Regulation of GABA_A Receptor Dynamics by Interaction with Purinergic P2X₂ Receptors*

γ-Aminobutyric acid type A receptors (GABA_ARs) in the spinal cord are evolving as an important target for drug development against pain. Purinergic P2X₂ receptors (P2X₂Rs) are also expressed in spinal cord neurons and are known to cross-talk with GABA_ARs. Here, we investigated a possible “dynamic” interaction between GABA_ARs and P2X₂Rs 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 P2X₂Rs forms a transient complex with GABA_ARs inside the cell, thus stabilizing these receptors and using them for co-trafficking to the cell surface, where P2X₂Rs and GABA_ARs are primarily located extra-synaptically. Furthermore, agonist-induced activation of P2X₂Rs results in a Ca²⁺-dependent as well as an apparently Ca²⁺-independent increase in the mobility and an enhanced degradation of GABA_ARs, whereas P2X₂Rs 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 P2X₂Rs and GABA_ARs 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_ARs.