SNARE-dependent upregulation of potassium chloride co-transporter 2 activity after metabotropic zinc receptor activation in rat cortical neurons in vitro.

The major outward chloride transporter in neurons is the potassium chloride co-transporter 2 (KCC2), critical for maintaining an inhibitory reversal potential for GABA(A) receptor channels. In a recent study, we showed that Zn(2+) regulates GABA(A) reversal potentials in the hippocampus by enhancing the activity of KCC2 through an increase in its surface expression. Zn(2+) initiates this process by activating the Gq-coupled metabotropic Zn(2+) receptor/G protein-linked receptor 39 (mZnR/GPR39). Here, we first demonstrated that mZnR/GPR39 is functional in cortical neurons in culture, and then tested the hypothesis that the increase in KCC2 activity is mediated through a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent process. We established the presence of functional mZnR in rat cultured cortical neurons by loading cells with a Ca(2+) indicator and exposing cells to Zn(2+), which triggered consistent Ca(2+) responses that were blocked by the Gq antagonist YM-254890, but not by the metabotropic glutamate receptor antagonist (RS)-α-methyl-4-carboxyphenylglycine (MCPG). Importantly, Zn(2+) treatment under these conditions did not increase the intracellular concentrations of Zn(2+) itself. We then measured KCC2 activity by monitoring both the rate and relative amount of furosemide-sensitive NH(4)(+) influx through the co-transporter using an intracellular pH-sensitive fluorescent indicator. We observed that Zn(2+) pretreatment induced a Ca(2+)-dependent increase in KCC2 activity. The effects of Zn(2+) on KCC2 activity were also observed in wild-type mouse cortical neurons in culture, but not in neurons obtained from mZnR/GPR39(-/-) mice, suggesting that Zn(2+) acts through mZnR/GPR39 activation to upregulate KCC2 activity. We next transfected rat cortical neurons with a plasmid encoding botulinum toxin C1 (Botox C1), which cleaves the SNARE proteins syntaxin 1 and synaptosomal-associated protein 25 (SNAP-25). Basal KCC2 activity was similar in both transfected and non-transfected neurons. Non-transfected cells, or cells transfected with marker vector alone, showed a Zn(2+)-dependent increase in KCC2 activity. In contrast, KCC2 activity in neurons expressing Botox C1 was unchanged by Zn(2+). These results suggest that SNARE proteins are necessary for the increased activity of KCC2 after Zn(2+) stimulation of mZnR/GPR39.

Complex role of zinc in methamphetamine toxicity in vitro.

Methamphetamine is a drug of abuse that can induce oxidative stress and neurotoxicity to dopaminergic neurons. We have previously reported that oxidative stress promotes the liberation of intracellular Zn(2+) from metal-binding proteins, which, in turn, can initiate neuronal injurious signaling processes. Here, we report that methamphetamine mobilizes Zn(2+) in catecholaminergic rat pheochromocytoma (PC12) cells, as measured by an increase in Zn(2+)-regulated gene expression driven by the metal response element transcription factor-1. Moreover, methamphetamine-liberated Zn(2+) was responsible for a pronounced enhancement in voltage-dependent K(+) currents in these cells, a process that normally accompanies Zn(2+)-dependent cell injury. Overnight exposure to methamphetamine induced PC12 cell death. This toxicity could be prevented by the cell-permeant zinc chelator N,N,N', N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), and by over-expression of the Zn(2+)-binding protein metallothionein 3 (MT3), but not by tricine, an extracellular Zn(2+) chelator. The toxicity of methamphetamine to PC12 cells was enhanced by the presence of co-cultured microglia. Remarkably, under these conditions, TPEN no longer protected but, in fact, dramatically exacerbated methamphetamine toxicity, tricine again being without effect. Over-expression of MT3 in PC12 cells did not mimic these toxicity-enhancing actions of TPEN, suggesting that the chelator affected microglial function. Interestingly, P2X receptor antagonists reversed the toxicity-enhancing effect of TPEN. As such, endogenous levels of intracellular Zn(2+) may normally interfere with the activation of P2X channels in microglia. We conclude that Zn(2+) plays a significant but complex role in modulating the cellular response of PC12 cells to methamphetamine exposure in both the absence and presence of microglia.

