Functional characterization of the Betaine/gamma-aminobutyric acid transporter BGT-1 expressed in Xenopus oocytes.

Betaine is an osmolyte accumulated in cells during osmotic cell shrinkage. The canine transporter mediating cellular accumulation of the osmolyte betaine and the neurotransmitter gamma-aminobutyric acid (BGT-1) was expressed in Xenopus oocytes and analyzed by two-electrode voltage clamp and tracer flux studies. Exposure of oocytes expressing BGT-1 to betaine or gamma-aminobutyric acid (GABA) depolarized the cell membrane in the current clamp mode and induced an inward current under voltage clamp conditions. At 1 mM substrate the induced currents decreased in the following order: betaine = GABA > diaminobutyric acid = beta-alanine > proline = quinidine > dimethylglycine > glycine > sarcosine. Both the Vmax and Km of GABA- and betaine-induced currents were voltage-dependent, and GABA- and betaine-induced currents and radioactive tracer uptake were strictly Na+-dependent but only partially dependent on the presence of Cl-. The apparent affinity of GABA decreased with decreasing Na+ concentrations. The Km of Na+ also depended on the GABA and Cl- concentration. A decrease of the Cl- concentration reduced the apparent affinity for Na+ and GABA, and a decrease of the Na+ concentration reduced the apparent affinity for Cl- and GABA. A comparison of 22Na+-, 36Cl--, and 14C-labeled GABA and 14C-labeled betaine fluxes and GABA- and betaine-induced currents yielded a coupling ratio of Na+/Cl-/organic substrate of 3:1:1 or 3:2:1. Based on the data, a transport model of ordered binding is proposed in which GABA binds first, Na+ second, and Cl- third. In conclusion, BGT-1 displays significant functional differences from the other members of the GABA transporter family.     

Interactions ofγ-aminobutyric acid (GABA), pentobarbital,and homopantothenic acid (HOPA) on internally perfused frog sensory neurons

Augmentatory actions among Cl- currents (ICl) induced by gamma-aminobutyric acid (GABA), pentobarbital (PB), and homopantothenic acid (HOPA) were investigated in isolated frog sensory neurons after suppression of Na+, K+, and Ca2+ currents using a suction pipette technique which combines internal perfusion with voltage clamp. GABA-sensitive neurons responded to both PB and HOPA, and the responses behaved as a simple Cl- electrode and reversed at the Cl- equilibrium potential (ECl). The dose-response curve for GABA-induced Cl- conductance was sigmoidal with the GABA concentration producing a half-maximum response (4.2 X 10(-5) M). Both GABA and HOPA dose-response curves shifted to the left in the presence of PB, though the facilitatory action of PB on GABA- and HOPA-induced ICl was more effective in the former. There was a significant facilitatory interaction between GABA- and HOPA-induced ICl. It is concluded that HOPA affects the GABA-GABA or PB-PB receptor interactions.     

Analysis of relations between NMDA receptors and GABA release at olfactory bulb reciprocal synapses

In the mammalian olfactory bulb, signal processing is mediated by synaptic interactions between dendrites. Glutamate released from mitral cell dendrites excites dendritic spines of granule cells, which in turn release GABA back onto the mitral cell dendrites, forming a reciprocal synaptic pair. This feedback synaptic circuit was shown to be mediated predominantly by NMDA receptors. We further utilized caged Ca2+ compounds to obtain insight into the mechanism that couples NMDA receptor activation to GABA release. Feedback inhibition elicited by photo-release of caged Ca2+ in mitral cell secondary dendrites persisted when voltage-gated Ca2+ channels were blocked by cadmium (Cd2+) and nickel (Ni2+). These results indicate that Ca2+ influx through NMDA receptors can directly trigger presynaptic GABA release for local dendrodendritic feedback inhibition.     

