The mitochondrial F1FO-ATPase exploits the dithiol redox state to modulate the permeability transition pore.

The dithiol reagents phenylarsine oxide (PAO) and dibromobimane (DBrB) have opposite effects on the F1FO-ATPase activity. PAO 20% increases ATP hydrolysis at 50 µM when the enzyme activity is activated by the natural cofactor Mg2+ and at 150 µM when it is activated by Ca2+. The PAO-driven F1FO-ATPase activation is reverted to the basal activity by 50 µM dithiothreitol (DTE). Conversely, 300 µM DBrB decreases the F1FO-ATPase activity by 25% when activated by Mg2+ and by 50% when activated by Ca2+. In both cases, the F1FO-ATPase inhibition by DBrB is insensitive to DTE. The mitochondrial permeability transition pore (mPTP) formation, related to the Ca2+-dependent F1FO-ATPase activity, is stimulated by PAO and desensitized by DBrB. Since PAO and DBrB apparently form adducts with different cysteine couples, the results highlight the crucial role of cross-linking of vicinal dithiols on the F1FO-ATPase, with (ir)reversible redox states, in the mPTP modulation.

Oxidative Modification of Redox Proteins: Role in the Regulation of HBL-100 Cell Proliferation.

HBL-100 breast epithelial cells were cultured with a blocker (N-ethylmaleimide) and protector (1,4-dithioerythritol) of SH groups. The study assessed changes in redox potential of glutathione and thioredoxin systems, intensity of oxidative modification of proteins, ROS production, and cell proliferation. The roles of thioredoxin system and protein oxidative modification in HBL-100 cell proliferation under redox status modulation were established. The role of carbonylated thioredoxin in arrest of the cell cycle in S-phase was demonstrated, which could be used for targeted therapy of the diseases accompanied by oxidative stress and disturbed redox status.

Attenuation of endothelial phosphatidylserine exposure decreases ischemia-reperfusion induced changes in microvascular permeability.

BACKGROUND: Translocation of phosphatidylserine from the inner leaflet to the outer leaflet of the endothelial membrane via phospholipid scramblase-1 (PLSCR1) is an apoptotic signal responsible for the loss of endothelial barrier integrity after ischemia-reperfusion injury (IRI). We hypothesized that inhibiting phosphatidylserine expression on endothelial cells would attenuate IRI induced increases in hydraulic permeability (Lp). METHODS: Mesenteric Lp was measured in rat post-capillary mesenteric venules subjected to IRI via superior mesenteric artery (SMA) occlusion (45 minutes) and release (300 minutes) in conjunction with several inhibitors of phosphatidylserine exposure as follows: (1) inhibition of PLSCR1 translocation (dithioerythritol, n = 3), (2) inhibition of PLSCR1 membrane trafficking (2-bromopalmitate [2-BP], n = 3), and (3) inhibition of ion exchange necessary for PLSCR1 function (4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid [DIDS], n = 3). Under the same IRI conditions, rats were also administered targeted inhibitors of phosphatidylserine exposure including knockdown of PLSCR1 (n = 3) using RNA interference (RNAi), and as a potential therapeutic tool Diannexin, a selective phosphatidylserine blocker (n = 3). RESULTS: During IRI net Lp increased by 80% (p < 0.01). Net reductions of Lp were accomplished by 2-BP (46% reduction, p = 0.005), combined DET + 2-BP + DIDS (32% reduction, p = 0.04), RNAi (55% reduction, p = 0.002), Diannexin administered pre-SMA artery occlusion (73% reduction, p = 0.001), and post-SMA occlusion (70% reduction, p = 0.002). CONCLUSION: Phosphatidylserine exposure is a key event in the pathogenesis of microvascular dysfunction during IRI. Clinically, inhibition of phosphatidylserine exposure is a promising strategy that may 1 day be used to mitigate the effects of IRI.

Nitric oxide inhibits the mitochondrial carnitine/acylcarnitine carrier through reversible S-nitrosylation of cysteine 136.

