Modulation of GABA flux across rat brain membranes resolved by a rapid quenched incubation technique

doi:10.1016/0304-3940(94)90209-7

Neuroscience Letters

Volume 182, Issue 1, 21 November 1994, Pages 73-76

https://doi.org/10.1016/0304-3940(94)90209-7 Get rights and content

Abstract

The progress and inhibition of [³H]GABA influx in native plasma membrane vesicles from the rat cerebral cortex was studied on a subsecond to minute time scale under different conditions by applying a rapid quenched incubation technique. In the absence of Ca²⁺ ([Ca²⁺]_free = 10⁻⁸ M), the progress of influx followed by the addition of 10 nM [³H]GABA to the membrane vesicle suspension with time (500 ms to 15 min) can be described by a first-order rate equation giving an overall rate constant, k, of 3.93 ± 0.48 × 10⁻³ s⁻¹ and equilibrium influx value, INF_e, of $8.84 ± 0.41 pmol [^{3} H]GABA/mg protein$ . In the presence of Ca²⁺ ([Ca²⁺]_free = 2.4 × 10⁻³ M) a significant increase in the INF_e value was observed (k = 4.64 ± 0.41 × 10⁻³ s⁻¹ and $INF_{e} = 13.9 ± 0.40 pmol [^{3} H]GABA /mg protein$ ). Multiplicity of GABA transporters was indicated in the time-dependent inhibition of [³H]GABA influx by different uptake blockers. In the absence of Ca²⁺, depolarization (75 mM KCl) inhibited the influx of [³H]GABA into the vesicles by ∼70% and initiated the efflux from vesicles loaded with [³H]GABA. Different uptake blockers inhibited the Ca²⁺-independent translocation of [³H]GABA in both directions with similar specificities.

References (13)

L.A. Borden et al.
Molecular heterogeneity of the γ-amino-butyric acid (GABA) transport system
J. Biol. Chem.
(1992)
D.J. Cash et al.
Quenched-flow technique with plasma membrane vesicles: acetylcholine-receptor-mediated transmembrane ion-flux
Anal. Biochem.
(1981)
J.L. Corey et al.
Protein kinase C modulates the activity of a cloned γ-aminobutyric acid transporter expressed in Xenopus Oocytes via regulated subcellular redistribution of the transporter
J. Biol. Chem.
(1994)
Z. Liron et al.
Studies on uptake of γ-aminobutyric acid by mouse brain particles; toward the development of a model
Brain Res.
(1988)
O.H. Lowry et al.
Protein measurements with the Folin phenol reagent
J. Biol. Chem.
(1951)
P. Serfózó et al.
Effect of a benzodiazepine (chlordiazepoxide) on a GABA receptor from rat brain: requirement of only one bound GABA molecule for channel opening
FEBS Lett.
(1992)

There are more references available in the full text version of this article.

Cited by (15)