Gamma-aminobutyric acid (GABA) and pentobarbital induce different conformational rearrangements in the GABA A receptor alpha1 and beta2 pre-M1 regions

Gamma-aminobutyric acid (GABA) binding to GABA(A) receptors (GABA(A)Rs) triggers conformational movements in the alpha(1) and beta(2) pre-M1 regions that are associated with channel gating. At high concentrations, the barbiturate pentobarbital opens GABA(A)R channels with similar conductances as GABA, suggesting that their open state structures are alike. Little, however, is known about the structural rearrangements induced by barbiturates. Here, we examined whether pentobarbital activation triggers movements in the GABA(A)R pre-M1 regions. Alpha(1)beta(2) GABA(A)Rs containing cysteine substitutions in the pre-M1 alpha(1) (K219C, K221C) and beta(2) (K213C, K215C) subunits were expressed in Xenopus oocytes and analyzed using two-electrode voltage clamp. The cysteine substitutions had little to no effect on GABA and pentobarbital EC(50) values. Tethering chemically diverse thiol-reactive methanethiosulfonate reagents onto alpha(1)K219C and alpha(1)K221C affected GABA- and pentobarbital-activated currents differently, suggesting that the pre-M1 structural elements important for GABA and pentobarbital current activation are distinct. Moreover, pentobarbital altered the rates of cysteine modification by methanethiosulfonate reagents differently than GABA. For alpha(1)K221Cbeta(2) receptors, pentobarbital decreased the rate of cysteine modification whereas GABA had no effect. For alpha(1)beta(2)K215C receptors, pentobarbital had no effect whereas GABA increased the modification rate. The competitive GABA antagonist SR-95531 and a low, non-activating concentration of pentobarbital did not alter their modification rates, suggesting that the GABA- and pentobarbital-mediated changes in rates reflect gating movements. Overall, the data indicate that the pre-M1 region is involved in both GABA- and pentobarbital-mediated gating transitions. Pentobarbital, however, triggers different movements in this region than GABA, suggesting their activation mechanisms differ.     

Characteristics of chloride-dependent effects of acetylcholine and neutral aminoacids on neurons of the snail (Achatina)

The response was investigated of neurons composing the cerebral ganglia inAchatina fulica (the Giant African snail) to application of acetylcholine (ACh), gamma-aminobutyric acid (GABA), and glycine (Gly). Chloride-dependent currents induced by these transmitters in 1 1/2-month old siblings were inhibited by dibutyryl-cAMP and strychnine. Inhibition of ACh response produced 10^–8 M GABA was mimicked by application of dibutyryl-cAMP and isobutylmethylxanthine. Complete cross-desensitization was characteristic of both GABA- and Bly-induced response, but this effect did not occur when ACh and GABA (or Gly) were applied. A conclusion was reached on the basis of the pharmacological relationship between GABA- and Gly-induced response that these amino acids act on a single receptor — channel complex in the neurons of infant snails, whereas ACh-, GABA-, and Gly-induced chloride currents were not so related in cells of 4 year-oldAchatina.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 1, pp. 35–43, January–February, 1991.     

A patch-clamp study of GABA- and strychnine-sensitive glycine-activated currents in post-natal tissue-cultured hippocampal neurons.

GABA- and strychnine-sensitive glycine-activated currents in post-natal tissue-cultured hippocampal neurons were studied by using patch-clamp techniques. Current-voltage relations for both agonists in symmetrical Cl- solutions showed outward rectification. Strychnine-sensitive glycine-activated currents have not been studied in detail before in post-natal tissue-cultured hippocampal neurons. Partial desensitization of whole-cell currents was observed in symmetrical Cl- solutions during bath application of GABA- and glycine. In F-/Cl- solutions, both agonists gave a PF/Pcl value of about 0.06. The reversal potentials in mixtures of Cl- and SO4(2-) solutions were close to the equilibrium potentials of Cl- in the presence of both GABA and glycine. Single channels in inside-out excised patches with 2 mumol l-1 GABA and 5 mumol l-1 glycine in the pipette showed outward current rectification. The dose-response curves for GABA and glycine gave Kd values of 52 and 61 mumol l-1, respectively, and Hill coefficients close to 2. GABA and glycine binding were competitively blocked by their respective antagonists (bicuculline and strychnine). The similarities between GABA- and glycine-activated currents and the response in a combination of saturating concentrations of both GABA and glycine implied that the two agonists activated comparable numbers of anatomically distinct channels with very similar permeation properties.     

