Regulation of GABAA Receptor Dynamics by Interaction with Purinergic P2X2 Receptors

Abstract

␥-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.GABA A Rs 2 are the major inhibitory transmitter receptors in the central nervous system and the site of action of benzodiazepines, barbiturates, neuroactive steroids, anesthetics, and convulsants. They are ligand-gated chloride channels composed of five subunits that can belong to different subunit classes. The majority of these receptors are composed of one ␥, two ␣, and two ␤ subunits (1-3). GABA A Rs 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 but also at extrasynaptic locations where they are mediating tonic inhibition (7). Using single particle tracking (SPT), it has been demonstrated that most neurotransmitter receptors, including GABA A Rs, are exchanged between synaptic and extrasynaptic domains by lateral diffusion (8 -10). The lateral mobility of receptors can be modulated by interaction with scaffolding molecules such as gephyrin for GABA A (7, 11) and glycine receptors (12, 13). In addition, receptor insertion and removal are considered major determinants in the regulation of receptor number at the cell surface and the strength of GABAergic transmission (14, 15).The P2X receptor superfamily includes seven different subunits (P2X 1 -P2X 7 ) (16). P2X 2 subunits mainly form homotrimeric receptors but also assemble with P2X 3 subunits to form heterotrimeric P2X 2/3 receptors (P...

Subtype-Specific Mechanisms for Functional Interaction between α6β4* Nicotinic Acetylcholine Receptors and P2X Receptors

“…A prerequisite for such a mechanism is the close proximity of receptors. Previous work confirmed physical interactions for combinations of P2X2-a4b2, P2X2-5-HT 3 , and P2X2-GABA C receptors (Khakh et al, 2000(Khakh et al, , 2005Boué-Grabot et al, 2003, 2004aToulmé et al, 2007;Decker and Galligan, 2010;Jo et al, 2011;Shrivastava et al, 2011). The evidence for physical contact suggests that there is no major role for second messengers generated by endogenous and electrophysiologically silent metabotropic P2Y receptors in the cross inhibition.…”

The Cell Adhesion Molecule Neuroplastin-65 Is a Novel Interaction Partner of γ-Aminobutyric Acid Type A Receptors

“…Rabbit polyclonal antibodies against both isoforms of np (np55/65) as well as the isoform-specific antibodies against np65 were generated and used as described previously (28), and goat antibodies against np65 were purchased from R&D Systems (Minneapolis, MN), and mouse monoclonal antibodies against both np isoforms (SMgp65) were a gift from Prof. P. W. Beesley (Royal Holloway University of London, United Kingdom). Rabbit polyclonal antibodies against full-length EGFP protein were generated as described elsewhere (30). Mouse monoclonal antibodies against gephyrin and guinea pig polyclonal antibodies against VIAAT were purchased from Synaptic Systems (Göttingen, Germany).…”

“…Constructs ␣1-ECFP or ␣1-EYFP and ␤2-ECFP or ␤2-EYFP were subcloned into the pECFP-C1 or EYFP-C1 vectors (Clontech), by incorporating the fluorescence tags ECFP or EYFP into the intracellular loops of the ␣1 or ␤2 subunit, and further characterization of these constructs was described elsewhere (30). The cDNAs of np55 and np65 were cloned into the expression vectors pRC/CMV (Invitrogen) as described previously (26).…”

“…To do so, we generated fluorescence-tagged np65-ECFP or np65-EYFP fusion proteins, as well as tagged GABA A ␣1-EYFP or ␤2-EYFP receptor subunit constructs (30). Different combinations of constructs were transfected into HEK cells (Table 1), and their expression was investigated using an epifluorescence microscope, and distinct regions where both fluorescence-tagged proteins were clearly present were chosen to calculate their N FRET values (see under "Experimental Procedures").…”

Imaging P2X4 Receptor Lateral Mobility in Microglia

“…Thus, quantum dot (QD) 3 -based single-molecule imaging and single particle tracking (SPT) have shed light on the regulation of several neurotransmitter receptors, including P2X2 receptors (26,31,32), which are known to display different types of lateral mobility in the plasma membrane with proposed and demonstrated signaling roles (26). In contrast, the lateral mobility of P2X4 receptors has not been measured, and little is known about this aspect of their function or even if mobility is regulated by ATP or in settings where P2X receptors are known to play critical roles, such as in resting and activated microglia.…”