GABA-gated Cl Research Articles

Neuroactive steroids modulate GABAA receptors in peptidergic nerve terminals.

Neuroactive steroid modulation of GABAA receptors was investigated in the peptidergic nerve terminals of the posterior pituitary using patch clamp techniques. In common with GABAA receptors in cell bodies, the nerve terminal GABAA receptor was potentiated by the synthetic steroid alphaxalone and by physiological concentrations of the progesterone metabolite allopregnanolone. Both of these agents enhanced Cl- currents elicited by GABA. Estradiol-17 beta had a weak inhibitory effect on GABA responses of nerve terminals, but only at high concentrations. The potentiating action was manifest as an increase in the probability of channel opening, with no effect on the rate of desensitization of the GABAA receptor. Neuroactive steroids enhanced GABA-gated Cl- channel activity in cell-free membrane patches, thus demonstrating a membrane delimited response. These results indicated that with regard to allosteric modulation by neuroactive steroids, the nerve terminal GABAA receptor is similar to the GABAA receptors of nerve cell bodies and endocrine cells. Neuroactive steroids are thus capable of altering the chemosensitivity of nerve terminal membranes by enhancing GABA inhibition at this location. The neuroactive steroid sensitivity of nerve terminal GABAA receptors provides a pathway by which gonadal steroid derivatives could regulate peptide secretion from neurosecretory neurons. Such a pathway could participate in the coordination of neuropeptide secretion during complex neuroendocrine functions. With specific regard to the neurohypophysis, neuroactive steroid-induced changes in the sensitivity of nerve terminal GABAA receptors could play a role in the initiation of oxytocin secretion during the transition between pregnancy and parturition.

Allosteric modulation of [ 35S]TBPS-binding in the cerebral cortex of the rat during postnatal development

The ontogenesis of the GABA-gated Cl − channel was investigated in the cerebral cortex of the rat by monitoring the binding parameters of [ 35 S]t- butylbicyclophosphorothionate ([ 35S]TBPS) at intervals after birth (1–90 days). To investigate the influence of the developmental changes in the content of GABA on [ 35S]TBPS-binding, the assays were carried out in unwashed membranes, in which the concentration of GABA was dependent on its content in vivo, and in repeatedly washed membranes in the presence of defined concentrations of exogenous GABA. At birth, the density ( B max) of [ 35S]TBPS-binding sites in unwashed membranes was similar to that found in well-washed membranes. However, in unwashed membranes, the number of [ 35S]TBPS-binding sites increased by two-fold within 10 days after birth whereas in washed membranes it increased by four-fold during the same period. The higher density of [ 35S]TBPS-binding sites in washed membranes as compared with the unwashed counterparts persisted throughout development. In unwashed membranes, the apparent K d for [ 35S]TBPS-binding increased with age whereas in washed membranes the affinity of [ 35S]TBPS for its binding sites remained constant throughout development. The binding of [ 35S]TBPS to the GABA-gated Cl − channel is allosterically modulated by drugs acting on different sites of the GABA A receptor complex. Thus, GABA and diazepam decrease [ 35S]TBPS-binding whereas the GABA A receptor antagonist, bicuculline, and the inverse agonist for benzodiazepine receptors, 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylic acid methyl ester, increase it. The magnitude of the effects of positive and negative modulators of the GABA A receptor on [ 35S]TBPS-binding did not change significantly from birth through adulthood, provided the concentration of GABA in the incubation medium was kept constant at 3 μM. These results reflect the early development of the functional interactions between the different components of the GABA A receptor.

Identification of the GABAA receptor alpha 3 subunit in the IMR-32 neuroblastoma cell line.

A previous report has described the presence of t-[35S]-butylbicyclophosphorothionate binding sites and GABA-gated Cl- flux in the human neuroblastoma IMR-32 cell line. We now report the further characterisation of this binding site and, even more important, the identification of the GABAA receptor alpha 3 sub-unit expressed in these cells. Cell membranes prepared from IMR-32 cells were screened by immunoblotting for reactivity with various GABAA receptor alpha subunit-specific antibodies. Of these, only anti-Cys alpha 3 454-467 antibodies recognised specifically and in a dose-dependent manner an immunoreactive band. This M(r) 58,000 immunoreactive species and the N-deglycosylated derivatives were both coincident with the respective homologues found in both calf cerebral cortex membranes and purified receptor preparations. This is the first report of the identification of a specific GABAA receptor subunit expressed in a human cell line, and it therefore provides a convenient model for the study of receptor structure and regulation.