Preimplantation genetic diagnosis for BRCA1/2--a novel clinical experience.

OBJECTIVE: To describe our 2-year experience with preimplantation genetic diagnosis (PGD) for carriers of mutations in the genes BRCA1 and BRCA2, the dilemmas incurred and the lessons learned. METHODS: We collected data on those carriers of BRCA1/2 mutations who applied for PGD counseling and who decided to proceed. We describe the PGD procedures that were conducted and their outcome. RESULTS: Ten carriers of BRCA1/2 mutations applied for PGD counseling, seven were healthy, and three were BC survivors. Eight women needed in vitro fertilization (IVF) because of coexisting infertility. After counseling, six opted for the procedure and five of them underwent PGD for the BRCA mutation. In one of these PGD, fluorescence in situ hybridization (FISH) analysis for chromosomes 21, X and Y was also performed. Three women conceived, each in the first treatment attempt. One of them gave birth to twins, the second to a singleton and the third is currently pregnant. During the pregnancies, dilemmas concerning PGD confirmation were discussed. CONCLUSIONS: PGD is an acceptable reproductive option for BRCA mutation carriers, especially for those who require IVF due to fertility problems. Discussion of this option should be carried out with sensitivity, taking into account the age of the woman, her health, fertility status and emotional state. Confirmatory prenatal diagnosis may not always be encouraged.

Laser-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis.

BACKGROUND: Human embryonic stem cells (hESCs) suitable for future transplantation therapy should preferably be developed in an animal-free system. Our objective was to develop a laser-based system for the isolation of the inner cell mass (ICM) that can develop into hESC lines, thereby circumventing immunosurgery that utilizes animal products. METHODS: Hatching was assisted by micromanipulation techniques through a laser-drilled orifice in the zona pellucida of 13 abnormal preimplantation genetic diagnosed blastocysts. ICMs were dissected from the trophectoderm by a laser beam and plated on feeders to derive hESC lines. RESULTS: eight ICMs were isolated from nine hatched blastocysts and gave rise to three hESC lines affected by myotonic dystrophy type 1, hemophilia A and a carrier of cystic fibrosis 405 + 1G > A mutation. Five blastocysts that collapsed during assisted hatching or ICM dissection were plated whole, giving rise to an additional line affected by fragile X. All cell lines expressed markers of pluripotent stem cells and differentiated in vitro and in vivo into the three germ layers. CONCLUSIONS: These hESC lines can serve as an important model of the genetic disorders that they carry. Laser-assisted isolation of the ICMs may be applied for the derivation of new hESC lines in a xeno-free system for future clinical applications.

A vital role for voltage-dependent potassium channels in dopamine transporter-mediated 6-hydroxydopamine neurotoxicity.

6-Hydroxydopamine (6-OHDA), a neurotoxic substrate of the dopamine transporter (DAT), is widely used in Parkinson's disease models. However, the molecular mechanisms underlying 6-OHDA's selectivity for dopamine neurons and the injurious sequelae that it triggers are not well understood. We tested whether ectopic expression of DAT induces sensitivity to 6-OHDA in non-dopaminergic rat cortical neurons and evaluated the contribution of voltage-dependent potassium channel (Kv)-dependent apoptosis to the toxicity of this compound in rat cortical and midbrain dopamine neurons. Cortical neurons expressing DAT accumulated dopamine and were highly vulnerable to 6-OHDA. Pharmacological inhibition of DAT completely blocked this toxicity. We also observed a p38-dependent Kv current surge in DAT-expressing cortical neurons exposed to 6-OHDA, and p38 antagonists and Kv channel blockers were neuroprotective in this model. Thus, DAT-mediated uptake of 6-OHDA recruited the oxidant-induced Kv channel dependent cell death pathway present in cortical neurons. Finally, we report that 6-OHDA also increased Kv currents in cultured midbrain dopamine neurons and this toxicity was blocked with Kv channel antagonists. We conclude that native DAT expression accounts for the dopamine neuron specific toxicity of 6-OHDA. Following uptake, 6-OHDA triggers the oxidant-associated Kv channel-dependent cell death pathway that is conserved in non-dopaminergic cortical neurons and midbrain dopamine neurons.

Apoptotic surface delivery of K+ channels.

