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Methylene blue inhibits GABAA receptors by interaction with GABA binding site
Abstract
Methylene blue (MB) is commonly used in diagnostic procedures and is also used to treat various medical conditions. Neurological effects of MB have been reported in clinical observations and experimental studies. Thus the modulation of GABAA receptor function by MB was investigated. Whole-cell GABA-activated currents were recorded from HEK293 cells expressing various GABAA receptor subunit configurations. MB inhibition of GABA currents was apparent at 3 μM, and it had an IC50 of 31 μM in human α1β2γ2 receptors. The MB action was rapid and reversible. MB inhibition was not mediated via the picrotoxin site, as a mutation (T6'F of the β2 subunit) known to confer resistance to picrotoxin had no effect on MB-induced inhibition. Blockade of GABAA receptors by MB was demonstrated across a range of receptors expressing varying subunits, including those expressed at extrasynaptic sites. The sensitivity of α1β2 receptors to MB was similar to that observed in α1β2γ2 receptors, indicating that MB's action via the benzodiazepine or Zn(2+) site is unlikely. MB-induced inhibition of GABA response was competitive with respect to GABA. Furthermore, mutation of α1 F64 to A and β2 Y205 to F in the extracellular N-terminus, both residues which are known to comprise GABA binding pocket, remarkably diminished MB inhibition of GABA currents. These data suggest that MB inhibits GABAA receptor function by direct or allosteric interaction with the GABA binding site. Finally, in mouse hippocampal CA1 pyramidal neurons, MB inhibited GABA-activated currents as well as GABAergic IPSCs. We demonstrate that MB directly inhibits GABAA receptor function, which may underlie some of the effects of MB on the CNS.
... Facilitation of LTD without disrupting LTP may also be useful in conditions, such as AD, in which LTP is impaired only [52,53]. Preventing of LTP of spike by MB does not agree with a previous study in which MB inhibits GABA A receptor function by direct or allosteric interaction with the GABA-binding site [54], rather suggest a role of cGMP for modulation of neuronal excitability [55]. In that study, tadalafil increased, MB decreased plasma cGMP levels, showing opposite effect on convulsions. ...
Background
The present study examined whether inhibition of guanylate cyclase (GC) is associated with the plasticity-related microtubule-stabilizing protein tau phosphorylation in the dentate gyrus (DG) of hippocampal formation.Methods
To address this issue, methylene blue (MB 50 μM) or saline was infused into the DG starting from the induction of long-term potentiation (LTP) or depression (LTD) for 1 h. Then, protein phosphatase 1 alpha (PP1α), glycogen synthase kinase 3 beta (GSK3β), and tau total and phosphorylated protein levels were measured in these hippocampi using western blotting. LTP and LTD were induced by application of high- and low-frequency stimulation protocols (HFS and LFS), respectively. 5-min averages of the excitatory postsynaptic potential (EPSP) slopes and population spike amplitudes at the end of recording were averaged to measure the magnitude of LTP or LTD.ResultsLow-frequency stimulation protocols was unable to phosphorylate thr181 and thr231epitopes of tau, but possessed kinase activity similar to the HFS in phosphorylation of ser396 and ser416 epitopes. MB infusion during LTD induction attenuated LTD, prevented EPSP/spike dissociation and increased tau phosphorylation at ser396 and ser416 epitopes, without changing tau phosphorylation at thr181 and thr231 epitopes. Neither LTP nor LTP-related tau phosphorylation state was changed by MB infusion.Conclusion
Although MB can benefit to stabilize the balance between LTP and LTD, and to fix the increased spike wave discharges, it might trigger deregulation of tau phosphorylation, leading to the development of Alzheimer’s disease by a mechanism that goes awry during induction of LTD. Thereby detailed studies to reveal more precise evidence for the use of MB in this disease are needed.
... The pharmacological properties of these receptors have been extensively characterized in our previous publications (Huang et al., 2001;Huang et al., 2004;Huang et al., 2017). On the other hand, hα1β2 and hρ1 GABA A Rs, and hα1 GlyRs, were transiently transfected in HEK293 cells using PolyJet™ DNA In Vitro and Trans-IT 293 transfection reagents (Snell and Gonzales, 2015;Chen et al., 2017). Briefly, hρ1 (2 μg with 2 μg GFP), hα1β2 (1 μg α1, 2 μg β2) GABA A Rs, or hα1 GlyRs (1 μg) subunit cDNAs were added to cells growing on glass coverslips. ...
Traumatic brain injury (TBI) is a major cause of disability and death worldwide. The initial mechanical insult results in tissue and vascular disruption with hemorrhages and cellular necrosis that is followed by a dynamic secondary brain damage that presumably results in additional destruction of the brain. In order to minimize deleterious consequences of the secondary brain damage-such as inflammation, bleeding or reduced oxygen supply. The old concept of the -staircase approach- has been updated in recent years by most guidelines and should be followed as it is considered the only validated approach for the treatment of TBI. Besides, a variety of novel therapies have been proposed as neuroprotectants. The molecular mechanisms of each drug involved in inhibition of secondary brain injury can result as potential target for the early and late treatment of TBI. However, no specific recommendation is available on their use in clinical setting. The administration of both synthetic and natural compounds, which act on specific pathways involved in the destructive processes after TBI, even if usually employed for the treatment of other diseases, can show potential benefits. This review represents a massive effort towards current and novel therapies for TBI that have been investigated in both pre-clinical and clinical settings. This review aims to summarize the advancement in therapeutic strategies basing on specific and distinct -target of therapies-: brain edema, ICP control, neuronal activity and plasticity, anti-inflammatory and immunomodulatory effects, cerebral autoregulation, antioxidant properties, and future perspectives with the adoption of mesenchymal stromal cells.
The GABA A receptor (GABA A R) is the predominant inhibitory neurotransmitter in the mammalian central nervous system, and is the site of action of numerous therapeutic agents. This receptor is part of a superfamily of ligand-gated ion channels, which also include receptors for acetylcholine (nicotinic), glycine and serotonin (5-HT 3 subtype). The first GABA A R subunits were cloned in 1987; since then, at least 18 additional subunits have been cloned. Advances in molecular biology and structural analysis, combined with biochemical and electrophysiological techniques, have led to significant advances in our understanding of the physiological and pharmacological properties of this important class of neurotransmitter receptors. This review summarizes our current understanding of the structure and function of the GABA A R, as well as pharmacologic and physiologic mechanisms by which its activity is modulated. Abbreviations 5α3α-THPROG, 5α-pregnan-3α-ol-20-one; 5-HT 3 R, 5-hydroxytryptamine type 3
Methylene blue USP (MB) is a FDA-grandfathered drug used in clinics to treat methemoglobinemia, carbon monoxide poisoning and cyanide poisoning that has been shown to increase fMRI evoked blood oxygenation level dependent (BOLD) response in rodents. Low dose MB also has memory enhancing effect in rodents and humans. However, the neural correlates of the effects of MB in the human brain are unknown. We tested the hypothesis that a single low oral dose of MB modulates the functional connectivity of neural networks in healthy adults. Task-based and task-free fMRI were performed before and one hour after MB or placebo administration utilizing a randomized, double-blinded, placebo-controlled design. MB administration was associated with a reduction in cerebral blood flow in a task-related network during a visuomotor task, and with stronger resting-state functional connectivity in multiple regions linking perception and memory functions. These findings demonstrate for the first time that low-dose MB can modulate task-related and resting-state neural networks in the human brain. These neuroimaging findings support further investigations in healthy and disease populations.
