Allosteric modulation of GABAA receptors by extracellular ATP

Jun Liu,Yu Wang

doi:10.1186/1756-6606-7-6

Abstract

BackgroundThe γ-aminobutyric acid type A receptor (GABAAR) is the primary receptor mediating fast synaptic inhibition in the brain and plays a critical role in modulation of neuronal excitability and neural networks. Previous studies have demonstrated that ATP and its nucleotide analogs may regulate the function of GABAARs via Ca2+-dependent intracellular mechanisms, which require activation of purinergic 2 (P2) receptors or cross-talk between two receptors.ResultsHere, we report a potentiation of GABAARs by extracellular ATP via a previously un-recognized allosteric mechanism. Using cultured hippocampal neurons as well as HEK293 cells transiently expressing GABAARs, we demonstrate that extracellular ATP potentiates GABAAR mediated currents in a dose-dependent manner with an EC50 of 2.1 ± 0.2 mM. The potentiation was mediated by a postsynaptic mechanism that was not dependent on activation of either ecto-protein kinase or P2 receptors. Single channel recordings from cell-free excised membrane patches under outside-out mode or isolated membrane patches under cell-attached mode suggest that the ATP modulation of GABA currents is achieved through a direct action of ATP on the channels themselves and manifested by increasing the single channel open probability without alteration of its conductance. Moreover, this ATP potentiation of GABAAR could be reconstituted in HEK293 cells that transiently expressed recombinant rat GABAARs.ConclusionsOur data strongly suggest that extracellular ATP allosterically potentiates GABAAR-gated chloride channels. This novel mode of ATP-mediated modulation of GABAARs may play an important role in regulating neuronal excitability and thereby in fine-tuning the excitation-inhibition balance under conditions where a high level of extracellular ATP is ensured.

Highlights

The γ-aminobutyric acid type A receptor (GABAAR) is the primary receptor mediating fast synaptic inhibition in the brain and plays a critical role in modulation of neuronal excitability and neural networks
Extracellular adenosine 5′-triphosphate (ATP) potentiates GABAAR-mediated currents Under whole-cell voltage-clamp recordings of hippocampal neurons at a holding membrane potential of -60 mV, repetitive short pulses of pressure ejections of GABA (100 μM) to the neuron evoked robust inward currents, and these currents were mediated through GABAAR-gated Cl- channels as they were completely blocked by bath application of the GABAAR antagonist bicuculline (10 μM; Figure 1A)
To examine if ATP can modulate GABAARs localized at the synapse, we examined effects of extracellularly applied ATP on whole-cell recordings of miniature postsynaptic inhibitory currents mediated by synaptic GABAARs. mIPSCs were recorded at a holding membrane potential of -60 mV after blocking ionotropic glutamate receptors with CNQX (20 μM) and AP-5 (50 μM), glycine receptor with strychnine (1 μM) and sodium channels with TTX (1 μM)

Summary

Introduction

The γ-aminobutyric acid type A receptor (GABAAR) is the primary receptor mediating fast synaptic inhibition in the brain and plays a critical role in modulation of neuronal excitability and neural networks. Previous studies have demonstrated that ATP and its nucleotide analogs may regulate the function of GABAARs via Ca2+-dependent intracellular mechanisms, which require activation of purinergic 2 (P2) receptors or cross-talk between two receptors. The γ-aminobutyric acid type A receptor (GABAAR) is a ligand-gated chloride ion channel, activation of which results in membrane hyperpolarization and inhibition of the neuronal excitability in the adult mammalian brain. Previous studies demonstrate that extracellular ATP can modulate GABAAR function by activation of P2 receptors [11,12,13]. Ortinau et al (2003) reported that extracellular ATP inhibits the function of N-methyl-Daspartate (NMDA) glutamate receptors by directly binding to the receptor, suggesting that extracellular ATP may function as an allosteric modulator for neurotransmitter receptors [17]

Results

Discussion

Conclusion