The colocalization of neurotransmitters in the presynaptic boutons of inhibitory synapses in the lamprey spinal cord]

Using the electron microscopy immunocytochemistry, the GABA and glycine immunoreactivity was studied in presynaptic axon terminals of the spinal cord central gray in the lamprey Lampetra fluviatilis. All immunopositive presynaptic terminals contacting motoneurones or non-identified post-synaptic profiles were divided into only GABA- (44%), only glycine-immunopositive terminals (26%), and both GABA- and glycine-containing terminals (30%). The glycine-immunopositive axon terminals contained flattened synaptic vesicles. Large dense core vesicles were co-localised with conventional synaptic vesicles in 74% of GABA-containing presynaptic terminals.     

Molecular and pharmacological properties of GABA-ρ subunits from white perch retina

Five γ-aminobutyric acid (GABA)-ρ subunits were cloned from a white perch retinal cDNA library and expressed in Xenopus oocytes. The deduced amino acid sequences indicated that all are highly homologous to the GABA-ρ subunits cloned from mammalian retinas; two clones (perch-ρ1A and perch-ρ1B) were in the ρ1 family, two (perch-ρ2A and perch-ρ2B) were in the ρ2 family, and one clone has been tentatively identified as a perch-ρ3 subunit. When expressed in Xenopus oocytes, all but one of the subunits (ρ3) formed functional homooligomeric receptors. However, the receptors expressed by each of the GABA-ρ subunits displayed unique response properties that distinguished one from the other. For example, receptors formed by perch-ρ1B subunits were more sensitive to GABA than the receptors formed by other GABA-ρ subunits, the dose–response curves for the various receptors revealed different Hill coefficients, and there were differences in the kinetics of the GABA-induced currents. In addition, the GABA-mediated current–voltage curve for ρ2 receptors was approximately linear, whereas the responses from ρ1 receptors showed outward rectification. A further division in the properties of the GABA-ρ subunits was revealed in their responses to imidazole-4-acetic acid (I4AA); the drug behaved as an antagonist on A-type ρ receptors and a partial agonist on the B-type ρ receptors. These results suggest that there is a large diversity of GABA_c receptors in the vertebrate retina, probably formed by homooligomeric and heterooligomeric combinations of GABA ρ subunits, that exhibit different functional properties. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 305–320, 1998     

Regulation of 3H-dopamine release by presynaptic GABA and glutamate heteroreceptors in the synaptosomes of the rat nucleus accumbens

The influence of GABA, muscimol, delta-aminolevulinic acid (DALA), baclofen and L-glutamate on K+-evoked release of 3H-dopamine (3H-DA) from the rat brain n. accumbens crude synaptosomal fraction was studied in superfusion experimental conditions. Both GABA-receptor agonists--GABA and muscimol (50 microM) depressed the 3H-DA release by bicuculline- and picrotoxin-sensitive manner. On the contrary, glutamate, DALA and baclofen led to the increase in 3H-DA efflux independently of the presence of GABA-receptor antagonists. While the action of glutamate was antagonized by glutamate-receptor blocker--glutamic acid diethyl ester (GDEE), the effects of DALA and baclofen were suppressed upon adding to superfusion medium of GABA uptake inhibitors (nipecotic acid and 2,4-diaminobutyric acid) but not GDEE. The data obtained demonstrate that 3-H-DA secretion from n. accumbens is inhibited by GABA- and stimulated by glutamate-heteroreceptors. At the same time the mechanism of interaction between baclofen, DALA and GABA-uptake blockers effects with 3H-DA release needs special investigations.     

Effects of serotonin-IA receptors on amino acid and dopaminergic responses of neurons]

By using intracellular technique the authors studied the responses of frog spinal motoneurones and rat dorsal root ganglion evoked by GABA, L-aspartate and dopamine in the presence of 5-hydroxytryptamine and its 1A-agonists or without them. It is shown that buspirone, campirone and serotonin increase the GABA effects but inhibit the effects of aspartate via the GABA- and NMDA-receptors modulation.     