Traumatic brain injury (TBI) leads to permanent neurological impairment, and methylene blue (MB) exerts central nervous system neuroprotective effects. However, only one previous study has investigated the effectiveness of MB in a controlled cortical impact injury model of TBI. In addition, the specific mechanisms underlying the effect of MB against TBI remain to be elucidated. Therefore, the present study investigated the neuroprotective effect of MB on TBI and the possible mechanisms involved. In a mouse model of TBI, the animals were randomly divided into sham, vehicle (normal saline) or MB groups. The treatment time‑points were 24 and 72 h (acute phase of TBI), and 14 days (chronic phase of TBI) post‑TBI. The brain water content (BWC), and levels of neuronal death, and autophagy were determined during the acute phase, and neurological deficit, injury volume and microglial activation were assessed at all time‑points. The injured hemisphere BWC was significantly increased 24 h post‑TBI, and this was attenuated following treatment with MB. There was a significantly higher number of surviving neurons in the MB group, compared with the Vehicle group at 24 and 72 h post‑TBI. In the acute phase, the MB‑treated animals exhibited significantly upregulated expression of Beclin 1 and increased LC3‑II to LC3‑I ratios, compared with the vehicle group, indicating an increased rate of autophagy. Neurological functional deficits, measured using the modified neurological severity score, were significantly lower in the acute phase in the MB‑treated animals and cerebral lesion volumes in the MB‑treated animals were significantly lower, compared with the other groups at all time‑points. Microglia were activated 24 h after TBI, peaked at 72 h and persisted until 14 days after TBI. Although the number of Iba‑1‑positive cells in the vehicle and MB groups 24 h post‑TBI were not significantly different, marked microglial inhibition was observed in the MB group 72 h and 14 days after ‑TBI. These results indicated that MB exerts a neuroprotective effect by increasing autophagy, decreasing brain edema and inhibiting microglial activation.
In Alzheimer's disease (AD), memory impairment is the most prominent feature that afflicts patients and their families. Although reactive astrocytes have been observed around amyloid plaques since the disease was first described, their role in memory impairment has been poorly understood. Here, we show that reactive astrocytes aberrantly and abundantly produce the inhibitory gliotransmitter GABA by monoamine oxidase-B (Maob) and abnormally release GABA through the bestrophin 1 channel. In the dentate gyrus of mouse models of AD, the released GABA reduces spike probability of granule cells by acting on presynaptic GABA receptors. Suppressing GABA production or release from reactive astrocytes fully restores the impaired spike probability, synaptic plasticity, and learning and memory in the mice. In the postmortem brain of individuals with AD, astrocytic GABA and MAOB are significantly upregulated. We propose that selective inhibition of astrocytic GABA synthesis or release may serve as an effective therapeutic strategy for treating memory impairment in AD.
Amyloid plaques and tau tangles are common pathological hallmarks for Alzheimer's disease (AD); however, reducing Aβ production failed to relieve the symptoms of AD patients. Here we report a high GABA (γ-aminobutyric acid) content in reactive astrocytes in the dentate gyrus (DG) of a mouse model for AD (5xFAD) that results in increased tonic inhibition and memory deficit. We also confirm in human AD patient brains that dentate astrocytes have a high GABA content, suggesting that high astrocytic GABA level may be a novel biomarker and a potential diagnostic tool for AD. The excessive GABA in 5xFAD astrocytes is released through an astrocyte-specific GABA transporter GAT3/4, and significantly enhances tonic GABA inhibition in dentate granule cells. Importantly, reducing tonic inhibition in 5xFAD mice rescues the impairment of long-term potentiation (LTP) and memory deficit. Thus, reducing tonic GABA inhibition in the DG may lead to a novel therapy for AD.
Methylene blue (MB), the first lead chemical structure of phenothiazine and other derivatives, is commonly used in diagnostic procedures and as a treatment for methemoglobinemia. We have previously demonstrated that MB could function as an alternative mitochondrial electron transfer carrier, enhance cellular oxygen consumption, and provide protection in vitro and in rodent models of Parkinson's disease and stroke. In the present study, we investigated the structure-activity relationships of MB in vitro using MB and six structurally related compounds. MB reduces mitochondrial superoxide production via alternative electron transfer that bypasses mitochondrial complexes I-III. MB mitigates reactive free radical production and provides neuroprotection in HT-22 cells against glutamate, IAA and rotenone toxicity. Distinctly, MB provides no protection against direct oxidative stress induced by glucose oxidase. Substitution of a side chain at MB's 10-nitrogen rendered a 1000-fold reduction of the protective potency against glutamate neurototoxicity. Compounds without side chains at positions 3 and 7, chlorophenothiazine and phenothiazine, have distinct redox potentials compared to MB and are incapable of enhancing mitochondrial electron transfer, while obtaining direct antioxidant actions against glutamate, IAA, and rotenone insults. Chlorophenothiazine exhibited direct antioxidant actions in mitochondria lysate assay compared to MB, which required reduction by NADH and mitochondria. MB increased complex IV expression and activity, while 2-chlorphenothiazine had no effect. Our study indicated that MB could attenuate superoxide production by functioning as an alternative mitochondrial electron transfer carrier and as a regenerable anti-oxidant in mitochondria.
By restoring mitochondrial function, methylene blue (MB) is an effective neuroprotectant in many neurological disorders (e.g., Parkinson's and Alzheimer's diseases). MB has also been proposed as a brain metabolic enhancer because of its action on mitochondrial cytochrome c oxidase. We used in vitro and in vivo approaches to determine how MB affects brain metabolism and hemodynamics. For in vitro, we evaluated the effect of MB on brain mitochondrial function, oxygen consumption, and glucose uptake. For in vivo, we applied neuroimaging and intravenous measurements to determine MB's effect on glucose uptake, cerebral blood flow (CBF), and cerebral metabolic rate of oxygen (CMRO(2)) under normoxic and hypoxic conditions in rats. MB significantly increases mitochondrial complex I-III activity in isolated mitochondria and enhances oxygen consumption and glucose uptake in HT-22 cells. Using positron emission tomography and magnetic resonance imaging (MRI), we observed significant increases in brain glucose uptake, CBF, and CMRO(2) under both normoxic and hypoxic conditions. Further, MRI revealed that MB dramatically increased CBF in the hippocampus and in the cingulate, motor, and frontoparietal cortices, areas of the brain affected by Alzheimer's and Parkinson's diseases. Our results suggest that MB can enhance brain metabolism and hemodynamics, and multimetric neuroimaging systems offer a noninvasive, nondestructive way to evaluate treatment efficacy.
Ionotropic GABA(A) receptors (GABA(A)Rs), which mediate inhibitory neurotransmission in the central nervous system, are implicated in the behavioral effects of alcohol and alcoholism. Site-directed mutagenesis studies support the presence of discrete molecular sites involved in alcohol enhancement and, more recently, inhibition of GABA(A)Rs. We used Xenopus laevis oocytes to investigate the 6' position in the second transmembrane region of GABA(A)Rs as a site influencing alcohol inhibition. We asked whether modification of the 6' position by substitution with larger residues or methanethiol labeling [using methyl methanethiosulfonate (MMTS)] of a substituted cysteine, reduced GABA action and/or blocked further inhibition by alcohols. Labeling of the 6' position in either α2 or β2 subunits reduced responses to GABA. In addition, methanol and ethanol potentiation increased after MMTS labeling or substitution with tryptophan or methionine, consistent with elimination of an inhibitory site for these alcohols. Specific alcohols, but not the anesthetic etomidate, competed with MMTS labeling at the 6' position. We verified a role for the 6' position in previously tested α2β2 as well as more physiologically relevant α2β2γ2s GABA(A)Rs. Finally, we built a novel molecular model based on the invertebrate glutamate-gated chloride channel receptor, a GABA(A)R homolog, revealing that the 6' position residue faces the channel pore, and modification of this residue alters volume and polarity of the pore-facing cavity in this region. These results indicate that the 6' positions in both α2 and β2 GABA(A)R subunits mediate inhibition by short-chain alcohols, which is consistent with the presence of multiple counteracting sites of action for alcohols on ligand-gated ion channels.