Apoptosis in cortical neurons requires efflux of cytoplasmic potassium mediated by a surge in Kv2.1 channel activity. Pharmacological blockade or molecular disruption of these channels in neurons prevents apoptotic cell death, while ectopic expression of Kv2.1 channels promotes apoptosis in non-neuronal cells. Here, we use a cysteine-containing mutant of Kv2.1 and a thiol-reactive covalent inhibitor to demonstrate that the increase in K+ current during apoptosis is due to de novo insertion of functional channels into the plasma membrane. Biotinylation experiments confirmed the delivery of additional Kv2.1 protein to the cell surface following an apoptotic stimulus. Finally, expression of botulinum neurotoxins that cleave syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) blocked upregulation of surface Kv2.1 channels in cortical neurons, suggesting that target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins support proapoptotic delivery of K+ channels. These data indicate that trafficking of Kv2.1 channels to the plasma membrane causes the apoptotic surge in K+ current.

Functional expression of muscle-type nicotinic acetylcholine receptors in rat forebrain neurons in vitro.

Molecular studies aimed at modifying ligand-gated ion channel function in primary neuronal cultures have been hampered by the limited efficacy of gene transfer techniques. Although viruses can effectively deliver genes to neurons, their preparation requires a substantial effort and their usefulness in delivering multiple subunits of a single protein has not been well established. In this study, we have successfully cotransfected mammalian expression plasmids for all subunits of the muscle nicotinic acetylcholine receptor (nAChR), along with a plasmid containing green fluorescent protein (GFP), into rat forebrain neurons using a calcium phosphate method. Immunostaining experiments and cell counting showed that the nAChR were successfully expressed in approximately 1% of the neurons in the culture. The functionality of the expressed receptors was demonstrated electrophysiologically as carbachol-induced currents were present in nearly 90% of the GFP-positive neurons tested. These currents could be completely inhibited by the irreversible antagonist alpha-cobratoxin. Surprisingly, no currents were obtained in any of the GFP-negative cells tested. These results demonstrate that gene delivery of complex, multi-subunit proteins can be successfully performed in cultured neurons, thereby opening new strategies for the pharmacological study of ligand-gated ion channels.

The neuroprotective agent ebselen modifies NMDA receptor function via the redox modulatory site.

Ebselen is a seleno-organic compound currently in clinical trials for the treatment of ischemic stroke and subarachnoid hemorrhage. Its putative mode of action as a neuroprotectant is via cyclical reduction and oxidation reactions, in a manner akin to glutathione peroxidase. For this reason, we have investigated the effects of ebselen on the redox-sensitive NMDA receptor. We have found that ebselen readily reversed dithiothreitol (DTT) potentiation of NMDA-mediated currents in cultured neurons and in Chinese hamster ovary (CHO) cells expressing wild-type NMDA NR1/NR2B receptors. In contrast, ebselen was unable to modulate NMDA-induced currents in neurons previously exposed to the thiol oxidant 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), or in CHO cells expressing a mutant receptor lacking the NR1 redox modulatory site, suggesting that ebselen oxidizes the NMDA receptor via this site. In addition, ebselen was substantially less effective in modifying NMDA responses in neurons exposed to alkylating agent N-ethylmaleimide (NEM) following DTT treatment. Ebselen also reversed DTT block of carbachol-mediated currents in Cos-7 cells expressing the alpha(2)beta delta epsilon subunits of the acetylcholine receptor, an additional redox-sensitive ion channel. Ebselen was observed to significantly increase cell viability following a 30-min NMDA exposure in cultured neurons. In contrast, other more typical antioxidant compounds did not afford neuroprotection in a similar paradigm. We conclude that ebselen may be neuroprotective in part due to its actions as a modulator of the NMDA receptor redox modulatory site.

p38 activation is required upstream of potassium current enhancement and caspase cleavage in thiol oxidant-induced neuronal apoptosis.