GABA-mediated positive autofeedback loop controls horizontal cell kinetics in tiger salamander retina

Horizontal cells (HCs) appear to release, and also to be sensitive to, GABA. The external GABA concentration is increased with depolarization of the HC membrane via an electrogenic GABA transporter. This extracellular GABA opens a GABAA-gated Cl- channel in the HC membrane. Since the equilibrium potential for Cl- (ECl) is near -20 mV, GABA released by the HC further depolarizes the HC. The GABA transporter and the GABAA receptor thus constitute a positive feedback loop in the HC membrane. This loop can slow down the kinetics of the light responses in HCs. GABA released via the GABA transporter can affect the GABAA receptor, probably because diffusion from the extracellular space is normally restricted by the intact retinal structure. We therefore used retinal slices rather than isolated HCs to maintain that structure. To measure single-cell currents in the slice, HCs were electrically uncoupled by including cAMP in the patch pipette. Under these conditions, bath application of GABA elicited two currents: (1) a picrotoxin-blocked current reversing near ECl, probably mediated by GABAA receptors, and (2) a picrotoxin-insensitive current similar to that elicited by cis-4-hydroxynipecotic acid (NIP) shown in other preparations to act at the GABA transporter. Under physiological conditions, the HC membrane potential is controlled by two major conductances, the GABAA-gated Cl- conductance described above, and the glutamate-gated conductance modulated by photoreceptor input. A bright light flash eliminates the glutamate-gated conductance, leaving only the GABA-gated Cl- conductance to control the membrane. With the Cl- conductance a significant fraction of the overall membrane conductance the GABAergic positive feedback loop can decrease the response kinetics. We increased the ambient extracellular GABA concentration by adding 50 microM GABA to the extracellular medium. This increased the ambient Cl- conductance, but the transporter still modulated Cl- conductance because responses to light stimuli were significantly slowed. The slowdown of the HC response could be reversed by interrupting the loop in two ways: (1) picrotoxin opened the loop and speeded the responses by uncoupling the GABA concentration from control of the membrane conductance, and (2) NIP opened the loop by uncoupling the extracellular GABA concentration from the Cl- conductance and therefore the membrane potential.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular mechanisms of the partial allosteric modulatory effects of bretazenil at gamma-aminobutyric acid type A receptor.

In central nervous system gamma-aminobutyric acid (GABA) inhibits neuronal activity by acting on GABA type A (GABAA) receptors. These heterooligomeric integral membrane proteins include a GABA-gated Cl- channel and various allosteric modulatory sites where endogenous modulators and anxiolytic drugs act to regulate GABA action. In vivo, various anxiolytic drugs exhibit a wide range of variability in their modulatory efficacy and potency of GABA action. For instance, bretazenil modulatory efficacy is much lower than that of diazepam. Such low efficacy could be due either to a preferential modulation of specific GABAA receptor subtypes or to a low modulatory efficacy at every GABAA receptor subtype. To address these questions we studied drug-induced modifications of GABA-activated Cl- currents in native GABAA receptors of cortical neurons in primary cultures and in recombinant GABAA receptors transiently expressed in transformed human embryonic kidney cells (293) after transfection with cDNAs encoding different molecular forms of alpha, beta, and gamma subunits of GABAA receptors. In cortical neurons the efficacy of bretazenil was lower than that of diazepam, whereas the potency of the two drugs was similar. In cells transfected with gamma 2 subunits and various molecular forms of alpha and beta subunits bretazenil efficacy was always lower than that of diazepam. However, in cells transfected with gamma 1 or gamma 3 subunits and various forms of alpha and beta subunits the efficacy of both diazepam and bretazenil was lower and always of similar magnitude. When bretazenil and diazepam were applied together to GABAA receptors including a gamma 2 subunit, the action of diazepam was curtailed in a manner related to the dose of bretazenil.