This paper provides the first review of the memory-enhancing and neuroprotective metabolic mechanisms of action of methylene blue in vivo. These mechanisms have important implications as a new neurobiological approach to improve normal memory and to treat memory impairment and neurodegeneration associated with mitochondrial dysfunction. Methylene blue's action is unique because its neurobiological effects are not determined by regular drug-receptor interactions or drug-response paradigms. Methylene blue shows a hormetic dose-response, with opposite effects at low and high doses. At low doses, methylene blue is an electron cycler in the mitochondrial electron transport chain, with unparalleled antioxidant and cell respiration-enhancing properties that affect the function of the nervous system in a versatile manner. A major role of the respiratory enzyme cytochrome oxidase on the memory-enhancing effects of methylene blue is supported by available data. The memory-enhancing effects have been associated with improvement of memory consolidation in a network-specific and use-dependent fashion. In addition, low doses of methylene blue have also been used for neuroprotection against mitochondrial dysfunction in humans and experimental models of disease. The unique auto-oxidizing property of methylene blue and its pleiotropic effects on a number of tissue oxidases explain its potent neuroprotective effects at low doses. The evidence reviewed supports a mechanistic role of low-dose methylene blue as a promising and safe intervention for improving memory and for the treatment of acute and chronic conditions characterized by increased oxidative stress, neurodegeneration and memory impairment.
The GABA type A receptor (GABA(A)R) is expressed ubiquitously throughout the brain and is a target for many therapeutic agents, including general anesthetics and benzodiazepines, which enhance receptor function by increasing the open probability (P(o)) of the ion channel. It is commonplace for in vitro studies of receptor pharmacological characteristics to use negative membrane holding potentials to mimic the resting potential of neurons and symmetrical chloride to eliminate Goldman rectification, which results in chloride flow in the opposite direction, compared with in vivo conditions. This critical difference is usually overlooked because the GABA(A)R has been reported to behave as an ohmic pore, but our results show that the current-voltage relationship is nonlinear with respect to P(o). Specifically, we found that currents were outwardly rectifying at low P(o) and linear at high P(o). We confirmed the correlation between P(o) and rectification with a partial agonist, piperidine-4-sulfonic acid, and a gating-impaired mutation, α1(L277A); both exhibited enhanced outward rectification. Furthermore, this correlation was independent of Goldman rectification and persisted under altered chloride gradient conditions, which suggests that rectification is linked to the direction of chloride flux. Finally, our results showed that the degree of potentiation by general anesthetics (etomidate, propofol, and isoflurane) was greater at negative membrane potentials. Traditional in vitro experiments thus overestimate the action of positive allosteric modulators of the GABA(A)R. Our results show that the direction of the driving force on the permeant ion, as well as P(o), must be considered together for a complete understanding of drug actions on ligand-gated ion channels.
The time course of synaptic currents is a crucial determinant of rapid signaling between neurons. Traditionally, the mechanisms underlying the shape of synaptic signals are classified as pre- and post-synaptic. Over the last two decades, an extensive body of evidence indicated that synaptic signals are critically shaped by the neurotransmitter time course which encompasses several phenomena including pre- and post-synaptic ones. The agonist transient depends on neurotransmitter release mechanisms, diffusion within the synaptic cleft, spill-over to the extra-synaptic space, uptake, and binding to post-synaptic receptors. Most estimates indicate that the neurotransmitter transient is very brief, lasting between one hundred up to several hundreds of microseconds, implying that post-synaptic activation is characterized by a high degree of non-equilibrium. Moreover, pharmacological studies provide evidence that the kinetics of agonist transient plays a crucial role in setting the susceptibility of synaptic currents to modulation by a variety of compounds of physiological or clinical relevance. More recently, the role of the neurotransmitter time course has been emphasized by studies carried out on brain slice models that revealed a striking, cell-dependent variability of synaptic agonist waveforms ranging from rapid pulses to slow volume transmission. In the present paper we review the advances on studies addressing the impact of synaptic neurotransmitter transient on kinetics and pharmacological modulation of synaptic currents at inhibitory synapses.
It has traditionally been thought that the pathological accumulation of tau in Alzheimer's disease and other tauopathies facilitates neurodegeneration, which in turn leads to cognitive impairment. However, recent evidence suggests that tau tangles are not the entity responsible for memory loss, rather it is an intermediate tau species that disrupts neuronal function. Thus, efforts to discover therapeutics for tauopathies emphasize soluble tau reductions as well as neuroprotection.
Here, we found that neuroprotection alone caused by methylene blue (MB), the parent compound of the anti-tau phenothiaziazine drug, Rember™, was insufficient to rescue cognition in a mouse model of the human tauopathy, progressive supranuclear palsy (PSP) and fronto-temporal dementia with parkinsonism linked to chromosome 17 (FTDP17): Only when levels of soluble tau protein were concomitantly reduced by a very high concentration of MB, was cognitive improvement observed. Thus, neurodegeneration can be decoupled from tau accumulation, but phenotypic improvement is only possible when soluble tau levels are also reduced.
Neuroprotection alone is not sufficient to rescue tau-induced memory loss in a transgenic mouse model. Development of neuroprotective agents is an area of intense investigation in the tauopathy drug discovery field. This may ultimately be an unsuccessful approach if soluble toxic tau intermediates are not also reduced. Thus, MB and related compounds, despite their pleiotropic nature, may be the proverbial "magic bullet" because they not only are neuroprotective, but are also able to facilitate soluble tau clearance. Moreover, this shows that neuroprotection is possible without reducing tau levels. This indicates that there is a definitive molecular link between tau and cell death cascades that can be disrupted.
Methylene blue (MB) has recently been reevaluated for malaria treatment. With the aim of excluding treatment failures due to low bioavailability, we have investigated the absolute bioavailability of MB given as an aqueous oral formulation and its interaction with chloroquine (CQ).
A phase I study in 16 healthy individuals was performed as a monocenter prospective open randomized intra-individual cross-over comparison of MB single doses [50 mg intravenous (i.v.), 500 mg orally, separated by a 1-week wash-out]. After a second week, the group was split for a randomized parallel group comparison of CQ 750 mg administered orally alone or combined with 500 mg MB orally.
Mean MB plasma area under the substrate concentration-time curve (AUC 0-infinity) was 7,639 +/- 3,384 ng/mL*h and 51,171 +/- 17,147 ng/mL*h after i.v. and oral administration, respectively (dosage 1:10), and 76,897 +/- 46,037 ng/mL*h after MB combined with CQ. The absolute bioavailability was 72.3 +/- 23.9%. Co-administration with CQ significantly increased MB plasma concentrations (p <or= 0.016); CQ kinetics remained unaffected.
The absolute bioavailability of MB is high. Co-administration of MB and CQ increases plasma, but not whole blood MB concentrations.
Ionotropic receptors for gamma-aminobutyric acid (GABA) are important to inhibitory neurotransmission in the mammalian retina, mediating GABAA and GABAC responses. In many species, these responses are blocked by the convulsant picrotoxinin (PTX), although the mechanism of block is not fully understood. In contrast, GABAC responses in the rat retina are extremely resistant to PTX. We hypothesized that this difference could be explained by molecular characterization of the receptors underlying the GABAC response. Here we report the cloning of two rat GABA receptor subunits, designated r rho 1 and r rho 2 after their previously identified human homologues. When coexpressed in Xenopus oocytes, r rho 1/r rho 2 heteromeric receptors mimicked PTX-resistant GABAC responses of the rat retina. PTX resistance is apparently conferred in native heteromeric receptors by r rho 2 subunits since homomeric r rho 1 receptors were sensitive to PTX; r rho 2 subunits alone were unable to form functional homomeric receptors. Site-directed mutagenesis confirmed that a single amino acid residue in the second membrane-spanning region (a methionine in r rho 2 in place of a threonine in r rho 1) is the predominant determinant of PTX resistance in the rat receptor. This study reveals not only the molecular mechanism underlying PTX blockade of GABA receptors but also the heteromeric nature of native receptors in the rat retina that underlie the PTX-resistant GABAC response.
