Regulation of GABAA Receptor Dynamics by Interaction with Purinergic P2X2 Receptors

Amulya Nidhi Shrivastava,Werner Sieghart,Isabella Sarto-Jackson,Antoine Triller

doi:10.1074/jbc.m110.165282

Abstract

γ-Aminobutyric acid type A receptors (GABA(A)Rs) in the spinal cord are evolving as an important target for drug development against pain. Purinergic P2X(2) receptors (P2X(2)Rs) are also expressed in spinal cord neurons and are known to cross-talk with GABA(A)Rs. Here, we investigated a possible "dynamic" interaction between GABA(A)Rs and P2X(2)Rs using co-immunoprecipitation and fluorescence resonance energy transfer (FRET) studies in human embryonic kidney (HEK) 293 cells along with co-localization and single particle tracking studies in spinal cord neurons. Our results suggest that a significant proportion of P2X(2)Rs forms a transient complex with GABA(A)Rs inside the cell, thus stabilizing these receptors and using them for co-trafficking to the cell surface, where P2X(2)Rs and GABA(A)Rs are primarily located extra-synaptically. Furthermore, agonist-induced activation of P2X(2)Rs results in a Ca(2+)-dependent as well as an apparently Ca(2+)-independent increase in the mobility and an enhanced degradation of GABA(A)Rs, whereas P2X(2)Rs are stabilized and form larger clusters. Antagonist-induced blocking of P2XRs results in co-stabilization of this receptor complex at the cell surface. These results suggest a novel mechanism where association of P2X(2)Rs and GABA(A)Rs could be used for specific targeting to neuronal membranes, thus providing an extrasynaptic receptor reserve that could regulate the excitability of neurons. We further conclude that blocking the excitatory activity of excessively released ATP under diseased state by P2XR antagonists could simultaneously enhance synaptic inhibition mediated by GABA(A)Rs.

Highlights

GABAARs2 are the major inhibitory transmitter receptors in the central nervous system and the site of action of benzodiazepines, barbiturates, neuroactive steroids, anesthetics, and con
Both receptors are co-localized in membranes and the cytoplasm of human embryonic kidney (HEK) cells co-transfected with ECFP-tagged GABAARs and EYFP-tagged P2X2 receptors (P2X2Rs) as demonstrated by confocal microscopy
fluorescence resonance energy transfer (FRET) experiments performed in these cells resulted in a similar FRET signal in the cytosol and at the membrane, suggesting that the same type of association of the GABAA-P2X2R complex is observed in these compartments (Fig. 2)

Summary

Introduction

GABAARs2 are the major inhibitory transmitter receptors in the central nervous system and the site of action of benzodiazepines, barbiturates, neuroactive steroids, anesthetics, and con-. They are ligand-gated chloride channels composed of five subunits that can belong to different subunit classes. GABAARs are widely distributed in the brain [4, 5] and spinal cord [6]. Clusters of these receptors can be found at inhibitory synapses mediating phasic inhibition and at extrasynaptic locations where they are mediating tonic inhibition [7]. GABAARs and P2X2Rs have been demonstrated to be co-localized and to functionally interact with each other in the spinal cord and dorsal root ganglion [20, 21]. Our studies identified a novel mechanism by which GABAAR distribution and dynamics can be modulated by P2X2Rs in the spinal cord

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