Mediation by calcium/calmodulin-dependent protein kinase II of suppression of GABAA receptors by NMDA

Using nystatin-perforated whole-cell recording configuration, the modulatory effect of N-methyl-D-aspartate (NMDA) on -aminobutyric acid (GABA)-activated whole-cell currents was investigated in neurons freshly dissociated from the rat sacral dorsal commissural nucleus (SDCN). The results showed that: (I) NMDA suppressed GABA- and muscimol (Mus)-activated currents (IGABA and Imus), respectively in the Mg2+-free external solution containing 1 mol/L glycine at a holding potential (VH) of 40 mV in SDCN neurons. The selective NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV, 100 mol/L), inhibited the NMDA-evoked currents and blocked the NMDA-induced suppression of IGABA; (ii) when the neurons were incubated in a Ca2+-free bath or pre-loaded with a membrane-permeable Ca2+ chelator, BAPTA AM (10 mol/L), the inhibitory effect of NMDA on IGABA disappeared. Cd2+ (10 mol/L) or La3+ (30 mol/L), the non-selective blockers of voltage-dependent calcium channels, did not affect the suppression of IGABA by NMDA application; (iii) the suppression of IGABA by NMDA was inhibited by KN-62, a calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor. These results indicated that the inhibition of GABA response by NMDA is Ca2+-dependent and CaMKII is involved in the process of the Ca2+-dependent inhibition.     

The cone pedicle, a complex synapse in the retina

Cone pedicles, the synaptic terminals of cone photoreceptors, are connected in the macaque monkey retina to several hundred postsynaptic dendrites. Using light and electron microscopy, we found underneath each cone pedicle a laminated distribution of dendritic processes of bipolar and horizontal cells. Superimposed were three strata of glutamate receptor (GluR) aggregates, including a novel layer of glutamate receptors clustered at desmosome-like junctions. They are, most likely, postsynaptic densities on horizontal cell dendrites. GABA(A) and GABA(C) receptors are aggregated on bipolar cell dendrites in a narrow band underneath the cone pedicle. Glutamate released from cone pedicles and GABA released from horizontal cell dendrites act not only through direct synaptic contacts but also (more so) through diffusion to the appropriate receptors.     

Gaba sensitivity of the somata and dendrites of pyramidal cells in isolated slices of rat hippocampus

During experiments on isolated slices slices of rat hippocampus the inhibitory action of -aminobutyric acid (GABA) was investigated on the excitation of field CA, pyramidal neurons, together with the effects of bicuculline, penicillin and thiopentone on this process. It was found that GABA effectively and reversibly reduced the amplitude of the antrodomic population spike in the area of both the somata and the dendrites of these cells. The sensitivity of apical dendrites to GABA exceeded that of the somata by one order, increasing in a proximal-distal direction. The somata of pyramidal neurons were marked by pronounced desensitization to GABA. Bicuculline and penicillin acted as GABA antagonists at all the levels of CA, pyramidal cells investigated. Bicuculline blocked the effects of GABA on somata and dendrites in almost equal measure. The antagonistic effects of penicillin were 10 times greater in the pyramidal layer than in the dendritic region. Thiopentone reinforced the inhibitory effects of GABA. The potentiating effects of thiopentone were exerted most strongly on the dendrites. It is postulated that the membrane of field CA, neurons contain two types of bicuculline-sensitive GABA receptors, differing in their location (mainly on the cell body or dendrite), their pharmacology, and degree of desensitization to GABA.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 17, No. 6, pp. 737–746, November–December, 1985.     

The Subcellular Distribution of T-Type Ca2+ Channels in Interneurons of the Lateral Geniculate Nucleus