The time course of EPSCs and IPSCs is at least partly determined by the concentration profile of neurotransmitter acting on postsynaptic receptors. Several recent reports have suggested that the peak synaptic cleft concentration of the inhibitory neurotransmitter GABA likely reaches at least 500 microM, a level that saturates the GABAA receptor. In the course of investigating the experimental anticonvulsant 3,3-diethyl-2-pyrrolidinone (diethyl-lactam), we have observed an important contribution to IPSC decay by subsaturating concentrations of GABA. Diethyl-lactam augments currents elicited by the exogenous application of subsaturating concentrations of GABA in voltage-clamped, cultured hippocampal neurons and significantly prolongs the decay of autaptic IPSCs and miniature IPSCs in our cultures. In addition, diethyl-lactam potentiates currents in excised outside-out membrane patches elicited by the prolonged application of low concentrations of GABA. However, when patches are exposed to 1-2 msec pulses of 1 mM GABA, diethyl-lactam does not alter current decay. Tiagabine, which blocks GABA reuptake, does not prolong IPSCs, so it is unlikely that uptake inhibition accounts for the enhancement of IPSCs. EPSCs and miniature IPSC frequency are unaffected by diethyl-lactam, again consistent with a postsynaptic site of action. We propose that during an IPSC, a substantial number of postsynaptic receptors must be exposed to subsaturating concentrations of GABA. A simplified model of GABAA receptor kinetics can account for the effects of diethyl-lactam on exogenous GABA and IPSCs if diethyl-lactam has its main effect on the monoliganded states of the GABAA receptor.
Pharmacological modulation of gamma-aminobutyric acid-A (GABAA) receptors can provide important information on the types of subunits composing these receptors. In recombinant studies, zinc more potently inhibits alphabeta subunits compared with the alphabetagamma combination, whereas modulation by nanomolar concentrations of the benzodiazepine type 1-selective agonist zolpidem is conferred by the alpha1betagamma2 subunit combination. We examined four properties of miniature inhibitory postsynaptic currents (mIPSCs) from identified necortical pyramidal cells in rat brain slices: decay time constant, peak amplitude, rate of rise, and interevent interval. Exposure to 50 microM zinc reduced the decay time constant, peak amplitude, and rate of rise with no effect on interevent interval. Zolpidem enhanced mIPSCs in a concentration-dependent manner. Both 20 and 100 nM zolpidem increased the decay time constants of mIPSCs. In some cells, both peak amplitude and rate of rise were also enhanced. All cells treated with zinc were also responsive to zolpidem. These results show that neocortical pyramidal cells have a population of GABAA receptors sensitive to both zinc and zolpidem.
GABAA receptors, along with the receptors for acetylcholine, glycine, and serotonin, are members of a ligand-gated ion channel superfamily (Ortells and Lunt, 1995). Because of the paucity of crystallographic information for these ligand-gated channels, little is known about the structure of their binding sites or how agonist binding is transduced into channel gating. We used the substituted cysteine accessibility method to obtain secondary structural information about the GABA binding site and to systematically identify residues that line its surface. Each residue from alpha1 Y59 to K70 was mutated to cysteine and expressed with wild-type beta2 subunits in Xenopus oocytes or HEK 293 cells. The sulfhydryl-specific reagent N-biotinylaminoethyl methanethiosulfonate (MTSEA-Biotin) was used to covalently modify the cysteine-substituted residues. Receptors with cysteines substituted at positions alpha1 T60, D62, F64, R66, and S68 reacted with MTSEA-Biotin, and alpha1 F64C, R66C, and S68C were protected from reaction by agonist. We conclude that alpha1 F64, R66, and S68 line part of the GABA binding site. The alternating pattern of accessibility of consecutive engineered cysteines to reaction with MTSEA-Biotin indicates that the region from alpha1 Y59 to S68 is a beta-strand.
We studied the effects of extracellular pH (pHo) on gamma-aminobutyric acid (GABA)-mediated Cl- current in rat hypothalamic neurons and recombinant type-A GABA (GABA(A)) receptors stably expressed in human embryonic kidney cells (HEK 293), using whole cell and outside-out patch-clamp recordings. In alpha3beta2gamma2s receptors, acidic pH decreased, whereas alkaline pH increased the response to GABA in a reversible and concentration-dependent manner. Acidification shifted the GABA concentration-response curve to the right, significantly increasing the EC50 for GABA without appreciably changing the slope or maximal current induced by GABA. We obtained similar effects of pH in alpha1beta2gamma2 receptors and in GABA-activated currents recorded from thin hypothalamic brain slices. In outside-out patches recorded from alpha3beta2gamma2 recombinant receptors, membrane patches were exposed to 5 microM GABA at control (7.3), acidic (6.4), or alkaline (8.4) pH. GABA activated main and subconductance states of 24 and 16 pS, respectively, in alpha3beta2gamma2 receptors. Alkaline pH(o) increased channel opening frequency and decreased the duration of the long closed state, resulting in an increase in open probability (from 0.0801 +/- 0.015 in pH 7.3 to 0.138 +/- 0.02 in pH 8.4). Exposure of the channels to acidic pH(o) had the opposite effects on open probability (decreased to 0.006 +/- 0.0001). Taken together, our results indicate that the function of GABA(A) receptors is modulated by extracellular pH. The proton effect is similar in recombinant and native receptors and is dependent on GABA concentration. In addition, the effect appears to be independent of the alpha-subunit isoform, and is due to the ability of H+ to alter the frequency of channel opening. Our findings indicate that GABAergic signaling in the CNS may be significantly altered during conditions that increase or decrease pH.
nAChRs are pentameric transmembrane proteins into the superfamily of ligand-gated ion channels that includes the 5HT3, glycine, GABAA, and GABAC receptors. Electron microscopy, affinity labeling, and mutagenesis experiments, together with secondary structure predictions and measurements, suggest an all-beta folding of the N-terminal extracellular domain, with the connecting loops contributing to the ACh binding pocket and to the subunit interfaces that mediate the allosteric transitions between conformational states. The ion channel consists of two distinct elements symmetrically organized along the fivefold axis of the molecule: a barrel of five M2 helices, and on the cytoplasmic side five loops contributing to the selectivity filter. The allosteric transitions of the protein underlying the physiological ACh-evoked activation and desensitization possibly involve rigid body motion of the extracellular domain of each subunit, linked to a global reorganization of the transmembrane domain responsible for channel gating.
Although gamma-aminobutyric acid type A receptor agonists and antagonists bind to a common site, they produce different conformational changes within the site because agonists cause channel opening and antagonists do not. We used the substituted cysteine accessibility method and two-electrode voltage clamping to identify residues within the binding pocket that are important for mediating these different actions. Each residue from alpha(1)T60 to alpha(1)K70 was mutated to cysteine and expressed with wild-type beta(2) subunits in Xenopus oocytes. Methanethiosulfonate reagents reacted with alpha(1)T60C, alpha(1)D62C, alpha(1)F64C, alpha(1)R66C, alpha(1)S68C, and alpha(1)K70C. gamma-Aminobutyric acid (GABA) slowed methanethiosulfonate modification of alpha(1)F64C, alpha(1)R66C, and alpha(1)S68C, whereas SR-95531 slowed modification of alpha(1)D62C, alpha(1)F64C, and alpha(1)R66C, demonstrating that different residues are important for mediating GABA and SR-95531 actions. In addition, methanethiosulfonate reaction rates were fastest for alpha(1)F64C and alpha(1)R66C, indicating that these residues are located in an open, aqueous environment lining the core of the binding pocket. Positively charged methanethiosulfonate reagents derivatized alpha(1)F64C and alpha(1)R66C significantly faster than a negatively charged reagent, suggesting that a negative subsite important for interacting with the ammonium group of GABA exists within the binding pocket. Pentobarbital activation of the receptor increased the rate of methanethiosulfonate modification of alpha(1)D62C and alpha(1)S68C, demonstrating that parts of the binding site undergo structural rearrangements during channel gating.