S-nitrosylation of the mitochondrial carnitine/acylcarnitine transporter (CACT) has been investigated on the native and the recombinant proteins reconstituted in proteoliposomes, and on intact mitochondria. The widely-used NO-releasing compound, GSNO, strongly inhibited the antiport measured in proteoliposomes reconstituted with the native CACT from rat liver mitochondria or the recombinant rat CACT over-expressed in E. coli. Inhibition was reversed by the reducing agent dithioerythritol, indicating a reaction mechanism based on nitrosylation of Cys residues of the CACT. The half inhibition constant (IC50) was very similar for the native and recombinant proteins, i.e., 74 and 71µM, respectively. The inhibition resulted to be competitive with respect the substrate, carnitine. NO competed also with NEM, correlating well with previous data showing interference of NEM with the substrate transport path. Using a site-directed mutagenesis approach on Cys residues of the recombinant CACT, the target of NO was identified. C136 plays a major role in the reaction mechanism. The occurrence of S-nitrosylation was demonstrated in intact mitochondria after treatment with GSNO, immunoprecipitation and immunostaining of CACT with a specific anti NO-Cys antibody. In parallel samples, transport activity of CACT measured in intact mitochondria, was strongly inhibited after GSNO treatment. The possible physiological and pathological implications of the post-translational modification of CACT are discussed.

[Redox-dependent mechanisms of regulation of breast epithelial cell proliferation]. / Redoks-zavisimye mekhanizmy reguliatsii proliferatsii kletok épiteliia molochnoi zhelezy.

Activation of free radical oxidation in different cell types, including breast epithelial cells, may result in damage to macromolecules, in particular, proteins taking part in regulation of cell proliferation and apoptosis. The glutathione, glutaredoxin and thioredoxin systems play an essential role in maintaining intracellular redox homeostasis. Due to this fact, modulation of cellular redox status under the effect of an SH group inhibitor and an SH group protector may be used as a model for studying the role of redox proteins and glutathione in regulating cell proliferation in different pathological processes. In this study we have evaluated the state of the thioredoxin, glutaredoxin and glutathione systems as well as their role in regulating proliferation of HBL-100 breast epithelial cells under redox status modulation with N-ethylmaleimide (NEM) and 1,4-dithioerythriol (DTE). Modulating the redox status of breast epithelial cells under the effect of NEM and DTE influences the functional activity of glutathione-dependent enzymes, glutaredoxin, thioredoxin, and thioredoxin reductase through changes in the GSH and GSSG concentrations. In HBL-100 cells under redox-status modulation, we have found an increase in the number of cells in the S-phase of the cell cycle and a decrease in the number of cells in the G0/G1 and G2/М phases, as opposed to the values in the intact culture. The proposed model of proliferative activity of cells under redox status modulation may be used for development of new therapeutic approaches for treatment of diseases accompanied by oxidative stress generation.

Preparation of core-crosslinked linear-dendritic copolymer micelles with enhanced stability and their application for drug solubilisation.

In this study we explore the preparation of core-crosslinked micelles of linear-dendritic methoxy-poly(ethylene glycol) (MPEG)-co-poly(ester-sulfide) (PES) polymers to improve the stability of such polymeric micelle systems against premature disintegration and drug release. A series of MPEG-PES copolymers were synthesised via stepwise reactions of acetylation and thiol-ene photoreaction. Surface tension measurement showed that the copolymers with ethenyl surface groups could self-associate in dilute aqueous solutions to form micelles. Crosslinking within the micelle cores in the presence of dithioerythritol (DTT) linker was initiated under UV radiation. The formation of core-crosslinked micelles was confirmed by HPLC in combination with charged aerosol detection (CAD). The copolymers were found to readily hydrolyse under acidic conditions due to the ester-containing dendrons. Drug solubilisation capacities of the micellar solutions were determined using griseofulvin as a poorly water-soluble model drug. The solubility of griseofulvin showed a 10-fold enhancement in 1% w/v micelle solution and increased with the concentration of the copolymers. Drug release studies indicated that a more sustained release of griseofulvin was achieved for the core-crosslinked micelles compared to the non-crosslinked micelles, attributable to greater stability of the crosslinked core structure. The findings of this study present a new pathway towards developing biodegradable polymeric nanocarriers.

Mercury and protein thiols: Stimulation of mitochondrial F1FO-ATPase and inhibition of respiration.