Oxidant-induced neuronal apoptosis has been shown to involve potassium and zinc dysregulation, energetic dysfunction, activation of stress-related kinases, and caspase cleavage. The temporal ordering and interdependence of these events was investigated in primary neuronal cultures exposed to the sulfhydryl oxidizing agent 2,2'-dithiodipyridine (DTDP), a compound that induces the intracellular release of zinc. We previously observed that tetraethylammonium (TEA), high extracellular potassium, or cysteine protease inhibitors block apoptosis induced by DTDP. We now report that both p38 and extracellular signal-regulated kinase phosphorylation are evident in neuronal cultures within 2 hr of a brief exposure to 100 microm DTDP. However, only p38 inhibition is capable of blocking oxidant-induced toxicity. Cyclohexamide or actinomycin D does not attenuate DTDP-induced cell death, suggesting that posttranslational modification of existing targets, rather than transcriptional activation, is responsible for the deleterious effects of p38. Indeed, an early robust increase in TEA-sensitive potassium channel currents induced by DTDP is attenuated by p38 inhibition but not by caspase inhibition. Moreover, we found that activation of p38 is required for caspase 3 and 9 cleavage, suggesting that potassium currents enhancement is required for caspase activation. Finally, we observed that DTDP toxicity could be blocked with niacinamide or benzamide, inhibitors of poly (ADP-ribose) synthetase. Based on these findings, we conclude that oxidation of sulfhydryl groups on intracellular targets results in intracellular zinc release, p38 phosphorylation, enhancement of potassium currents, caspase cleavage, energetic dysfunction, and translationally independent apoptotic cell death.

The regulation of the Escherichia coli mazEF promoter involves an unusual alternating palindrome.

The Escherichia coli mazEF system is a chromosomal "addiction module" that, under starvation conditions in which guanosine-3',5'-bispyrophosphate (ppGpp) is produced, is responsible for programmed cell death. This module specifies for the toxic stable protein MazF and the labile antitoxic protein MazE. Upstream from the mazEF module are two promoters, P(2) and P(3) that are strongly negatively autoregulated by MazE and MazF. We show that the expression of this module is positively regulated by the factor for inversion stimulation. What seems to be responsible for the negative autoregulation of mazEF is an unusual DNA structure, which we have called an "alternating palindrome." The middle part, "a," of this structure may complement either the downstream fragment, "b," or the upstream fragment, "c". When the MazE.MazF complex binds either of these arms of the alternating palindrome, strong negative autoregulation results. We suggest that the combined presence of the two promoters, the alternating palindrome structure and the factor for inversion stimulation-binding site, all permit the expression of the mazEF module to be sensitively regulated under various growth conditions.

Induction of neuronal apoptosis by thiol oxidation: putative role of intracellular zinc release.

The membrane-permeant oxidizing agent 2,2'-dithiodipyridine (DTDP) can induce Zn(2+) release from metalloproteins in cell-free systems. Here, we report that brief exposure to DTDP triggers apoptotic cell death in cultured neurons, detected by the presence of both DNA laddering and asymmetric chromatin formation. Neuronal death was blocked by increased extracellular potassium levels, by tetraethylammonium, and by the broad-spectrum cysteine protease inhibitor butoxy-carbonyl-aspartate-fluoromethylketone. N,N,N', N'-Tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) and other cell-permeant metal chelators also effectively blocked DTDP-induced toxicity in neurons. Cell death, however, was not abolished by the NMDA receptor blocker MK-801, by the intracellular calcium release antagonist dantrolene, or by high concentrations of ryanodine. DTDP generated increases in fluorescence signals in cultured neurons loaded with the zinc-selective dye Newport Green. The fluorescence signals following DTDP treatment also increased in fura-2- and magfura-2-loaded neurons. These responses were completely reversed by TPEN, consistent with a DTDP-mediated increase in intracellular free Zn(2+) concentrations. Our studies suggest that under conditions of oxidative stress, Zn(2+) released from intracellular stores may contribute to the initiation of neuronal apoptosis.

Alterations of N-methyl-D-aspartate receptor properties after chemical ischemia.

Sublethal ischemic challenges can protect neurons against a second, more severe hypoxic insult. We report here that nonlethal chemical ischemia induces a transient alteration of NMDA receptors in rat cortical neurons in culture. Cells were incubated with 3 mM KCN in a glucose-free solution for 90 min. Analysis of NMDA receptor unitary events in patches excised from KCN-treated neurons showed an increased incidence of a small conductance channel 24 h after chemical ischemia. Whole-cell recordings of NMDA-induced currents 1 day after cyanide exposure revealed a significant increase in voltage-dependent extracellular Mg(2+) block compared with untreated neurons. The block reverted to control levels within 48 h. Both of these changes in the NMDA receptor could decrease the overall current flowing through the channel. Message levels for the NMDA receptor subunits NR1, NR2A, and NR2B were not different between the chemically challenged neurons and control cells, whereas NR2C message was barely detectable in either group. These results suggest that the alterations in NMDA receptor properties after KCN exposure may contribute to the molecular mechanisms that are activated in neurons to withstand lethal ischemic events in the brain after preconditioning.