Influence of extracellular and intracellular pH on GABA-gated chloride conductance in crayfish muscle fibres

The effect of intracellular and extracellular pH on GABA-gated Cl − conductance was studied using H +-selective microelectrodes and a three-microelectrode voltage clamp in crayfish leg opener muscle fibres in bicarbonate-free solutions. Experimental variation of intracellular pH in the range 6.4–8.0 did not affect the GABA-gated conductance. In contrast to this, the GABA-gated conductance was sensitive to changes in external pH. Raising the external pH from 7.4 to 8.4 decreased the GABA-gated peak conductance observed immediately following application of GABA by 30%, and a change from 7.4 to 6.4 produced an increase of 26%. The effect of extracellular pH on the GABA-gated peak conductance was approximately linear in the pH range 6.4–8.9. A slight decrease in the slope of the pH-conductance relationship was evident in the pH range 5.4–6.4. The desensitization of the GABA-gated conductance was also affected by external pH. At pH 6.9 the conductance produced by 1 mM GABA showed a desensitization of about 15%, and at pH 8.9 this value was 34%. Raising the external pH in the presence of GABA decreased the GABA-gated peak conductance and increased the fractional desensitization, while lowering the external pH produced opposite effects, and was capable of repriming the conductance from a desensitized state to the non-desensitized state. The above results show that the GABA-gated conductance is sensitive to changes in external pH in the physiological range, and suggest that pH-dependent changes in the postsynaptic efficacy of GABA-mediated inhibition may contribute to H + modulation of neuronal excitability.

Quinolones and fenbufen interact with GABAA receptor in dissociated hippocampal cells of rat

1. Interaction of quinolone antibiotics and the anti-inflammatory agent fenbufen with the gamma-aminobutyric acid-A (GABAA) receptor-chloride channel complex in pyramidal neurons freshly dissociated from the hippocampal CA1 region of the rats was investigated in whole-cell mode, using the patch-clamp technique under voltage-clamp conditions. 2. Quinolones in clinical doses had no effects on the GABA-gated Cl- current (ICl) but slightly suppressed the response at concentrations greater than 10(-5) M. A metabolite of fenbufen, 4-biphenylacetic acid (BPA), also had little effect on the GABA response at therapeutic concentrations. 3. Coadministration of one of quinolones and BPA suppressed the GABA-gated ICl with increase in each of them in a concentration-dependent manner, and there was a parallel shift of the concentration-response curve for GABA to the right but with no effect on the maximum response, thereby indicating a competitive antagonism. The inhibitory potency of antibiotics in combination with BPA was in the order of norfloxacin much greater than enoxacin greater than cyprofloxacin greater than pipemidic acid much greater than ofloxacin greater than cinoxacin = piromidic acid = nalidixic acid = 0. 4. Norfloxacin and BPA, administered simultaneously, also strongly suppressed pentobarbital sodium (PB)-gated ICl, but they did not act on benzodiazepine (BZP) receptors. 5. Both GABA- and PB-induced ICls reversed at the Cl- equilibrium potential (ECl). In the presence of BPA, the quinolone-induced inhibition of GABA-gated ICls showed no voltage dependence. 6. It was concluded that, in the presence of an anti-inflammatory agent, the quinolone antibiotics decrease the affinity of GABAA receptors, the result being induction of epileptogenic neurotoxicities.

Site-specific acylation of GABA-gated Cl − channels: effects on 36Cl − uptake

Radioligand binding studies indicate that p-isothiocyanato-t-butylbicycloorthobenzoate (p-NCS-TBOB) specifically acylates GABA-gated chloride channels. Preincubation of synaptoneurosomes with p-NCS-TBOB followed by washing resulted in a concentration dependent (63–500 nM) inhibition of both muscimol-stimulated chloride uptake and [355]t-butylbicyclophosphorothionate (TBPS) binding. The extent of acylation (assessed by inhibition of [ 35S]TBPS binding) was highly correlated (r = 0.89; p < 0.0001) with the inhibition of muscimol-stimulated Cl − uptake. Neither basal Cl − uptake nor [ 3H]muscimol binding to GABA A receptors were affected by p-NCS-TBOB. Preincubation with the nonacylating ‘cage’ convulsant t-butylbicycloorthobenzoate (500 nM) followed by washing had no effect on either muscimol-stimulated Cl − uptake or [ 35S]TBPS binding. These findings indicate that p-NCS-TBOB interferes with the efficacy of muscimol promoted channel openings, but does not affect the recognition qualities of GABA A receptors. p-NCS-TBOB should prove useful in electrophysiological and biochemical studies examining the properties of GABA-gated Cl − channels.