In spite of the known widespread toxicity of mercury, its impact on mitochondrial bioenergetics is a still poorly explored topic. Even if many studies have dealt with mercury poisoning of mitochondrial respiration, as far as we are aware Hg2+ effects on individual complexes are not so clear. In the present study changes in swine heart mitochondrial respiration and F1FO-ATPase (F-ATPase) activity promoted by micromolar Hg2+ concentrations were investigated. Hg2+ was found to inhibit the respiration of NADH-energized mitochondria, whereas it was ineffective when the substrate was succinate. Interestingly, the same micromolar Hg2+ doses which inhibited the NADH-O2 activity stimulated the F-ATPase, most likely by interacting with adjacent thiol residues. Accordingly, Hg2+ dose-dependently decreased protein thiols and all the elicited effects on mitochondrial complexes were reversed by the thiol reducing agent DTE. These findings clearly indicate that Hg2+ interacts with Cys residues of these complexes and differently modulate their functionality by modifying the redox state of thiol groups. The results, which cast light on some implications of metal-thiol interactions up to now not fully explored, may contribute to clarify the molecular mechanisms of mercury toxicity to mitochondria.

Sulfone-stabilized carbanions for the reversible covalent capture of a posttranslationally-generated cysteine oxoform found in protein tyrosine phosphatase 1B (PTP1B).

Redox regulation of protein tyrosine phosphatase 1B (PTP1B) involves oxidative conversion of the active site cysteine thiolate into an electrophilic sulfenyl amide residue. Reduction of the sulfenyl amide by biological thiols regenerates the native cysteine residue. Here we explored fundamental chemical reactions that may enable covalent capture of the sulfenyl amide residue in oxidized PTP1B. Various sulfone-containing carbon acids were found to react readily with a model peptide sulfenyl amide via attack of the sulfonyl carbanion on the electrophilic sulfur center in the sulfenyl amide. Both the products and the rates of these reactions were characterized. The results suggest that capture of a peptide sulfenyl amide residue by sulfone-stabilized carbanions can slow, but not completely prevent, thiol-mediated generation of the corresponding cysteine-containing peptide. Sulfone-containing carbon acids may be useful components in the construction of agents that knock down PTP1B activity in cells via transient covalent capture of the sulfenyl amide oxoform generated during insulin signaling processes.

[The role of oxidative protein modification and the gluthatione system in modulation of the redox status of breast epithelial cells].

The effects of the SH-group blocker N-ethylmaleimide (NEM) and thiol group protector 1,4-dithioerythritol (DTE) on the redox status of cells HBL-100 cells, oxidative modification of their proteins and the state of glutathione and thioredoxin systems have been investigated. Breast epithelial cells cultivated in the presence of NEM were characterized by decreased redox status, increased glutathione reductase activity, and increased concentrations of products of irreversible oxidative modification of protein and amino acids. Cultivation of HBL-100 cells in the presence of DTE resulted in a shift of the redox status towards reduction processes and increased reversible protein modification by glutathionylation. The proposed model of intracellular redox modulation may be used in the development of new therapeutic approaches to treat diseases accompanied by impaired redox homeostasis (e.g. oncologic, inflammatory, cardiovascular and neurodegenerative disease).

The Role of Protein Oxidative Modification and the Cellular Redox Status in Realization of Apoptosis of MCF-7 Breast Adenocarcmoma Cells.

The aim of this study was to establish the role of redox modification of proteins and redox status in the realization of apoptosis of MCF-7 breast adenocarcinoma cells du-ing cultivation with the SH-group blocker N-ethylmaleimide (NEM) and the SH-group protector 1,4-dithioerythritol (DTE). The activation of apoptosis in MCF-7 breast adenocarcinoma cells was shown to be due to the irreversible modification of redox sensitive protein molecules. The presence of DTE in the culture medium of cancer.cells caused reversible glutathionylation of protein molecules and did not change the: number of apoptotic MCF-7 cells.

Structural aspects of a protein-surfactant assembly: native and reduced States of human serum albumin.

The inherently present seventeen disulfide bonds of the circulatory protein, human serum albumin (HSA) provide the necessary structural stability. Various spectroscopic approaches were used to investigate the effect of reduction of these disulfide bonds and its binding with the anionic surfactant, sodium dodecyl sulfate (SDS). Based on several spectroscopic analyses, our investigations highlight the following interesting aspects: (1) HSA on reduction loses not only its tertiary structure but also a significant amount of secondary structure as well. However, the reduced state of the protein is not like the molten-globule, (2) this structural loss of the protein due to reduction is more prominent than that caused by higher SDS concentrations alone and can certainly be attributed to the role of disulfide bonds, (3) lower surfactant concentrations provide marginal structural rigidity to the native state of the protein, whereas, higher concentrations of SDS induces secondary structure to the reduced state of HSA, (4) the binding of SDS with both the native and reduced states of HSA, occurred in three distinct stages which was followed by a saturation stage. However, the nature of such binding is different for both the states as investigated by using the Stern-Volmer equations and estimating the thermodynamic parameters. Besides, in contrast to the native state, the reduced state of HSA shows that the lone tryptophan residue gets more buried. However, there occurs a sudden decrement in the lifetime of the tryptophan and the hydrodynamic diameter increases by twofold.

