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.