitivity to the NR2B subunit-selective NMDA antagonist ifenprodil. These changes are paralleled by a developmental increase in the expression of NR2A. Thus, in mature animals a population of NMDA receptors appears with a lower affinity for glycine that might not be saturated under normal physiological conditions. Ifenprodil (10 microM) inhibits virtually all of the NMDA receptor-evoked current in very young neurons that contain a single population of receptors exhibiting a high affinity for glycine (mKD = approximately 20 nM). In older neurons, which contain NMDA receptors with both high and low affinities for glycine, ifenprodil (10 microM) inhibits both the high-affinity population and a significant proportion of the low-affinity component, thus revealing three pharmacologically distinct populations of NMDA receptors in single neurons. Moreover, these observations suggest that ifenprodil might bind with high affinity to NMDA receptors containing both NR2A and NR2B subunits as well as those containing only NR2B.

Seawater microorganisms have a high affinity glycine betaine uptake system which also recognizes dimethylsulfoniopropionate

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 15:39-51 (1998) - doi:10.3354/ame015039 Seawater microorganisms have a high affinity glycine betaine uptake system which also recognizes dimethylsulfoniopropionate Ronald P. Kiene1,2,*, Lynn P. Hoffmann Williams1,2, Joel E. Walker1,2 1Department of Marine Sciences, University of South Alabama, Mobile, Alabama 36688, USA 2Dauphin Island Sea Lab, Dauphin Island, Alabama 36528, USA *E-mail: rkiene@jaguar1.usouthal.edu In 24 h old seawater filtrate cultures, comprised mostly of free-living bacteria, the uptake of methyl-14C-glycine betaine (GBT) displayed Michaelis-Menten-type saturation kinetics with half-saturation constants (Kt) of <5 nM. The uptake of 5 nM 14C-GBT was strongly inhibited by additions of 1 to 30 nM unlabeled b-dimethylsulfoniopropionate (DMSP), a naturally occurring analog of GBT. The dose response to DMSP was consistent with simple dilution of labeled GBT. Conversely, the uptake of 35S-DMSP was inhibited by the presence of GBT over a similar concentration range. Comparison of 14C-GBT and 35S-DMSP uptake kinetics in the presence and absence of unlabeled analogs suggested that GBT and DMSP act as competitive inhibitors of their respective uptake by seawater microbes. Direct comparisons of 14C-GBT and 35S-DMSP uptake in filtrate cultures yielded nearly identical kinetic patterns. It was also found that a portion of the 14C-GBT taken up into particulate material could be chased from the particles by a 200-fold excess of unlabeled GBT or DMSP, whereas it was retained as untransformed 14C-GBT in the particles for several hours in unchased samples. A screening of organic compounds revealed that compounds with close structural similarity to GBT (DMSP, dimethylsulfonioacetate, proline betaine and dimethylglycine) strongly inhibited (<36% of controls) 14C-GBT uptake. Compounds showing moderate inhibition (48 to 89% of controls) included trigonelline, proline, glutamic acid, carnitine and choline, while compounds bearing no structural or chemical similarity to GBT (glycine, glucose, acrylic acid, and 3-methiolpropionate) had no effects on 14C-GBT uptake. Our results indicate that the GBT uptake system expressed by natural populations of microorganisms is multifunctional, displaying high affinity for both GBT and DMSP and possibly other naturally occurring betaine-like compounds. This microbial uptake system is likely to play an important role in the biogeochemical dynamics of GBT and DMSP. Osmolytes · Transport · Bacteria · Dimethylsulfoniopropionate · Inhibitors Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 15, No. 1. Publication date: May 22, 1998 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 1998 Inter-Research.