Expression and regulation of GABA receptors in the brain

GABA receptors are classified into two receptor subtypes: GABAA and GABAB receptors. The GABAA receptor, one of the ionotropic type receptors, is formed by various subunits (alpha, beta, gamma and delta subunits) and constitutes the GABA-gated Cl- channel. The different combinations of these subunits are known to produce functionally heterogeneous GABAA receptors both pharmacologically and physiologically. On the other hand, GABAB receptor is known to be metabotropic type which is negatively coupled with adenylate cyclase and inositol phosphate turnover systems via inhibitory GTP binding protein.

Involvement of phosphoinositide metabolism in GABA-induced catecholamine release from cultured bovine adrenal chromaffin cells

The effects of GABA on catecholamine release and phosphoinositide metabolism were studied in cultured bovine adrenal chromaffin cells. GABA and muscimol, a specific agonist for the GABA A receptor, each evoked a gradual secretion of catecholamines from the cells in the presence of ouabain, an inhibitor of Na +,K +-ATPase. This release was inhibited by bicuculline, a specific antagonist for the GABA A receptor, or by picrotoxin, a blocker of GABA-gated Cl − channels, and was potentiated by diazepam or pentobarbital. GABA or muscimol induced a concentration-dependent formation of inositol phosphates. This accumulation of inositol phosphates was also inhibited by bicuculline, picrotoxin or removal of extracellular Ca 2+, and also potentiated by diazepam and pentobarbital. Nicardipine suppressed GABA-induced catecholamine release in the presence of ouabain and accumulation of inositol phosphates, while verapamil, diltiazem, and ω-conotoxin failed to inhibit these responses to GABA. The phosphoinositide-specific phospholipase C inhibitor neomycin also inhibited both GABA-induced accumulation of inositol phosphates and stimulation of catecholamine release in the presence of ouabain. These results taken together indicate that GABA evoked catecholamine release from the chromaffin cells in the presence of ouabain by stimulation of phosphoinositide metabolism in a Ca 2+-sensitive manner via activation of GABA A receptor-coupled Cl − channels.

Effect of temperature on the inhibition of the GABA-gated response by intracellular calcium

The effects of the voltage-dependent Ca 2+inward current (I Ca) on γ-aminobutyric acid (GABA)-induced Cl − current (I Cl) in isolated sensory neurons were examined at different temperatures and with different concentrations of external Ca 2+ using the ‘concentration-clamp’ technique. The total amount of inhibition of GABA-induced I Cl by a preceding I Ca increased in a hyperbolic manner with increasing Ca 2+ influx. The time course of recovery of the GABA response after inhibition by Ca 2+influx followed a single exponential and was facilitated by warming but slowed dramatically by a slight cooling from 20 to 15 °C in spite of a decrease in Ca 2+ influx. It is discussed that the energy-dependent, temperature-sensitive ionic pump and exchange systems at the surface membrane and intracellular organelles regulate the cytoplasmic free Ca 2+, thus explaining the quantitative effects of Ca 2+ influx and temperature on the inhibition of the GABA-gated Cl − channel.