Inhibitory interneurons (INs) in the lateral geniculate nucleus (LGN) provide both axonal and dendritic GABA output to thalamocortical relay cells (TCs). Distal parts of the IN dendrites often enter into complex arrangements known as triadic synapses, where the IN dendrite plays a dual role as postsynaptic to retinal input and presynaptic to TC dendrites. Dendritic GABA release can be triggered by retinal input, in a highly localized process that is functionally isolated from the soma, but can also be triggered by somatically elicited Ca²⁺-spikes and possibly by backpropagating action potentials. Ca²⁺-spikes in INs are predominantly mediated by T-type Ca²⁺-channels (T-channels). Due to the complex nature of the dendritic signalling, the function of the IN is likely to depend critically on how T-channels are distributed over the somatodendritic membrane (T-distribution). To study the relationship between the T-distribution and several IN response properties, we here run a series of simulations where we vary the T-distribution in a multicompartmental IN model with a realistic morphology. We find that the somatic response to somatic current injection is facilitated by a high T-channel density in the soma-region. Conversely, a high T-channel density in the distal dendritic region is found to facilitate dendritic signalling in both the outward direction (increases the response in distal dendrites to somatic input) and the inward direction (the soma responds stronger to distal synaptic input). The real T-distribution is likely to reflect a compromise between several neural functions, involving somatic response patterns and dendritic signalling.     

Release of γ-[3H]Aminobutyric Acid from Rat Olfactory Bulb and Substantia Nigra: Differential Modulation by Glutamic Acid

We have studied the glutamate modulation of gamma-[3H]aminobutyric acid ([3H]GABA) release from GABAergic dendrites of the external plexiform layer of the olfactory bulb and from GABAergic axons of the substantia nigra. In the olfactory bulb, [3H]GABA release was induced by high K+ and kainate, and not by aspartate and glutamate alone. However, when the tissue was conditioned by a previous K+ depolarization, glutamate and aspartate caused [3H]GABA release. The effect of glutamate was significantly enhanced when the GABA uptake mechanism was blocked by nipecotic acid. N-Methyl-D-aspartate and quisqualate did not cause [3H]GABA release under the same conditions. The acidic amino acid receptor antagonist 2-amino-4-phosphonobutyric acid and the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid significantly inhibited the K+-glutamate- and the kainate-induced [3H]GABA release. Mg2+ (5 mM), which blocks the N-methyl-D-aspartate receptors, significantly inhibited the K+-glutamate-induced but not the kainic acid-induced [3H]GABA release. The K+-glutamate-stimulated release, but not the K+-stimulated [3H]GABA release, was strongly inhibited by Na+-free solutions or by 300 nM tetrodotoxin. Apparently the glutamate-induced release of [3H]GABA occurs through an interneuron because it is dependent on the presence of nerve conduction. In the substantia nigra no [3H]GABA release was elicited by any of the glutamate agonists tested. The present results clearly differentiate between the effects of glutamate on the release of [3H]GABA from the substantia nigra and from the olfactory bulb.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ontogeny of K-stimulated release of [H]GABA in rat cerebral cortex studied by a simple technique in vitro

Ontogenetic development and Ca²⁺-dependence of the K⁺-stimulated release of [³H]γ-aminobutyric acid (GABA) were studied by two different methods using tissue slices in vitro. The results indicate that, in the developing rat cortex, the K⁺-stimulated release of [³H]GABA is initially very low but it develops rapidly during the second and third postnatal weeks. This supports an earlier study which concluded that, during the cortical ontogeny, the ratio of stimulated: resting release of [³H]GABA increased at the fastest rate about 9–12 days after the birth, thus preceding the formation of GABAergic synapses by about 10 days. Furthermore, most of the early postnatal release observed in the present experiments is Ca²⁺-independent. An important Ca²⁺-dependent component of the release appears at later developmental stages and it also seems to develop faster than the GABAergic synapses. The present study suggests that the stimulus-coupled release of GABA in the rat cortex profoundly changes during the ontogeny, both quantitatively (the period of rapid development) and qualitatively (with respect to Ca²⁺-dependence). These observations, possibly reflecting changes in the association of GABA release with different structures (e.g. initially axonal growth cones, then neuronal dendrites and only at later stages GABAergic synapses) may be important in the evaluation of the putative role of GABA in synaptogenesis.     