Enhancement of NMDA receptor-mediated currents by light in rat neurones in vitro.

1. N-Methyl-D-aspartate (NMDA) receptor function can be modified by the action of several endogenous and exogenous modulatory processes. In the present study, we report that brief pulses of light potentiate NMDA, but not non-NMDA glutamatergic receptor-mediated whole-cell and single channel currents in rat cortical neurones in vitro. In addition, light also potentiated NMDA receptor-mediated whole-cell responses in isolated rat retinal neurones. 2. Potentiation of NMDA whole-cell currents in cortical neurones was readily observed during and following a brief (< 2 s) exposure of neurones to wavelengths of less than 324 nm of relatively bright light (0.09 microW microm-2). In addition, prolonged exposures (> 30 s) to visible wavelengths (> 380 nm) or to attenuated light (1-3 % transmittance of non-attenuated light) were also sufficient to enhance NMDA receptor-mediated responses. 3. The light-induced potentiation of NMDA receptor-mediated currents persisted for several minutes, slowly reversing to control levels with a time constant of approximately 5 min. A subsequent exposure to light could potentiate NMDA receptor-mediated currents for a second time. 4. Light did not alter the apparent affinity of the NMDA receptor for the co-agonists NMDA and glycine. Additionally, potentiation of the NMDA-induced currents was not mediated by a change in the pH sensitivity of the receptor. In excised outside-out membrane patches, the effects of light on NMDA-activated unitary currents were manifested as a twofold increase in channel open frequency without alterations in single channel amplitude or open time. 5. Our results suggest the presence of a light-sensitive moiety within the NMDA receptor, or in a closely associated structure, which affects channel properties. This previously unrecognized form of NMDA receptor modulation may provide a tool for understanding the conformational changes associated with its gating. In addition, it is possible that light may affect NMDA receptor-mediated function or dysfunction in the retina.

Novel role for the NMDA receptor redox modulatory site in the pathophysiology of seizures.

Redox-active compounds modulate NMDA receptors (NMDARs) such that reduction of NMDAR redox sites increases, and oxidation decreases, NMDAR-mediated activity. Because NMDARs contribute to the pathophysiology of seizures, redox-active compounds also may modulate seizure activity. We report that the oxidant 5, 5'-dithio-bis(2-nitrobenzoic acid) (DTNB) and the redox cofactor pyrroloquinoline quinone (PQQ) suppressed low Mg(2+)-induced hippocampal epileptiform activity in vitro. Additionally, in slices exposed to 4-7 microM bicuculline, DTNB and PQQ reversed the potentiation of evoked epileptiform responses by the reductants dithiothreitol and Tris(2-carboxyethyl)phosphine (TCEP). NMDA-evoked whole-cell currents in CA1 neurons in slices were increased by TCEP and subsequently decreased by DTNB or PQQ at the same concentrations that modulated epileptiform activity. However, DTNB and PQQ had little effect on baseline NMDA-evoked currents in control medium, and PQQ did not alter NMDAR-dependent long-term potentiation. In contrast, in slices returned to control medium after low Mg(2+)-induced ictal activity, DTNB significantly inhibited NMDAR-mediated currents, indicating endogenous reduction of NMDAR redox sites under this epileptogenic condition. These data suggested that PQQ and DTNB suppressed spontaneous ictal activity by reversing pathological NMDAR redox potentiation without inhibiting physiological NMDAR function. In vivo, PQQ decreased the duration of chemoconvulsant-induced seizures in rat pups with no effect on baseline behavior. Our results reveal endogenous potentiation of NMDAR function via mass reduction of redox sites as a novel mechanism that may enhance epileptogenesis and facilitate the transition to status epilepticus. The results further suggest that redox-active compounds may have therapeutic use by reversing NMDAR-mediated pathophysiology without blocking physiological NMDAR function.