The GABA-binding site undergoes structural rearrangements during the transition from agonist binding to channel opening. To
define possible roles of the GABAAreceptor α1 subunit Pro174–Asp191 segment in these processes, we used the substituted cysteine accessibility method to characterize this region. Each residue
was individually mutated to cysteine, expressed with wild-type β2 subunits inXenopus laevis oocytes, and examined using two-electrode voltage clamp. Most mutations did not alter GABA EC50 values. The D183C mutation produced a 7-fold reduction in GABA sensitivity. There were no significant changes in theKI
values for the competitive antagonist, SR-95531. N-Biotinylaminoethyl methanethiosulfonate modified P174C-, R176C-, S177C-, V178C-, V180C-, A181C-, D183C-, R186C- and N188C-containing
receptors. The pattern of accessibility suggests that this protein segment is aqueous-exposed and adopts a random coil conformation.
Both GABA and SR-95531 slowed covalent modification of V178C, V180C, and D183C, indicating that these residues may line the
GABA-binding site. Further, pentobarbital-induced channel activation accelerated modification of V180C and A181C and slowed
the modification of R186C, suggesting that this region of the α1 subunit may act as a dynamic element during channel-gating transitions.
Purpose To investigate the sustained-attention and memory-enhancing neural correlates of the oral administration of methylene blue in the healthy human brain. Materials and Methods The institutional review board approved this prospective, HIPAA-compliant, randomized, double-blinded, placebo-controlled clinical trial, and all patients provided informed consent. Twenty-six subjects (age range, 22-62 years) were enrolled. Functional magnetic resonance (MR) imaging was performed with a psychomotor vigilance task (sustained attention) and delayed match-to-sample tasks (short-term memory) before and 1 hour after administration of low-dose methylene blue or a placebo. Cerebrovascular reactivity effects were also measured with the carbon dioxide challenge, in which a 2 × 2 repeated-measures analysis of variance was performed with a drug (methylene blue vs placebo) and time (before vs after administration of the drug) as factors to assess drug × time between group interactions. Multiple comparison correction was applied, with cluster-corrected P < .05 indicating a significant difference. Results Administration of methylene blue increased response in the bilateral insular cortex during a psychomotor vigilance task (Z = 2.9-3.4, P = .01-.008) and functional MR imaging response during a short-term memory task involving the prefrontal, parietal, and occipital cortex (Z = 2.9-4.2, P = .03-.0003). Methylene blue was also associated with a 7% increase in correct responses during memory retrieval (P = .01). Conclusion Low-dose methylene blue can increase functional MR imaging activity during sustained attention and short-term memory tasks and enhance memory retrieval. (©) RSNA, 2016 Online supplemental material is available for this article.
Pharmacological modulation of gamma-aminobutyric acid-A (GABA(A)) receptors can provide important information on the types of subunits composing these receptors. In recombinant studies, zinc more potently inhibits alpha beta subunits compared with the alpha beta gamma combination, whereas modulation by nanomolar concentrations of the benzodiazepine type 1-selective agonist zolpidem is conferred by the alpha 1 beta gamma 2 subunit combination. We examined four properties of miniature inhibitory postsynaptic currents (mIPSCs) from identified necortical pyramidal cells in rat brain slices: decay time constant, peak amplitude, rate of rise, and interevent interval. Exposure to 50 mu M zinc reduced the decay time constant, peak amplitude, and rate of rise with no effect on interevent interval. Zolpidem enhanced mIPSCs in a concentration-dependent manner. Both 20 and 100 nM zolpidem increased the decay time constants of mIPSCs. In some cells, both peak amplitude and rate of rise were also enhanced. An cells treated with zinc were also responsive to zolpidem. These results show that neocortical pyramidal cells have a population of GABA, receptors sensitive to both zinc and zolpidem.
Type-A γ-aminobutyric acid receptors (GABAARs) are the principal mediators of rapid inhibitory synaptic transmission in the human brain. A decline in GABAAR signalling triggers hyperactive neurological disorders such as insomnia, anxiety and epilepsy. Here we present the first three-dimensional structure of a GABAAR, the human β3 homopentamer, at 3 Å resolution. This structure reveals architectural elements unique to eukaryotic Cys-loop receptors, explains the mechanistic consequences of multiple human disease mutations and shows an unexpected structural role for a conserved N-linked glycan. The receptor was crystallized bound to a previously unknown agonist, benzamidine, opening a new avenue for the rational design of GABAAR modulators. The channel region forms a closed gate at the base of the pore, representative of a desensitized state. These results offer new insights into the signalling mechanisms of pentameric ligand-gated ion channels and enhance current understanding of GABAergic neurotransmission.
Changes in extracellular pH have a modulatory effect on GABAA receptor function. It has been reported that pH sensitivity of the GABA receptor is dependent on subunit composition and GABA concentration. Most of previous investigations focused on GABA-evoked currents, which only reflect the postsynaptic receptors. The physiological relevance of pH modulation of GABAergic neurotransmission is not fully elucidated. In the present studies, we examined the influence of extracellular pH on the GABAA receptor-mediated inhibitory neurotransmission in rat hypothalamic neurons. The inhibitory postsynaptic currents (IPSCs), tonic currents, and the GABA-evoked currents were recorded with whole-cell patch techniques on the hypothalamic slices from Sprague-Dawley rats at 15-26 postnatal days. The amplitude and frequency of spontaneous GABA IPSCs were significantly increased while the external pH was changed from 7.3 to 8.4. In the acidic pH (6.4), the spontaneous GABA IPSCs were reduced in amplitude and frequency. The pH induced changes in miniature GABA IPSCs (mIPSCs) similar to that in spontaneous IPSCs. The pH effect on the postsynaptic GABA receptors was assessed with exogenously applied varying concentrations of GABA. The tonic currents and the currents evoked by sub-saturating concentration of GABA ([GABA]) (10 M) were inhibited by acidic pH and potentiated by alkaline pH. In contrast, the currents evoked by saturating [GABA] (1 mM) were not affected by pH changes. We also investigated the influence of pH buffers and buffering capacity on pH sensitivity of GABAA receptors on human recombinant 122 GABAA receptors stably expressed in HEK 293 cells, The pH influence on GABAA receptors was similar in HEPES- and MES-buffered medium, and not dependent on protonated buffers, suggesting that the observed pH effect on GABA response is a specific consequence of changes in extracellular protons. Our data suggest that the hydrogen ions suppress the GABAergic neurotransmission, which is mediated by both presynaptic and postsynaptic mechanisms.
C57BL/6NNia and autoimmune NZB/B1NJ mice aged 12–14 months were tested for acquisition and retention of an active avoidance response following vehicle or flumazenil (40 mg/kg), a benzodiazepine antagonist. Acquisition and retention performance was improved in flumazenil-treated mice when compared with vehicle-treated mice, although the degree of improvement varied with the level of performance in vehicle-treated mice of each strain. The NZB/B1NJ mice, which generally performed more poorly than the C57BL/6NNia mice, showed the greater improvements following flumazenil. These results suggest that antagonism of benzodiazepine receptors leads to improved learning and/or memory performance in mice with spontaneous age-associated deficits.
Methylene blue was administered orally to seven normal human subjects at a dose of 10 mg. in capsule form. Total urinary recovery ranged from 53 to 97% of the dose, with an average of 74%. Of the material recovered, an average of 78% was excreted as Ieucomethylene blue (stabilized in some salt, complex, or combination form) and the remainder as methylene blue. Some excretion rate-time plots of both methylene blue and leucomethylene blue showed evidence of a circadian rhythm. In a male dog and a female dog, administered 15 mg./kg. methylene blue orally, no drug was detected in blood. The female dog was catheterized and urine was collected for 10 hr. postdosing; recovery was 2.4% of the dose. The female dog was also administered a 10-mg. dose of methylene blue orally, and urine was collected by catheter over 14 hr. Recovery was 3.8% of the dose. It was concluded that methylene blue is well absorbed in man and poorly absorbed in the dog after oral administration.