GABA-stimulated chloride uptake and enhancement by diazepam in synaptoneurosomes from rat brain during prenatal and postnatal development

The present study was undertaken to evaluate the ability of diazepam (DZ) to elicit a response in the brains of developing animals. γ-Aminobutyric acid (GABA)-stimulated 36Cl − uptake in the presence and absence of DZ was measured in synaptoneurosomal preparations from whole brain of fetal rats at 20 and 21 days gestation and from cerebral cortex of rats at 7, 14, 21, 28 and 60–90 days postnatal age. The ability of GABA to stimulate 36Cl − uptake in a concentration-dependent manner was evident from gestational day 20. The EC 50 for GABA stimulation but not the maximum stimulation increased significantly from day 20 to day 21 of gestation. Postnatally, only moderate changes in the EC 50 were evident, but the maximum 36Cl − transported increased significantly from 7 to 14 days and remained stable thereafter. DZ enhanced GABA-mediated 36Cl − influx from 20 days gestation, and this enhancement was seen as a decrease in the EC 50 for GABA stimulation. However, DZ also significantly increased maximum GABA-stimulated 36Cl − transport in synaptoneurosomal preparations at 21 days gestation and at 7 days postnatal age, a response to DZ not seen at older ages. DZ had a less robust effect on GABA-mediated 36Cl − transport at 28 days postnatal age than at any other age, a result consistent with functional observations of decreased responsivity to DZ in late juvenile, early adolescent rats. The benzodiazepine antagonist Ro 15-1788 prevented the effect of DA on GABA-stimulated 36Cl − uptake in tissue from 21-day fetuses and from 60- to 90-day-old adults. These studies demonstrate the early appearance of functional GABA-gated Cl − channels as well as the early developmental appearance of a functional link between the DZ recognition site and GABA-mediated Cl − influx, thus providing evidence that long-term consequences of prenatal exposure to DZ could result from a direct action of the drug on the fetal brain.

Augmentation of GABA-induced chloride current in frog sensory neurons by diazepam.

The effect of diazepam (DZP) on the GABA-induced macroscopic and microscopic Cl- current was investigated in isolated frog sensory neurons using both 'concentration-clamp' and patch-clamp techniques. At concentration range between 10(-9) and 10(-4) M, DZP itself evoked no response but potentiated time- and dose-dependently the subthreshold GABA responses, though at high DZP concentrations beyond 10(-5) M the potentiation ratio decreased. The potentiation effect was long-lasting and desensitized slowly over the course of several 10 minutes after washing-out of DZP. DZP potentiated GABA response without shifting the GABA reversal potential. The entire GABA dose-response curve was shifted in a parallel manner to the left by adding DZP without changing cooperatively: the Hill slope was 2.0. The potentiation of GABA response by DZP did not depend on either inward or outward direction of the Cl- current but slightly on the membrane potential. The time constants of activation of desensitization of GABA-gated Cl- current consisted of fast and slow components, respectively. The slow components were concentration-dependent, and significantly changed in the presence of DZP, while DZP had little effects on fast components. In the 'inside-out' configuration, the addition of DZP activated GABA-receptor ionophore complexes under subthreshold without changing the single Cl- channel conductance. It is concluded that DZP may act at a site to modulate GABA binding, in which DZP increases GABA binding affinity and also affects the kinetics of GABA-gated Cl- channels, indicating that DZP has dual action on the GABA-induced responses.

A comparison of the effects of midazolam and pentobarbital on the dose-response of GABA-gated Cl − influx in rat brain microsacs

γ-Aminobutyric acid (GABA)-gated Cl − influx was studied in a rat brain ‘microsac’ preparation. Midazolam (a benzodiazepine), at 0.1–10 μM, increased GABA potency (maximum 2-fold) without affecting its efficacy or exerting GABA-mimetic effect. Pentobarbital (10–500 μM) increased GABA potency (maximum 12-fold) and efficacy (maximum 40%). Pentobarbital exhibited GABA-mimetic effect at concentrations above 200 μM. The differential effects of midazolam and pentobarbital in modulating GABA-mediated responses may account for some of the differences between these two classes of drugs.

Ca2+-mediated suppression of the GABA-response through modulation of chloride channel gating in frog sensory neurones

A suppressant effect of intracellular free Ca2+ on the gamma-aminobutyric acid (GABA)-induced chloride inward current (ICI(GABA)) was studied in isolated frog sensory neurones under whole cell voltage clamp. Voltage-dependent Ca2+ influx elicited during the steady state of ICI(GABA)-induced a fast, slowly recovering current relaxation in the outward direction, the amplitude of which was dependent on total Ca2+ influx. This suppressant effect showed specificity for different divalent cations, suggesting action at a specific intracellular effector. Single channel recording revealed a Ca2+-dependent decrease in the duration of the open time of the GABA-gated Cl- channel without change in single channel conductance.