Inhibition by intracellular Mg(2+) of recombinant N-methyl-D-aspartate receptors expressed in Chinese hamster ovary cells.

Intracellular Mg(2+) (Mg(i)(2+)) inhibits the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors in cultured cortical neurons. To examine the effects of Mg(i)(2+) on recombinant NMDA receptors composed of subunit combinations found in cortical neurons, we expressed heteromeric receptors composed of NR1/NR2A and of NR1/NR2B subunits in Chinese hamster ovary (CHO) cells. We recorded whole-cell currents from the recombinant receptors in the absence and presence of Mg(i)(2+). The voltage dependence of control (0 Mg(i)(2+)) NMDA-activated currents obtained from CHO cells transfected with NR1/NR2A and with NR1/NR2B receptors showed outward rectification, a property that has been observed previously in native cortical NMDA receptors. The magnitude and voltage dependence of inhibition by Mg(i)(2+) of NMDA-activated currents were similar in CHO cells transfected with NR1/NR2A receptors, CHO cells transfected with NR1/NR2B receptors, and in cultured neurons expressing native NMDA receptors. These observations suggest that Mg(i)(2+) has uniform effects on the native NMDA receptors expressed in cortical neurons. Furthermore, inhibition by Mg(i)(2+) must not depend on intracellular factors or post-translational receptor modifications that are specific to neurons. Finally, the results indicate that the previously observed differences between whole-cell and outside-out patch measurements of Mg(i)(2+) inhibition could not result from poor control of voltage or Mg(i)(2+) concentration in the dendrites of neurons. The most likely alternative explanation is that patch excision causes an alteration in NMDA receptors that results in more effective inhibition by Mg(i)(2+).

NMDA and glutamate evoke excitotoxicity at distinct cellular locations in rat cortical neurons in vitro.

The development of cortical neurons in vivo and in vitro is accompanied by alterations in NMDA receptor subunit expression and concomitant modifications in the pharmacological profile of NMDA-activated ionic currents. For example, we observed that with decreasing NR2B/NR2A subunit expression ratio, the block of NMDA receptor-mediated whole-cell responses by the NR2B-selective antagonist haloperidol was also decreased. In mature cultures (>22 d in vitro), however, NMDA responses obtained from excised nucleated macropatches, which comprised a large portion of the soma, remained strongly antagonized by haloperidol. These results suggest that in more mature neurons NR1/NR2B receptors appear to be preferentially expressed in the cell body. As predicted from the whole-cell recording pharmacological profile, NMDA-induced toxicity was largely unaffected by haloperidol in mature cultures. However, haloperidol effectively blocked glutamate toxicity in the same cultures, suggesting that the neurotoxic actions of this amino acid were mostly due to the activation of somatic NMDA receptors. In experiments in which the potency of glutamate toxicity was increased by the transport inhibitor l-trans-pyrrolidine-2,4-dicarboxylic acid, the neuroprotective effects of haloperidol were significantly diminished. This was likely because of the fact that glutamate, now toxic at much lower concentrations, was able to reach and activate dendritic receptors under these conditions. These results strongly argue that exogenous glutamate and NMDA normally induce excitotoxicity at distinct cellular locations in mature mixed neuronal cultures and that NR1/NR2B receptors remain an important component in the expression of glutamate, but not NMDA-induced excitotoxicity.

A critical role of the N-methyl-D-aspartate (NMDA) receptor subunit (NR) 2A in the expression of redox sensitivity of NR1/NR2A recombinant NMDA receptors.