Mechanisms of interactions between glycine- and GABA-mediated responses in spinal cord neurons

The interactions of GABA- and glycine-mediated responses have been analyzed, the whole cell patch-clamp method being used. The response induced by co-application of glycine and GABA was a lesser one than the sum of responses induced by applying two transmitters separately. The molecular mechanisms underlying this effect have been determined. Due to applications of high concentrations of neurotransmitters it was revealed that GABA could activate glycine receptors in frog spinal motoneurons with relatively high efficiency (EC50 = 1.2 mM). The sequential application of neurotransmitters showed that even a single application of glycine could significantly boost the "run-down" of the GABA-mediated current, suggesting that there was a strong phosphorylation-dependent mechanism of GABAa-receptors inhibition. These mechanisms are likely to take place in frog spinal motoneurons when GABA and glycine are co-released from the same synaptic terminal.     

Differential blocking actions of 4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) and γ-hexachlorocyclohexane (γ-HCH) on γ-aminobutyric acid- and glutamate-induced responses of American cockroach neurons

4′-Ethynyl-4-n-propylbicycloorthobenzoate (EBOB) has been employed extensively as a radioligand in binding assays to evaluate the pharmacology of γ-aminobutyric acid (GABA)-gated Cl⁻ channels (GABARs) of insects and mammals, and γ-hexachlorocyclohexane (γ-HCH) was used as an insecticide targeting insect GABARs. Since recent studies have shown that not only GABARs but also glutamate-gated chloride channels (GluCls) are blocked by picrotoxinin, dieldrin and fipronil, the actions of EBOB and γ-HCH on native GABARs and GluCls of terminal abdominal ganglion neurons in American cockroach (Periplaneta americana) were tested using patch-clamp electrophysiology. A marked run-down of the GABA- and glutamate-induced responses of the cockroach neurons occurred, when a standard pipette solution was employed, but addition of pyruvate to the solution permitted stable recordings of these responses. With this solution, EBOB and γ-HCH were found to block not only the GABA- but also glutamate-gated responses, with the actions augmented by repeated co-application with the agonists. It was also found that prolonged pre-application of EBOB and γ-HCH prior to co-application with GABA and glutamate resulted in enhanced blocking actions, indicating resting-state actions of the blockers. The blocking actions of EBOB and γ-HCH on the GABA- and glutamate-induced responses were compared by determining IC₅₀ values under steady state condition. The IC₅₀ values for the actions of EBOB on GABAR and GluCls differed less than those of γ-HCH.     

Comparison of the effect of gamma-aminobutyric acid and its derivative baclofen on the activity of Purkinje cells of frog cerebellum in vitro

The inhibitory effect of gamma-aminobutyric acid (GABA) and its synthetic derivative baclofen were compared in frog cerebellum in vitro. Baclofen inhibited synaptic transmission from parallel fibres to the Purkinje cells in EC50 concentrations approximately 200-fold lower than for GABA. In addition to its inhibitory effect, GABA induced temporary facilitation of responses in the dendrite zone by a mechanism dependent on the presence of a normal Cl- concentration; the inhibitory phase was only partly sensitive to reduction of the Cl- concentration in the medium and to the administration of picrotoxin. The action of baclofen, which was unaffected by these treatments, requires an intact catecholamine and serotonin pool, since it is ineffective in reserpine-treated animals. Both substances also influence the excitability of parallel fibres. In solutions with a high Mg2+ and a low Ca2+ concentrations GABA inhibits the spontaneous activity of Purkinje cells by acting on the postsynaptic membrane of the soma and the primary dendrites. The effect of baclofen is evidently the outcome of inhibition of transmitter release from presynaptic endings.     

Metabolism of glutamate and {gamma}-aminobutyrate in cultured tobacco cells

In cultured tobacco cells glutamate-U-¹⁴C administrated wasreadily converted to -aminobutyrate (GABA) by decarboxylation,however, GABA-1-¹⁴C remained unchanged. Glutamate decarboxylasewas found in tobacco cells and reached its maximum activityin the rapidly growing stage during culture. Enzyme activityparalleled formation of GABA from glutamate-U-¹⁴C. A high contentof GABA in tobacco cells seems to be due to the rapid decarboxylationof glutamate by glutamate decarboxylase and a slow turn overof GABA. ¹ Present address: The Okayama Tobacco Experiment Station, JapanMonopoly Corp., Tamashima, Kurashiki, Japan. (Received November 20, 1971; )