Feedback adaptation of synaptic excitability via Glu:Na<sup>+</sup> symport driven astrocytic GABA and Gln release
2019, Neuropharmacology
Citation Excerpt :
Thus, understanding the mechanisms of extracellular release of GABA through the astrocytic GAT-3 isoform needs further clarifications. First, distinct modes of transporter operation, i.e. high-affinity influx and low-affinity efflux have been identified [Kardos et al., 1997; Mabjeesh and Kanner, 1989], which was characterized by similar inhibitor specificity [Kardos et al., 1994]. Furthermore, we propose that in efflux mode, the cytoplasmic binding of the thermodynamically favoured H-bond ring-forming GABA conformer to GAT-3 is of low-affinity [Kardos et al., 2010; Kardos and Héja, 2015; Simon et al., 2014; Palló et al., 2007, 2009; Skovstrup et al., 2010].
Glutamatergic transmission composed of the arriving of action potential at the axon terminal, fast vesicular Glu release, postsynaptic Glu receptor activation, astrocytic Glu clearance and Glu→Gln shuttle is an abundantly investigated phenomenon. Despite its essential role, however, much less is known about the consequences of the mechanistic connotations of Glu:Na⁺ symport. Due to the coupled Na⁺ transport, Glu uptake results in significantly elevated intracellular astrocytic [Na⁺] that markedly alters the driving force of other Na⁺-coupled astrocytic transporters. The resulting GABA and Gln release by reverse transport through the respective GAT-3 and SNAT3 transporters help to re-establish the physiological Na⁺ homeostasis without ATP dissipation and consequently leads to enhanced tonic inhibition and replenishment of axonal glutamate pool. Here, we place this emerging astrocytic adjustment of synaptic excitability into the centre of future perspectives.
This article is part of the issue entitled ‘Special Issue on Neurotransmitter Transporters’.
Major human γ-aminobutyrate transporter: In silico prediction of substrate efficacy
2007, Biochemical and Biophysical Research Communications
The inhibitory γ-aminobutyric acid transporter subtype 1 (GAT1) maintains low resting synaptic GABA level, and is a potential target for antiepileptic drugs. Here we report a high scored binding mode that associates GABA with gating in a homology model of the human GAT1. Docking and molecular dynamics calculations recognize the amino function of GABA in the H-bonding state favoring TM1 and TM8 helix residues Y60 and S396, respectively. This ligand binding mode visibly ensures the passage of GABA and substrate inhibitors (R)-homo-β-Pro, (R)-nipecotic acid, and guvacine. It might therefore represent the principle, sufficient for sorting out less-effective or non-GAT ligands such as β-Pro, (S)-nipecotic acid, (R)-baclofen, Glu, and Leu.
Suppression of neuronal network excitability and seizure-like events by 2-methyl-4-oxo-3H-quinazoline-3-acetyl piperidine in juvenile rat hippocampus: Involvement of a metabotropic glutamate receptor
2006, Neurochemistry International
We present data on the antiepileptic potency of 2-methyl-4-oxo-3H-quinazoline-3-acetyl piperidine (Q5) in juvenile (P9-13) rat hippocampal slices and in particular Q5's action mechanism and target. Q5 (200–500 μM), but not α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/Kainate receptor antagonists blocked low-[Mg²⁺]-induced seizure-like events (SLE) in the CA3 region. Q5 (100 μM) decreased Glu-induced [³⁵S]guanosine 5′-O-(3-thiotriphosphate) binding enhancement in brain homogenates, without interaction with ionotropic Glu receptor sites and Glu transport. In voltage-clamped CA3 pyramidal cells, Q5 (500 μM) depressed activities of spontaneous excitatory and inhibitory postsynaptic currents without affecting miniature inhibitory currents. Metabotropic Glu receptor (mGluR) subtype antagonists affected network excitability dissimilarly. Intracellular Ca²⁺ ion transients induced by the mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) were suppressed by Q5. Agreeing predictions obtained by modelling Q5 binding to different experimental conformations of mGlu1, Q5 was bound partially to an mGluR binding site in the presence of 1 mM ACPD. Findings suggest the apparent involvement of a novel phenotype of action or a new mGluR subtype in the specific suppression of epileptiform activity by Q5 through the depression of network excitability.
Cyclothiazide binding to functionally active AMPA receptor reveals genuine allosteric interaction with agonist binding sites
2004, Neurochemistry International
The agonist, [ $^{3} H$ ](−)[S]-1-(2-amino-2-carboxyethyl)-5-fluoro-pyrimidine-2,4-dione ([ $^{3} H$ ](S)F-Willardiine) binding to functional alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors of resealed plasma membrane vesicles and nerve endings freshly isolated from the rat cerebral cortex displayed two binding sites (K_D1=33±7 nM, B_MAX1=1.6±0.3 pmol/mg protein, K_D2=720±250 nM and B_MAX2=7.8±4.0 pmol/mg protein). The drug which impairs AMPA receptor desensitisation, 6-chloro-3,4-dihydro-3-(2-norbornene-5-yl)-2H-1,2,4-benzothiadiazine-7-sulphonamide-1,1-dioxide (cyclothiazide, CTZ) fully displaced the [ $^{3} H$ ](S)F-Willardiine binding at a concentration of 500 μM. In the presence of 100 μM CTZ (K_I(CTZ)=60±6 μM), both the antagonist [ $^{3} H$ ]-1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo(F)quinoxaline-7-sulfonamide ([ $^{3} H$ ]NBQX: K_D=24±4 nM, B_MAX=12.0±0.1 pmol/mg protein) and the high-affinity agonist binding showed similar affinity reduction ([ $^{3} H$ ](S)F-Willardiine: K_D=140±19 nM, B_MAX=2.9±0.5 pmol/mg protein; [ $^{3} H$ ]NBQX: K_D=111±34 nM, B_MAX=12±3 pmol/mg protein). To disclose structural correlates underlying genuine allosteric binding interactions, molecular mechanics calculations of CTZ-induced structural changes were performed with the use of PDB data on extracellular GluR2 binding domain dimeric crystals available by now. Hydrogen-bonding and root mean square (rms) values of amino acid residues recognising receptor agonists showed minor alterations in the agonist binding sites itself. Moreover, CTZ binding did not affect dimeric subunit structures significantly. These findings indicated that the structural changes featuring the non-desensitised state could possibly occur to a further site of the extracellular GluR2 binding domain. The increase of agonist efficacy on allosteric CTZ binding may be interpreted in terms of a mechanism involving AMPA receptor desensitisation sequential to activation.
Chapter 8 Sorption (binding) and transport phenomena in biomembranes
2004, Interface Science and Technology
This chapter focuses on the principles that have applicability to a great variety of biological membranes. The study of the movement of solutes across biological membranes has progressed from the collection of phenomenological observations on cellular transport and electrical behavior to the identification, purification, characterization, and reconstitution of some of the integral membrane proteins responsible for the phenomena. To overcome the time-consuming separation of bound from free ligands, that cannot be automated, and that often can lead to increased non-specific binding, a scintillation proximity assay has been developed. The principle of this assay is that only radiolabeled ligands, which are bound to the receptor molecule generate a signal on the fluomicrosphere coated with receptors. The technique that is carried out in aqueous media (buffer) eliminates the use of liquid scintillants required for tritium-based assays. The scintillation proximity assay requires radiolabels that emit low-energy radiation, which is dissipated easily into an aqueous media.
Characterisation of an uridine-specific binding site in rat cerebrocortical homogenates
2003, Neurochemistry International
Parameters of [ $^{3} H$ ]uridine binding to synaptic membranes isolated from rat brain cortex (K_D=71±4 nM, B_max=1.37±0.13 pmol/mg protein) were obtained. Pyrimidine and purine analogues displayed different rank order of potency in displacement of specifically bound [ $^{3} H$ ]uridine (uridine>5-F-uridine>5-Br-uridine∼adenosine⪢5-ethyl-uridine∼suramin>theophylline) and in the inhibition of [ $^{14} C$ ]uridine uptake (adenosine>uridine>5-Br-uridine∼5-F-uridine∼5-ethyl-uridine) into purified cerebrocortical synaptosomes. Furthermore, the effective ligand concentration for the inhibition of [ $^{14} C$ ]uridine uptake was about two order of magnitude higher than that for the displacement of specifically bound [ $^{3} H$ ]uridine. Adenosine evoked the transmembrane Na⁺ ion influx, whereas uridine the transmembrane Ca²⁺ ion influx much more effectively. Also, uridine was shown to increase free intracellular Ca²⁺ ion levels in hippocampal slices by measuring Calcium-Green fluorescence. Uridine analogues were found to be ineffective in displacing radioligands that were bound to various glutamate and adenosine—recognition and modulatory—binding sites, however, increased [ $^{35} S$ ]GTPgammaS binding to membranes isolated from the rat cerebral cortex. These findings provide evidence for a rather specific, G-protein-coupled site of excitatory action for uridine in the brain.