Methylene Blue (MB) is being investigated in clinical studies for its beneficial effects in the treatment of Alzheimer disease. However, its exact mechanisms of action have not been fully elucidated. The modulation of nicotinic acetylcholine receptors (nAChRs) has been suggested to play a role in the pathogenesis of various neurodegenerative diseases. Therefore, in the present study, the effect of MB on the function of the cloned α7 subunit of the human nAChR expressed in Xenopus oocytes was investigated using the two-electrode voltage-clamp technique. MB reversibly inhibited ACh (100 μM)-induced currents in a concentration-dependent manner with an IC50 value of 3.4 ± 0.3 µM. The effect of MB was not dependent on the membrane potential. MB did not affect the activity of endogenous Ca2+-dependent Cl- channels, since the inhibition by MB was unaltered in oocytes injected with the Ca2+ chelator 1,2-bis (o-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid and perfused with Ca2+-free bathing solution containing 2 mM Ba2+. MB decreased the maximal ACh-induced responses without significantly affecting ACh potency. Furthermore, specific binding of [125I] α-bungarotoxin, a radioligand selective for the α7 nAChR, was not altered by MB (10 µM), indicating that MB acts as a noncompetitive antagonist on α7 nAChRs. In hippocampal slices, whole-cell recordings from CA1 pyramidal neurons indicated that the increases in the frequency and amplitudes of the γ-aminobutyric acid-mediated spontaneous postsynaptic currents induced by bath application of 2 mM choline, a specific agonist for α7 nAChRs, were abolished after 10 min application of 3 μM MB. These results demonstrate that MB inhibits the function of human α7 nAChRs expressed in Xenopus oocytes and of α7 nAChR-mediated responses in rat hippocampal neurons.
Methylene Blue (MB), following its introduction to biology in the 19th century by Ehrlich, has found uses in various areas of medicine and biology. At present, MB is the first line of treatment in methemoglobinemias, is used frequently in the treatment of ifosfamide-induced encephalopathy, and is routinely employed as a diagnostic tool in surgical procedures. Furthermore, recent studies suggest that MB has beneficial effects in Alzheimer's disease and memory improvement. Although the modulation of the cGMP pathway is considered the most significant effect of MB, mediating its pharmacological actions, recent studies indicate that it has multiple cellular and molecular targets. In the majority of cases, biological effects and clinical applications of MB are dictated by its unique physicochemical properties including its planar structure, redox chemistry, ionic charges, and light spectrum characteristics. In this review article, these physicochemical features and the actions of MB on multiple cellular and molecular targets are discussed with regard to their relevance to the nervous system.
General anesthetics, once thought to exert their effects through non-specific membrane effects, have highly specific ion channel targets that can silence neuronal populations in the nervous system, thereby causing unconsciousness and immobility, characteristic of general anesthesia. Inhibitory GABA(A) receptors (GABA(A)Rs), particularly highly GABA-sensitive extrasynaptic receptor subtypes that give rise to sustained inhibitory currents, are uniquely sensitive to GABA(A)R-active anesthetics. A prominent population of extrasynaptic GABA(A)Rs is made up of alpha4, beta2 or beta3, and delta subunits. Considering the demonstrated importance of GABA receptor beta3 subunits for in vivo anesthetic effects of etomidate and propofol, we decided to investigate the effects of GABA anesthetics on "extrasynaptic" alpha4beta3delta and also binary alpha4beta3 receptors expressed in human embryonic kidney (HEK) cells. Consistent with previous work on similar receptor subtypes we show that maximal GABA currents through "extrasynaptic" alpha4beta3delta receptors, receptors defined by sensitivity to EtOH (30mM) and the beta-carboline beta-CCE (1microM), are enhanced by the GABA(A)R-active anesthetics etomidate, propofol, and the neurosteroid anesthetic THDOC. Furthermore, we show that receptors formed by alpha4beta3 subunits alone also show high GABA sensitivity and that saturating GABA responses of alpha4beta3 receptors are increased to the same extent by etomidate, propofol, and THDOC as are alpha4beta3delta receptors. Therefore, both alpha4beta3 and alpha4beta3delta receptors show low GABA efficacy, and GABA is also a partial agonist on certain binary alphabeta receptor subtypes. Increasing GABA efficacy on alpha4/6beta3delta and alpha4beta3 receptors is likely to make an important contribution to the anesthetic effects of etomidate, propofol and the neurosteroid THDOC.
The effect of flumazenil, a benzodiazepine-receptor antagonist, was evaluated in a spatial-reference memory procedure in a water maze. Flumazenil (1.0, 3.0, and 10.0 mg/kg, ip) did not modify acquisition of spatial information. Retention was similar between control and experimental rats 24 h after the training phase, as all groups showed bias to the target quadrant in a free swim trial. However, 10 days later, only flumazenil-injected rats (3.0 mg/kg) showed bias to the target quadrant. Flumazenil did not affect retrieval of spatial information in a group of well-trained rats. These results suggest that a benzodiazepine-receptor mediated endogenous mechanism is activated during learning of spatial tasks and that its blockade facilitates retention of spatial information.
Long-lasting potentiation (LLP) of synaptic transmission in the CAI region of the hippocampal slice preparation has been examined. The effects of reduced postsynaptic inhibition given by application of gamma-aminobutyric acid (GABA) antagonists (mainly picrotoxin) on the generation of LLP were investigated. It was first demonstrated that picrotoxin had little effect on excitatory synaptic transmission itself as judged by the rising phase of the field EPSP. Moreover, there were largely no actions on short-lasting synaptic effects such as paired pulse facilitation and frequency potentiation. On the other hand, following drug application, much fewer afferent volleys were needed to generate a given amount of LLP. Long-lasting potentiation could be produced by trains containing as few as 2-5 impulses, trains that normally give rise to only short-lasting effects. There was no apparent difference in the maximal amount of LLP that could be produced for a given input, suggesting that the GABA antagonists do not operate by enhancing the capacity for LLP production but by facilitating its induction. As in normal solution, the LLP in the presence of the drugs was confined to the tetanized pathway. Tetanization in the treated slices was associated with enhanced somatic firing as well as an increase of the negative extracellular potential recorded in the dendritic layer. It is proposed that part of this increased negativity represents current through synaptically opened N-methyl-D-aspartate (NMDA) receptor channels. Furthermore, it is suggested that the facilitated induction of LLP in the presence of GABA antagonists is related to a facilitated activation of these NMDA receptor channels which is secondary to the higher levels of dendritic depolarization attained during tetanization under conditions of reduced postsynaptic inhibition.
A long-lasting potentiation of synaptic transmission can be induced in the hippocampus by high-frequency stimulation of the afferent fibres1,2. This process has been regarded as a possible physiological substrate for long-term memory. From theoretical considerations on memory function it has been proposed that synaptic strengthening occurs as a result of simultaneous pre- and postsynaptic activation3, a principle which has been used in several theoretical models (for references see refs 4, 5). It thus seems important to study factors which control hippocampal long-lasting potentiation and, in particular, whether these factors act pre- or postsynaptically. A presynaptic control of long-lasting potentiation was established in experiments where two separate inputs to the same population of CA1 pyramidal cells were used6,7. Studies on the granule cell population of area dentata however, revealed, a heterosynaptic modulation of the generation of potentiation, suggesting a possible postsynaptic control also8,9. We have now studied long-lasting potentiation in the hippocampal slice preparation (CA1 region) in conditions where postsynaptic inhibition was reduced by application of gamma-aminobutyric acid (GABA) receptor blockers. We found that in addition to their direct effect on inhibition, GABA blockers dramatically facilitated the induction of long-lasting potentiation. The results indicate involvement of postsynaptic mechanisms in the generation of long-lasting potentiation.