Neurotrophin Modulation of NMDA Receptors in Cultured Murine and Isolated Rat Neurons

Neurotrophin modulation of NMDA receptors in cultured murine and isolated rat neurons. J. Neurophysiol. 78: 2363-2371, 1997. Patch-clamp and calcium imaging techniques were used to assess the acute effects of the neurotrophins, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and nerve growth factor (NGF), on the responses of cultured and acutely isolated hippocampal and cultured striatal neurons to the glutamate receptor agonist N-methyl--aspartic acid (NMDA). The effects of BDNF on NMDA-activated currents were examined in greater detail. Currents evoked by NMDA, and the accompanying changes in intracellular calcium, were enhanced by low concentrations of the neurotrophins (1-20 ng/ml). The potentiation by the neurotrophins was rapid in onset and offset (<1 s). The neurotrophins also reduced desensitization of these currents in most cells. The enhancement of NMDA-activated currents by BDNF was observed using both perforated and whole cell patch recording techniques and could be demonstrated in outside-out patches. Furthermore, its effects were not attenuated by pretreatment with the protein kinase inhibitors genistein or 1-(5-isoquinolynesulfony)2-methylpiperazine (H7). Therefore, the actions of BDNF do not appear to be mediated by phosphorylation. Similar enhancements were observed with NT-3 and NT-4 and with NGF despite the fact that hippocampal neurons lack TrkA receptors. All together this evidence suggests that the enhancement of NMDA-evoked currents is unlikely to be mediated through the activation of growth factor receptors. Modulation of NMDA responses by BDNF was dependent on the concentration of extracellular glycine. The most pronounced potentiation by BDNF was observed at low concentrations, whereas no potentiation was observed in saturating concentrations of glycine, suggesting that BDNF may have increased the affinity of the NMDA receptor for glycine. However, the competitive glycine-site antagonist 7-chloro-kynurenic acid blocked the enhancement by BDNF without shifting the dose-inhibition relationship for this antagonist, and Mg2+ consistently depressed the potentiation of NMDA-evoked currents by BDNF, indicating that BDNF does not alter glycine affinity. BDNF also reversibly increased the probability of opening of NMDA channels recorded from outside-out patches taken from cultured hippocampal neurons. Other unrelated peptides including dynorphin and somatostatin also caused a glycine-dependent enhancement of NMDA currents and depressed the currents in saturating concentrations of glycine. In contrast, a shortened analogue dynorphin (6-17), which lacks N-terminus glycine residues, and another peptide met-enkephalin were without effects on NMDA currents recorded in low concentrations of glycine. Our results suggest that neurotrophins and other peptides can serve as glycine-like ligands for the NMDA receptor.

The unimolecular chemistry of protonated glycine and the proton affinity of glycine: a computational model

The potential energy hypersurface of protonated glycine, GH+, has been investigated. The calculated G2(MP2) value for the proton affinity (PA) of glycine, PA calc=895kJ mol−1, is in good agreement with the experimental value which has been estimated to lie in the range 864kJ mol−1 < PA exp <891kJ mol−1. Ab initio quantum chemical calculations of relevant parts of the potential energy surface of GH+ give a reaction model which is consistent with the observed mass spectrometric fragmentation pattern. The lowest energy unimolecular reactions of GH+ are two distinct processes: (1) loss of CO, which has a substantial barrier for the reverse reaction, and (2) loss of CO plus H2O, which has no barrier for the reverse reaction.

Permeation and gating of α1 glycine-gated channels expressed at low and high density in Xenopus oocyte

When a high density of α1-subunit glycine receptor (GlyR) is expressed in Xenopus oocytes, two populations of channels can be distinguished according to their apparent affinity for glycine which differs 5- to 6-fold. To compare the open pore diameter of these channels, the relative permeability of formate with respect to chloride ( P formate/ P Cl) was determined in bionic conditions. For the low-affinity GlyR P formate/ P Cl was comparable to that reported for glycine-gated channels in cultured spinal cord and hippocampal neurons. In contrast, the high-affinity GlyR had a 56% larger P formate/ P Cl. In addition, the open probability of the channels was differentially sensitive to voltage. These results show that the high expression of α1 GlyR resulted in two populations of GlyR which differed not only in the affinity to agonists but also in permeation and gating mechanisms.