Inhibition of Na +-dependent GABA uptake in isolated frog sensory nerve cell bodies by extra- and intracellular Li +

The effect of Li + on the Na +-dependent γ-aminobutyric acid (GABA) uptake was investigated by examining the effects of external and internal Li + ([Li] o and [Li] i) on GABA-gated Cl − current ( I Cl) in the frog dorsal root ganglion cells using the suction pipette technique which allows the internal perfusion under voltage-clamp condition. The suppression of GABA responses in the presence of external Na + ([Na] o) was larger at lower than at higher GABA concentration. Replacement of [Na] o with Li + completely removed the Na + suppression, and GABA dose-response curve in Li + external solution agreed well with that in Na +-free (Tris +) external solution. Either increasing [Li] i or internal Na + ([Na] i) at a constant [Na] o equally reduced the Na +-dependent suppression of GABA-gated I Cl. The results indicate that both the [Li] o and [Li] i remove in different manner the [Na] o-dependent suppression of GABA-induced I Cl: i.e. the [Li] o acts as a blocker of Na +-GABA co-transport mechanism while the increase of [Li] i decreases the Na + electrochemical potential gradient across the soma membrane as well as [Na] i does.

Different actions of intracellular free calcium on resting and GABA-gated chloride conductances

The effects of voltage-dependent Ca 2+ current (I Ca) on resting and γ-aminobutyric acid (GABA)-gated Cl - conductance were studied in isolated frog sensory neurons. The amplitude of GABA-gated Cl - current (I Cl) was greatly suppressed by a preceding I Ca. The GABA dose—response curve was shifted to the right without changing the maximum response by increasing [Ca 2+] i. An I Ca-activated I Cl was observed as an inward tail current on the ‘off’ phase of I Ca. This tail current was easily saturated by increasing the amount of Ca 2+ influx.

Characterization of barbiturate-stimulated chloride efflux from rat brain synaptoneurosomes

Membrane chloride (Cl-) permeability was studied in a novel subcellular brain preparation, the synaptoneurosome. Using a radioactive tracer exchange technique, Cl- transport was determined by measuring 36Cl- efflux from rat cerebral cortical synaptoneurosomes. Barbiturates increased 36Cl- efflux in a dose-dependent manner with the following relative order of potency: 5-(1,3-dimethylbutyl)-5-ethyl barbituric acid ((-)-DMBB) greater than pentobarbital greater than secobarbital greater than (+)-DMBB greater than hexobarbital greater than amobarbital greater than mephobarbital. Phenobarbital and barbital were virtually inactive. A good correlation was observed between the potencies of these barbiturates in stimulating 36Cl- efflux and their anesthetic potencies in mice (r = 0.90, p less than 0.01) and their abilities to enhance [3H] diazepam binding to brain membranes (r = 0.77, p less than 0.05). The effect of pentobarbital in enhancing 36Cl- efflux was reversed by the gamma-aminobutyric acid (GABA) antagonists picrotoxin and bicuculline. Picrotoxin and bicuculline both decreased 36Cl- efflux in the absence of pentobarbital, suggesting the presence of endogenous GABA. Incubation of synaptoneurosomes with 4,4'-di-isothiocyano- or dinitro-2,2'-disulfonic acid stilbene, inhibitors of anion transport, also decreased both basal and pentobarbital-induced 36Cl- efflux. Pentobarbital (500 microM) was most effective in inducing 36Cl- efflux in the cerebellum, hippocampus, and cortex (23.7, 23.6, and 22.5%, respectively), and was less effective in stimulating 36Cl- efflux in the striatum (15.1%) and pons-medulla (6.2%). The relative efficacy of pentobarbital in enhancing 36Cl- efflux among these various brain regions was highly correlated (r = 0.96, p 0.01) with the relative densities of [35S]-t-butylbicyclophosphorothionate-binding sites, a measure of GABA-gated Cl- channel density.(ABSTRACT TRUNCATED AT 250 WORDS)