The predominant inhibitory neurotransmitter of the brain, GABA (gamma-aminobutyric acid), activates chloride-selective ion pores integral to the receptor complex. Subunits comprising the presumed hetero-pentameric GABA channel have been cloned, but little information is available on the domains important for activation. Rat wild-type or mutated alpha 1-, beta 2- and gamma 2-subunits (designated alpha, beta and gamma) were coexpressed in Xenopus oocytes and examined electrophysiologically. We report here the identification of two separate and homologous domains of the beta-subunit, each of which contributes a tyrosine and threonine essential for activation by GABA. Conservative substitution of each of these four amino acids dramatically decreased GABA channel sensitivity to activation by GABA and the GABA agonist muscimol. These substitutions, however, did not impair activation by the barbiturate pentobarbital, indicating these two different classes of agonists activate GABA channels through distinct mechanisms. We also present evidence suggesting that the two identified domains of the beta-subunit contribute a major component of the GABA receptor.
In the preceding paper it was found that infusions of chlordiazepoxide (CDP) into the medial septal region, but not several other regions possessing a high density of benzodiazepine receptors, impaired spatial learning, but not cue learning or swim speed, in the Morris water maze. The present investigation sought to further characterize the neuropharmacological profile of this effect. Initially, it was reconfirmed that systemically administered CDP impaired spatial learning, but not cue learning or swim speed, in the water maze. Additionally, it was found that systemically administered scopolamine, a muscarinic antagonist, impaired both spatial and cue learning, but not swim speed, confirming the detrimental effects of cholinergic hypofunction on maze learning. In new rats, a dose-response assessment revealed that 60 and 30 nmol, but not 10 nmol, CDP infused into the medial septum impaired spatial learning, but not cue learning or swim speed. On the following day, rats from each dose group, now undrugged, acquired a reversed platform location at control levels, suggesting that the previously observed impairment was not due to a neurotoxic effect. Additionally, it was found that systemically administered flumazenil (10 mg/kg) blocked the spatial learning deficit produced by the 60 nmol dose of CDP infused into the medial septum. However, intraseptal infusions of flumazenil (10, 20, or 30 nmol) failed to attenuate the spatial learning deficit produced by systemically administered CDP. Finally, systemically administered tetrahydroaminoacridine (1 or 3 mg/kg), an acetylcholinesterase inhibitor, failed to attenuate the spatial learning deficit produced by intraseptal CDP (60 nmol). Together these results implicate benzodiazepine receptors in the medial septum in the amnesic actions of CDP but suggest that additional sites also mediate this action. The present results fail to support the idea that the spatial learning deficit produced by intraseptal infusions of CDP is due to a suppression of septo-hippocampal cholinergic activity and it is proposed that CDP impairs spatial learning by exacerbating hippocampal inhibition by inhibiting septo-hippocampal GABAergic projection neurons.
Site-directed mutagenesis and the two-electrode voltage-clamp techniques were used to evaluate the site of action of picrotoxin on rat alpha 1 beta 2 gamma 2 containing GABAA receptors expressed in Xenopus oocytes. Following a sequence comparison between GABAA subunits and the picrotoxin-insensitive glycine beta subunit, the following mutations were made near the center of the M2 region of the alpha 1, beta 2, and gamma 2 GABAA subunits: alpha 1(T261F/T267A), beta 2(T246F/T252A), and gamma 2(T271F/T277A). Wild type (alpha 1 beta 2 gamma 2) GABA channels had an IC50 for picrotoxin of 1.3 +/- O.3 microM. In contrast, alpha 1 beta 2 gamma 2 channels that contained any one of the mutated alpha 1, beta 2, or gamma 2 subunits produced currents that were insensitive to picrotoxin (0.1-100 microM). The single mutant beta 2(T246F), in combination with wild type alpha and gamma subunits, also conferred picrotoxin-insensitivity. In contrast, combinations containing beta 2(T252A) were blocked by picrotoxin with an IC50 of 1.4 +/- 0.4 microM. In some instances, the EC50 to GABA was slightly altered in the mutant receptors; but no change was observed in EC50 or potentiation by the allosteric modulator, alprazolam. The data in this study suggest that picrotoxin's site of action is within the channel pore; however the mechanism by which picrotoxin blocks current remains unknown.
The GABAA receptor belongs, along with the nicotinic acetylcholine receptor, the glycine receptor and the 5-HT3 receptor, to a family of homologous transmitter-gated ion channels mediating fast synaptic transmission. Many classes of drug interact with the GABAA receptor, which is the major inhibitory ion channel in the mammalian brain. Among these drugs are the allosteric modulators acting at the benzodiazepine binding site. In this article, Erwin Sigel and Andreas Buhr discuss recent studies that have identified amino acid residues that are thought to form the binding pocket for these compounds. These residues are probably located at subunit interfaces of the protein pentamer and at least some of them are homologous to residues implicated in channel agonist binding. This implies pseudosymmetry of channel agonist and channel modulatory sites, which may be, as recent data indicate, a general principle realized in other pseudosymmetric protein complexes.
Until 1987, when the first GABA-A receptor subunit cDNAs were cloned and sequenced, it was thought that there were perhaps two subtypes of receptor in the brain. These were defined by the fact that benzodiazepines, which act through the GABA-A receptor, had two binding sites with different affinities. By 1991 it was known that the GABA-A receptor family existed as a family of subunits which coassembled to form a family of receptor subtypes in the brain. More recently, two additional GABA-A receptor subunits have been identified, epsilon and theta. The identification of these new members of the gene family, and the characterisation of the receptor subtypes into which they are incorporated, is reviewed.
We have assessed the interaction of picrotoxin and a putative picrotoxin-site ligand [4-dimethyl-3-t-butylcarboxyl-4,5-dihydro (1, 5-a) quinoxaline] (U-93631) with varying configurations of recombinant GABA(A) receptors, using the whole-cell patch clamp technique. In alpha2beta2gamma2 GABA(A) receptors, coapplication of picrotoxin with GABA had minimal effects on initial GABA-activated Cl(-) current amplitude, and subsequently enhanced decay of GABA-activated Cl(-) currents. The half-maximal inhibitory concentration (IC(50)) for picrotoxin in alpha2beta2gamma2 receptors was 10.3+/-1.6 microM. The alpha subunit isoform did not affect picrotoxin-induced inhibition, as IC(50) values for alpha3beta2gamma2 (5.1+/-0.7 microM) and alpha6beta2gamma2 receptors (7.2+/-0.4 microM) were comparable to those obtained in alpha2beta2gamma2 receptors. Interestingly, in receptors lacking an alpha subunit (beta2gamma2 configuration), picrotoxin had a markedly lower IC(50) (0.5+/-0.05 microM) compared to alpha-containing receptors. The inhibitory profile was generally similar for the presumed picrotoxin-site ligand U-93631, i.e., IC(50) values were comparable in all alphabetagamma-containing receptors, but the IC(50) in beta2gamma2 receptors was greater than 10-fold lower. In addition, a modest but significant initial stimulation of GABA-activated current by U-93631 was observed in alpha2beta2gamma2 and beta2gamma2 receptors. A mutation in the second transmembrane domain, shown previously to abolish picrotoxin sensitivity, also greatly attenuated sensitivity to U-93631. Moreover, incubation of receptors with excess U-93631 hindered picrotoxin's ability to gain access to its binding site; both results indicate that U-93631 interacts at the picrotoxin site of the receptor. Our results indicate the presence of an alpha subunit hinders the ability of picrotoxin to block the GABA(A) receptor, and thus provides additional insight into the site of action of picrotoxin. In addition, we have shown that domains important for the actions of picrotoxin also affect U-93631. Thus, this compound should prove to be a useful ligand for analysis of the convulsant site of this receptor.