Tauroursodeoxycholic acid prevents stress induced aggregation of proteins in vitro and promotes PERK activation in HepG2 cells.

Tauroursodeoxycholic acid (TUDCA) a bile salt and chemical chaperone reduces stress-induced aggregation of proteins; activates PERK [PKR (RNA-dependent protein kinase)-like ER (endoplasmic reticulum) kinase] or EIF2AK3, one of the hall marks of ER stress induced unfolded protein response (UPR) in human hepatoblastoma HepG2 cells; prevents heat and dithiothreitol (DTT) induced aggregation of BSA (bovine serum albumin), and reduces ANS (1-anilino-naphthalene-8-sulfonate) bound BSA fluorescence in vitro. TUDCA inactivates heat treated, but not the native EcoR1 enzyme, and reduces heat-induced aggregation and activity of COX-1 (cyclooxygenase enzyme-1) in vitro. These findings suggest that TUDCA binds to the hydrophobic regions of proteins and prevents their subsequent aggregation. This may stabilize unfolded proteins that can mount UPR or facilitate their degradation through cellular degradation pathways.

Reversible native chemical ligation: a facile access to dynamic covalent peptides.

The broad interest of using reversible covalent bonds in chemistry, in particular at its interfaces with biology and materials science, has been recently established through numerous examples in the literature. However, the challenging exchange of peptide fragments using a dynamic covalent peptide bond has not yet been achieved without enzymatic catalysis because of its high thermodynamic stability. Here we show that peptide fragments can be exchanged by a chemoselective and reversible native chemical ligation (NCL) which can take place at N-(methyl)-cysteine residues. This very mild reaction is efficient in aqueous solution, is buffered at physiological pH in the presence of dithiothreitol (DTT), and shows typical half-times of equilibration in the 10 h range.

The mitochondrial F1FO-ATPase desensitization to oligomycin by tributyltin is due to thiol oxidation.

The antibiotic oligomycin is known to inhibit mitochondrial F-type ATP synthases. The antibiotic inhibits both ATP synthesis and hydrolysis by blocking the H(+) translocation through FO which is coupled to the catalytic activity of F1. The amphiphilic organotin tri-n-butyltin (TBT), a known mitochondrial poison, can penetrate into biological membranes and covalently bind to electron-donor atoms of biomolecules such as sulfur. This study aims at exploring the mechanism(s) involved in the enzyme desensitization to oligomycin which occurs at concentrations >1 µM TBT. This poorly known effect of TBT, which only appeared at temperatures above the break in the Arrhenius plot of the enzyme activity, was found to be accompanied by the oxidation of isolated thiol groups of the mitochondrial complex. The oligomycin sensitivity was restored by the reducing agents glutathione and dithioerythritol and not influenced by antioxidants. The whole of data is consistent with the hypothesis that thiol oxidation is caused by TBT covalent binding to cysteine residues in a low-affinity site on FO and not by other possible oxidative events. According to this putative model, the onset of tin-sulfur bonds would trigger conformational changes and weaken the oligomycin interaction with FO.

High levels of inactive thymidine kinase 1 polypeptide detected in sera from dogs with solid tumours by immunoaffinity methods: implications for in vitro diagnostics.