Differential expression of five N-methyl- d-aspartate receptor subunit mRNAs in vasopressin and oxytocin neuroendocrine cells

Vasopressin and oxytocin neuroendocrine cells within the supraoptic nucleus display distinctive electrophysiological properties and differential responses to selected NMDA receptor (NR) antagonists. To determine if these differences might be due to NMDA receptor composition, we compared the expression of NR1, NR2A, NR2B, NR2C and NR2D subunit mRNAs in immunocytochemically identified vasopressin and oxytocin neuroendocrine cells. In contrast to NR1 subunit mRNA which was equally expressed in both vasopressin and oxytocin cells, NR2B and NR2C displayed very different expression patterns. In oxytocin cells, the NR2B subunit comprised the majority (65%) of the total NR2 expression with NR2C and NR2D contributing 6% and 27%, respectively. Vasopressin cells exhibited 5-fold higher NR2C (32%), approximately half as much NR2B mRNA (39%) and equivalent NR2D (31%). In vitro expression studies have shown that the NR1-NR2C subunit combination exhibits weaker magnesium block and higher affinity for glycine than NR1-NR2B. Thus, the high expression of NR2C in vasopressin cells relative to oxytocin cells may make these cells more susceptible to glutamatergic activation. These observations in vasopressin and oxytocin cells provide the basis for a working model to investigate how differential NMDA receptor composition may shape the neurophysiological properties of neurons.

Absolute Affinities of α-Amino Acids for Cu+ in the Gas Phase. A Theoretical Study

Ab initio calculations have been carried out on the [glycine−Cu]+, [serine−Cu]+, and [cysteine−Cu]+ complexes. Investigation of several types of structures for each complex shows that the preferred binding site of Cu+ involves chelation between the carbonyl oxygen and the amino nitrogen. With glycine, this leads to a complexation energy (best estimate of D0) of 64.3 kcal/mol. Additional chelation with the alcohol group of serine or the thiol group of cysteine leads to larger binding energies, with cysteine binding more strongly than serine, in good agreement with a recent experimental scale of relative Cu+ affinities of all α-amino acids present in natural peptides. Combining this scale to the accurate determination of the Cu+ affinity of glycine from the present work leads to absolute values of Cu+ affinities of all amino acids. Calculations were also carried out on the complexes of Cu+ with water, ammonia, formaldehyde, and hydrogen sulfide. The geometrical and electronic structures of these complexes are used to analyze the binding of Cu+ to amino acids.

An Alanine Residue in the M3-M4 Linker Lines the Glycine Binding Pocket of the N-Methyl-D-aspartate Receptor

While attempting to map a central region in the M3-M4 linker of the N-methyl-D-aspartate receptor NR1 subunit, we found that mutation of a single position, Ala-714, greatly reduced the apparent affinity for glycine. Proximal N-glycosylation localized this region to the extracellular space. Glycine affinities of additional Ala-714 mutations correlated with side chain volume. Substitution of alanine 714 with cysteine did not alter glycine sensitivity, although this mutant was rapidly inhibited by dithionitrobenzoate. Glycine protected the A714C mutant from modification by dithionitrobenzoate, whereas the co-agonist L-glutamate was ineffective. These experiments place Ala-714 in the glycine binding pocket of the N-methyl-D-aspartate receptor, a determination not predicted by previous structural models based on bacterial periplasmic binding protein homology.

Effects of NMDA-R1 antisense oligodeoxynucleotide administration: behavioral and radioligand binding studies

The effects of an antisense phosphodiester oligodeoxynucleotide (ODN) directed to the NR1 subunit of the NMDA receptor mRNA and of its corresponding sense ODN were investigated in mice. Treatment with the antisense ODN significantly increased the time mice spent in the open arms of an elevated maze while the total number of arm entries was unaltered. Furthermore, seizure latencies after the administration of an ED 100 dose of NMDA (150 mg/kg) were significantly higher in antisense treated animals compared to vehicle controls. At the same time, treatment with NR1 antisense ODN significantly reduced the B max of [ 3H]CGS-19755 binding (2101 fmol/mg protein) compared to both vehicle (2787 fmol/mg protein) and sense (2832±39 fmol/mg protein) controls without any significant change in K D (33 nM). A corresponding reduction of [ 3H]CGP-39653 binding was also observed after treatment with NR1 antisense compared to both sense and vehicle controls. In contrast, neither antisense nor sense ODNs altered the proportion of high affinity glycine sites or the potency of glycine at either high or low affinity glycine binding sites to inhibit [ 3H]CGP-39653 binding. These results show that in vivo treatment with NR1 antisense ODNs to the NMDA receptor complex reduces antagonist binding at NMDA receptors and has pharmacological effects similar to those observed with some NMDA receptor antagonists. These results also suggest that treatment with antisense ODNs may provide another means to investigate allosteric modulation of receptor subtypes in vivo.