We tested the ability of the central nervous system convulsant pentylenetetrazole (PTZ) to inhibit gamma-aminobutyric acid (GABA)-gated current in receptors expressing a mutation that rendered them resistant to picrotoxin. Consistent with previous reports, receptors expressing beta2(T246F), along with alpha3 and gamma2 subunits, resulted in a greatly diminished sensitivity to picrotoxin. Sensitivity to PTZ was completely abolished in the mutant receptor, confirming the hypothesis that PTZ acts at the picrotoxin site. Quite unexpected, however, was our finding that PTZ elicited marked stimulation (up to 400% of control) in the mutated receptors. This stimulatory effect was not mediated via an interaction with the benzodiazepine site, as preincubation with the benzodiazepine antagonist flumazenil did not block the PTZ-induced stimulation. Our results reveal the existence of a novel stimulatory domain of PTZ in GABA(A) receptors.
To determine the pharmacokinetics and organ distribution of i.v. and oral methylene blue, which is used to prevent ifosfamide-induced encephalopathy in oncology.
The concentration of methylene blue in whole blood was measured using high-performance liquid chromatography in seven volunteers after i.v. and oral administration of 100 mg methylene blue with and without mesna. The distribution of methylene blue in different tissues was measured in rats after intraduodenal and i.v. application.
The time course of methylene blue in whole blood after i.v. administration showed a multiphasic time course with an estimated terminal half-life of 5.25 h. Following oral administration, the area under the concentration-time curve was much lower (9 nmol/min/ml vs 137 nmol/min/ml). Co-administration of mesna, which could influence distribution by ion-pairing, did not alter the pharmacokinetics. The urinary excretion of methylene blue and its leucoform was only moderately higher after i.v. administration (18% vs 28% dose). Intraduodenal administration to rats resulted in higher concentrations in intestinal wall and liver but lower concentrations in whole blood and brain than i.v. methylene blue.
Differences in organ distribution of methylene blue are mainly responsible for the different pharmacokinetics after oral and i.v. administration. If methylene blue acts in the liver, where ifosfamide is primarily activated to reactive and potentially toxic metabolites, oral and i.v. methylene blue are likely to be equally effective. However, if the site of action is the central nervous system, i.v. methylene blue which results in much higher concentrations in brain seems preferable.
The cellular and subcellular distribution of four GABA(A) receptor subtypes, identified by the presence of the alpha1, alpha2, alpha3, or alpha5 subunit, was investigated immunocytochemically in dissociated cultures of hippocampal neurons. We addressed the questions whether (1) cell-type specific expression, (2) axonal/somatodendritic targeting, and (3) synaptic/extrasynaptic clustering of GABA(A) receptor subtypes was retained in vitro. For comparison, the in vivo distribution pattern was assessed in sections from adult rat brain. The differential expression of GABA(A) receptor subunits allowed to identify five morphologically distinct cell types in culture: the alpha1 subunit was observed in glutamic acid decarboxylase-positive interneurons, the alpha2 and alpha5 subunits marked pyramidal-like cells, and the alpha3 subunit labeled three additional cell types, including presumptive hilar cells. All subunits were found in the somatodendritic compartment. In addition, appropriate axonal targeting was evidenced by the intense alpha2, and sometimes alpha3 subunit labeling of axon-initial segments (AIS) of pyramidal cells and hilar cells, respectively. Accordingly, both receptor subtypes were targeted to AIS in vivo, as well. Synaptic receptors were identified by colocalization with gephyrin, a postsynaptic clustering protein, and apposition to presynaptic terminals labeled with synapsin I. In vitro and in vivo, alpha1- and alpha2-receptor subtypes formed numerous synaptic clusters, alpha3-GABA(A) receptors were located either synaptically or extrasynaptically depending on the cell type, whereas alpha5-GABA(A) receptors were extrasynaptic. We conclude that receptor targeting to broad subcellular locations does not require specific GABAergic innervation patterns, which are disturbed in vitro, but depends on protein-protein interactions in the postsynaptic cell that are both subunit- and neuron-specific.
Neurotransmitter receptor systems have been the focus of intensive pharmacological research for more than 20 years for basic and applied scientific reasons, but only recently has there been a better understanding of their key features. One of these systems includes the type A receptor for the gamma-aminobutyric acid (GABA), which forms an integral anion channel from a pentameric subunit assembly and mediates most of the fast inhibitory neurotransmission in the adult vertebrate central nervous system. Up to now, depending on the definition, 16-19 mammalian subunits have been cloned and localized on different genes. Their assembly into proteins in a poorly defined stoichiometry forms the basis of functional and pharmacological GABA(A) receptor diversity, i.e. the receptor subtypes. The latter has been well documented in autoradiographic studies using ligands that label some of the receptors' various binding sites, corroborated by recombinant expression studies using the same tools. Significantly less heterogeneity has been found at the physiological level in native receptors, where the subunit combinations have been difficult to dissect. This review focuses on the characteristics, use and usefulness of various ligands and their binding sites to probe GABA(A) receptor properties and to gain insight into the biological function from fish to man and into evolutionary conserved GABA(A) receptor heterogeneity. We also summarize the properties of the novel mouse models created for the study of various brain functions and review the state-of-the-art imaging of brain GABA(A) receptors in various human neuropsychiatric conditions. The data indicate that the present ligands are only partly satisfactory tools and further ligands with subtype-selective properties are needed for imaging purposes and for confirming the behavioral and functional results of the studies presently carried out in gene-targeted mice with other species, including man.
gamma-Aminobutyric acid(A) (GABA(A)) receptors mediate most of the fast inhibitory neurotransmission in the CNS. They represent a major site of action for clinically relevant drugs, such as benzodiazepines and ethanol, and endogenous modulators, including neuroactive steroids. Alterations in GABA(A) receptor expression and function are thought to contribute to prevalent neurological and psychiatric diseases. Molecular cloning and immunochemical characterization of GABA(A) receptor subunits revealed a multiplicity of receptor subtypes with specific functional and pharmacological properties. A major tenet of these studies is that GABA(A) receptor heterogeneity represents a key factor for fine-tuning of inhibitory transmission under physiological and pathophysiological conditions. The aim of this review is to highlight recent findings on the regulation of GABA(A) receptor expression and function, focusing on the mechanisms of sorting, targeting, and synaptic clustering of GABA(A) receptor subtypes and their associated proteins, on trafficking of cell-surface receptors as a means of regulating synaptic (and extrasynaptic) transmission on a short-time basis, on the role of endogenous neurosteroids for GABA(A) receptor plasticity, and on alterations of GABA(A) receptor expression and localization in major neurological disorders. Altogether, the findings presented in this review underscore the necessity of considering GABA(A) receptor-mediated neurotransmission as a dynamic and highly flexible process controlled by multiple mechanisms operating at the molecular, cellular, and systemic level. Furthermore, the selected topics highlight the relevance of concepts derived from experimental studies for understanding GABA(A) receptor alterations in disease states and for designing improved therapeutic strategies based on subtype-selective drugs.
The nicotinic acetylcholine receptor controls electrical signalling between nerve and muscle cells by opening and closing a gated, membrane-spanning pore. Here we present an atomic model of the closed pore, obtained by electron microscopy of crystalline postsynaptic membranes. The pore is shaped by an inner ring of 5 alpha-helices, which curve radially to create a tapering path for the ions, and an outer ring of 15 alpha-helices, which coil around each other and shield the inner ring from the lipids. The gate is a constricting hydrophobic girdle at the middle of the lipid bilayer, formed by weak interactions between neighbouring inner helices. When acetylcholine enters the ligand-binding domain, it triggers rotations of the protein chains on opposite sides of the entrance to the pore. These rotations are communicated through the inner helices, and open the pore by breaking the girdle apart.