Determination of serum thymidine kinase 1 (STK1) activity has been used as a proliferation marker for neoplastic diseases in both human and veterinary medicine. The purpose of this study was to determine STK1 activity and enzyme levels in different dog tumours. Serum samples from three dogs with leukaemia, five with lymphoma, 21 with solid tumours and 18 healthy dogs were analyzed for STK1 activity, using an optimized [(3)H]-deoxythymidine (dThd) phosphorylation assay, and for STK1 protein levels using an immunoaffinity/western blot assay. STK1 activity in dogs with haematological tumours was significantly higher than in the solid tumour and healthy dog groups (mean ± standard deviation [SD] = 65 ± 79, 1.1 ± 0.5, and 1.0 ± 0.4 pmol/min/mL, respectively). Serum samples were analyzed after immunoaffinity isolation by western blot and the TK1 26 kDa band intensities quantified revealing that concentrations were significantly higher in dogs with haematological tumours and solid tumours compared to healthy dogs (mean ± SD=33 ± 12, 30 ± 13, and 10 ± 5 ng/mL, respectively). Pre-incubation with the reducing agent dithioerythritol (DTE) showed a decrease in STK1 activity and protein levels in most samples, but an increase of about 20% in sera from healthy dogs and from those with haematological malignancies. Compared to animals with solid tumours, the specific STK1 activity (nmol [(3)H]-deoxythymidine monophosphate (dTMP)/min/mg of TK1 protein of 26 kDa) was 30-fold higher in haematological malignancies and 2.5-fold higher in healthy dogs, respectively. The results demonstrate that there is a large fraction of inactive TK1 protein, particularly in sera from dogs with solid tumours. The findings are important in the use of STK1 as a biomarker.

Inhibition of mitochondrial carnitine/acylcarnitine transporter by H(2)O(2): molecular mechanism and possible implication in pathophysiology.

H(2)O(2) inhibits the [(3)H]carnitine/carnitine antiport catalysed by the mitochondrial carnitine/acylcarnitine transporter reconstituted in proteoliposomes. The inhibition was reversed by dithioerythritol, N-acetylcysteine and L-cysteine. Inhibition time-dependence revealed a faster and a slower reaction stages with orders of reaction of 1.0 and 1.9, respectively. Inhibition was tested on mutants in which one or more of the six Cys residues had been substituted with Ser or with Val. The four replacement mutant C23S/C58S/C89S/C283S containing C136 and C155 was inhibited as the wild-type. Mutants C23V/C58V/C155V/C89S/C283S and C23V/C58V/C136V/C89S/C283S containing only C136 or C155, respectively, were inhibited at a much lower extent respect to the wild-type, while the mutant C136S/C155S in which the two Cys were substituted and the C-less protein were virtually insensitive to inhibition. DTE reversed the inhibition of the H(2)O(2) sensitive proteins except that in the case of the mutants containing only C136 or C155 after long time of incubation with H(2)O(2). The IC(50) values obtained by dose-response curves of H(2)O(2) inhibition were 0.17 mM for the wild-type, 0.39 mM for the four replacement mutant containing C136 and C155, 2.23 or 1.8mM in the five replacement mutants containing the single C136 or C155, respectively. Carnitine and acetylcarnitine protected the protein from the inhibition by H(2)O(2). Inhibition kinetics showed a competitive behaviour of H(2)O(2) respect to carnitine. All the data concur to demonstrate that H(2)O(2) interacts with C136 and C155 and completely inactivates the transporter by inducing the formation of a disulphide.

Platelet adhesion and human umbilical vein endothelial cell cytocompatibility of biodegradable segmented polyurethanes prepared with 4,4'-methylene bis(cyclohexyl isocyanate), poly(caprolactone) diol and butanediol or dithioerythritol as chain extenders.

Biodegradable segmented polyurethanes were prepared with poly(caprolactone) diol as a soft segment, 4,4'-methylene bis(cyclohexyl isocyanate) (HMDI) and either butanediol or dithioerythritol as chain extenders. Platelet adhesion was similar in all segmented polyurethanes studied and not different from Tecoflex® although an early stage of activation was observed on biodegradable segmented polyurethane prepared with dithioerythritol. Relative viability was higher than 80% on human umbilical vein endothelial cells in contact with biodegradable segmented polyurethane extracts after 1, 2 and 7 days. Furthermore, both biodegradable segmented polyurethane materials supported human umbilical vein endothelial cell adhesion, spreading, and viability similar to Tecoflex® medical-grade polyurethane. These biodegradable segmented polyurethanes represent promising materials for cardiovascular applications.

Determination of biothiols by a novel on-line HPLC-DTNB assay with post-column detection.