View all citing articles on Scopus

View full text

Article preview

Neuroscience Letters

Abstract

References (13)

Molecular heterogeneity of the γ-amino-butyric acid (GABA) transport system

J. Biol. Chem.

Quenched-flow technique with plasma membrane vesicles: acetylcholine-receptor-mediated transmembrane ion-flux

Anal. Biochem.

Protein kinase C modulates the activity of a cloned γ-aminobutyric acid transporter expressed in Xenopus Oocytes via regulated subcellular redistribution of the transporter

J. Biol. Chem.

Studies on uptake of γ-aminobutyric acid by mouse brain particles; toward the development of a model

Brain Res.

Protein measurements with the Folin phenol reagent

J. Biol. Chem.

Effect of a benzodiazepine (chlordiazepoxide) on a GABA receptor from rat brain: requirement of only one bound GABA molecule for channel opening

FEBS Lett.

Cited by (15)

Feedback adaptation of synaptic excitability via Glu:Na<sup>+</sup> symport driven astrocytic GABA and Gln release

Major human γ-aminobutyrate transporter: In silico prediction of substrate efficacy

Suppression of neuronal network excitability and seizure-like events by 2-methyl-4-oxo-3H-quinazoline-3-acetyl piperidine in juvenile rat hippocampus: Involvement of a metabotropic glutamate receptor

Cyclothiazide binding to functionally active AMPA receptor reveals genuine allosteric interaction with agonist binding sites

Chapter 8 Sorption (binding) and transport phenomena in biomembranes

Characterisation of an uridine-specific binding site in rat cerebrocortical homogenates