In recombinant N-methyl-D-aspartate (NMDA) receptors, two redox modulatory sites are thought to exist, one formed by Cys744 and Cys798 on NMDA receptor subunit (NR) 1, and a second one, not yet localized, on NR2A. Reductants increase the open dwell-time and opening frequency of NR1/NR2A channels. In contrast, NR1/NR2B and NR1/NR2C channels exhibit changes only in opening frequency after redox treatments. Here, we evaluated whether the two redox sites act independently of each other, with the NR1 site affecting the opening frequency and the NR2A site altering open dwell-time. Unitary and whole-cell currents mediated by NMDA receptors composed of a cysteine-mutated NR1 subunit, NR1(C744A, C798A) were thus investigated. Dithiothreitol increased the open dwell-time and opening frequency of NR1(C744A, C798A)/NR2A receptors in a manner indistinguishable from that previously seen in wild-type channels. Marginal redox-induced changes in opening frequency of NR1(C744A, C798A)/NR2B receptors were noted. Redox modulation was completely abolished in NR1(C744A, C798A)/NR2C channels. Whole-cell recordings confirmed the single-channel results. Sulfhydryl reagents modulated NR1(C744A, C798A)/NR2A receptors identically to wild-type NR1/NR2A channels, whereas NR1(C744A, C798A)/NR2C receptors were insensitive to redox modulation. The oxidant 5,5'-dithio-bis-(2-nitrobenzoate) attenuated NR1(C744A, C798A)/NR2B receptor-mediated responses in a dithiothreitol-reversible manner. We conclude that cysteines 744 and 798 on the NR1 subunit are not involved in the redox modulation of NR1/NR2A receptors, but are crucial for the modulation of NR1/NR2C-containing receptors. This suggests that the NR2A subunit is necessary and sufficient for the expression of redox sensitivity in NR1/NR2A channels. The slight, but measurable residual redox sensitivity of the mutant NR1(C744A, C798A)/NR2B receptors suggests the existence of an additional redox-sensitive site on NR2B.

Lack of interaction between nitric oxide and the redox modulatory site of the NMDA receptor.

1. The inhibitory effects of nitric oxide (NO) on N-methyl-D-aspartate (NMDA) receptor function have been proposed to be mediated via the interaction of this gas with a redox-sensitive thiol moiety on the receptor. Here, we evaluated this suggested mechanism by examining the actions of various NO donors on native neuronal receptors as well as in wild-type and cysteine-mutated recombinant NMDA receptors expressed in Chinese hamster ovary (CHO) cells. 2. The NO donor N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydraxino)ethanamine (NOC-12; 100 microM) produced a rapid and readily reversible inhibition of whole-cell currents induced by NMDA (30 microM) in cultured cortical neurons. The inhibition was apparent at all holding potentials, though a more pronounced block was observed at negative voltages. The effects of NOC-12 disappeared when the donor was allowed to expire. A similar receptor block was observed with another NO-releasing agent, S-nitroso-N-acetylpenicillamine (SNAP; 1 mM). 3. The blocking effects of NO released by SNAP, 3-morpholinosydnonimine (SIN-1; 1 mM), and 3-[2-hydroxy-1-(1-methylethyl)-2-nitrosohydrazino]-1-propanamin e (NOC-5; 100 microM) on currents mediated by recombinant NRI/NR2B receptors were virtually indistinguishable from those observed on native receptors. Furthermore, mutating cysteines 744 and 798 of NR1, which constitute the principal redox modulatory site of the NR1/NR2B receptor configuration, did not affect the inhibition produced by NO. 4. The NR2A subunit may contribute its own redox-sensitive site. However, the effects of NO on NR1/NR2A receptors were very similar to those seen for all other receptor configurations evaluated. Hence, we conclude that NO does not exert its inhibition of NMDA-induced responses via a modification of any of the previously described redox-sensitive sites on the receptor.

rexB of bacteriophage lambda is an anti-cell death gene.

In Escherichia coli, programmed cell death is mediated through "addiction modules" consisting of two genes; the product of one gene is long-lived and toxic, whereas the product of the other is short-lived and antagonizes the toxic effect. Here we show that the product of lambdarexB, one of the few genes expressed in the lysogenic state of bacteriophage lambda, prevents cell death directed by each of two addiction modules, phd-doc of plasmid prophage P1 and the rel mazEF of E. coli, which is induced by the signal molecule guanosine 3',5'-bispyrophosphate (ppGpp) and thus by amino acid starvation. lambdaRexB inhibits the degradation of the antitoxic labile components Phd and MazE of these systems, which are substrates of ClpP proteases. We present a model for this anti-cell death effect of lambdaRexB through its action on the ClpP proteolytic subunit. We also propose that the lambdarex operon has an additional function to the well known phenomenon of exclusion of other phages; it can prevent the death of lysogenized cells under conditions of nutrient starvation. Thus, the rex operon may be considered as the "survival operon" of phage lambda.