A novel on-line HPLC-DTNB method was developed for the selective determination of biologically important thiols (biothiols) such as L-cysteine (Cys), glutathione (GSH), homocysteine (HCys), N-acetylcysteine (NAC), and 1,4-dithioerythritol (DTE) in pharmaceuticals and tissue homogenates. The biothiols were separated on C18 column using gradient elution, reacted with the postcolumn reagent, DTNB in 0.5% M-ß-CD (w/v) solution at pH 8, to form yellow-colored 5-thio-2-nitrobenzoic acid (TNB), and monitored with a PDA detector (λ=410 nm). With the optimized conditions for chromatography and the post-column derivatization, 40 nM of NAC, 40 nM of Cys, and 50 nM of GSH can be determined. The relative standard deviations of the recommended method were in the range of 3.2-5.4% for 50 µM biothiols. The negative peaks of biothiol constituents were monitored by measuring the increase in absorbance due to TNB chromophore. The detection limits of biothiols at 410 nm (in the range of 0.04-0.58 µM) after post-column derivatization with DTNB+M-ß-CD were much lower than those at 205 nm UV-detection without derivatization, and were distinctly lower than those with post-column DTNB alone. The method is rapid, inexpensive, versatile, nonlaborious, uses stable reagents, and enables the on-line qualitative and quantitative estimation of biothiol constituents of biological fluids and pharmaceuticals.

Shift in the equilibrium between on and off states of the allosteric switch in Ras-GppNHp affected by small molecules and bulk solvent composition.

Ras GTPase cycles between its active GTP-bound form promoted by GEFs and its inactive GDP-bound form promoted by GAPs to affect the control of various cellular functions. It is becoming increasingly apparent that subtle regulation of the GTP-bound active state may occur through promotion of substates mediated by an allosteric switch mechanism that induces a disorder to order transition in switch II upon ligand binding at an allosteric site. We show with high-resolution structures that calcium acetate and either dithioerythritol (DTE) or dithiothreitol (DTT) soaked into H-Ras-GppNHp crystals in the presence of a moderate amount of poly(ethylene glycol) (PEG) can selectively shift the equilibrium to the "on" state, where the active site appears to be poised for catalysis (calcium acetate), or to what we call the "ordered off" state, which is associated with an anticatalytic conformation (DTE or DTT). We also show that the equilibrium is reversible in our crystals and dependent on the nature of the small molecule present. Calcium acetate binding in the allosteric site stabilizes the conformation observed in the H-Ras-GppNHp/NOR1A complex, and PEG, DTE, and DTT stabilize the anticatalytic conformation observed in the complex between the Ras homologue Ran and Importin-ß. The small molecules are therefore selecting biologically relevant conformations in the crystal that are sampled by the disordered switch II in the uncomplexed GTP-bound form of H-Ras. In the presence of a large amount of PEG, the ordered off conformation predominates, whereas in solution, in the absence of PEG, switch regions appear to remain disordered in what we call the off state, unable to bind DTE.

An alternating GluN1-2-1-2 subunit arrangement in mature NMDA receptors.

NMDA receptors (NMDARs) form glutamate-gated ion channels that play a critical role in CNS physiology and pathology. Together with AMPA and kainate receptors, NMDARs are known to operate as tetrameric complexes with four membrane-embedded subunits associating to form a single central ion-conducting pore. While AMPA and some kainate receptors can function as homomers, NMDARs are obligatory heteromers composed of homologous but distinct subunits, most usually of the GluN1 and GluN2 types. A fundamental structural feature of NMDARs, that of the subunit arrangement around the ion pore, is still controversial. Thus, in a typical NMDAR associating two GluN1 and two GluN2 subunits, there is evidence for both alternating 1/2/1/2 and non-alternating 1/1/2/2 arrangements. Here, using a combination of electrophysiological and cross-linking experiments, we provide evidence that functional GluN1/GluN2A receptors adopt the 1/2/1/2 arrangement in which like subunits are diagonal to one another. Moreover, based on the recent crystal structure of an AMPA receptor, we show that in the agonist-binding and pore regions, the GluN1 subunits occupy a "proximal" position, closer to the central axis of the channel pore than that of GluN2 subunits. Finally, results obtained with reducing agents that differ in their membrane permeability indicate that immature (intracellular) and functional (plasma-membrane inserted) pools of NMDARs can adopt different subunit arrangements, thus stressing the importance of discriminating between the two receptor pools in assembly studies. Elucidating the quaternary arrangement of NMDARs helps to define the interface between the subunits and to understand the mechanism and pharmacology of these key signaling receptors.