GABAA Receptors: Post-Synaptic Co-Localization and Cross-Talk with Other Receptors

γ-Aminobutyric acid type A receptors (GABAARs) are the major inhibitory neurotransmitter receptors in the central nervous system, and importantly contribute to the functional regulation of the nervous system. Several studies in the last few decades have convincingly shown that GABA can be co-localized with other neurotransmitters in the same synapse, and can be co-released with these neurotransmitters either from the same vesicles or from different vesicle pools. The co-released transmitters may act on post-synaptically co-localized receptors resulting in a simultaneous activation of both receptors. Most of the studies investigating such co-activation observed a reduced efficacy of GABA for activating GABAARs and thus, a reduced inhibition of the post-synaptic neuron. Similarly, in several cases activation of GABAARs has been reported to suppress the response of the associated receptors. Such a receptor cross-talk is either mediated via a direct coupling between the two receptors or via the activation of intracellular signaling pathways and is used for fine tuning of inhibition in the nervous system. Recently, it was demonstrated that a direct interaction of different receptors might already occur in intracellular compartments and might also be used to specifically target the receptors to the cell membrane. In this article, we provide an overview on such cross-talks between GABAARs and several other neurotransmitter receptors and briefly discuss their possible physiological and clinical importance

Bidirectional Control of Synaptic GABAAR Clustering by Glutamate and Calcium

SummaryGABAergic synaptic transmission regulates brain function by establishing the appropriate excitation-inhibition (E/I) balance in neural circuits. The structure and function of GABAergic synapses are sensitive to destabilization by impinging neurotransmitters. However, signaling mechanisms that promote the restorative homeostatic stabilization of GABAergic synapses remain unknown. Here, by quantum dot single-particle tracking, we characterize a signaling pathway that promotes the stability of GABAA receptor (GABAAR) postsynaptic organization. Slow metabotropic glutamate receptor signaling activates IP3 receptor-dependent calcium release and protein kinase C to promote GABAAR clustering and GABAergic transmission. This GABAAR stabilization pathway counteracts the rapid cluster dispersion caused by glutamate-driven NMDA receptor-dependent calcium influx and calcineurin dephosphorylation, including in conditions of pathological glutamate toxicity. These findings show that glutamate activates distinct receptors and spatiotemporal patterns of calcium signaling for opposing control of GABAergic synapses

Interaction of GABAA receptors with purinergic P2X2 receptors

GABAA Rezeptoren im Rückenmark werden zu einem immer wichtigeren Angriffspunkt für die Entwicklung von Arzneimittel gegen den Schmerz. Purinerge P2X2 Rezeptoren finden sich ebenfalls im Rückenmark und es ist bekannt, dass sie in direkter Wechselwirkung (Cross-talk) mit GABAA Rezeptoren stehen. Hier untersuchten wir eine mögliche "dynamische" Interaktion zwischen GABAA Rezeptoren und P2X2 Rezeptoren mittels Co-Immunopräzipitation und FRET Studien in HEK Zellen sowie mittels Co-Lokalisationsstudien und "Single Particle Tracking" Untersuchungen in Rückenmarksneuronen. Unsere Resultate weisen darauf hin, dass ein signifikanter Anteil von P2X2 Rezeptoren einen intrazellulären, transienten Komplex mit GABAA Rezeptoren bilden, indem sie diese zur Stabilisation und zum gemeinsamen Transport nutzen. GABAA Rezeptoren und P2X2 Rezeptoren werden anschließend gemeinsam in die Zellmembran eingebaut, wo sie vor allem extrasynaptisch lokalisiert sind. Darüber hinaus konnten wir zeigen, dass eine durch Agonisten induzierte Aktivierung von P2X2 Rezeptoren zu einer Dissoziation des Komplexes und einer Destabilisierung von GABAA Rezeptoren führt, während P2X2 Rezeptoren stabilisiert werden und größere Cluster bilden. Eine durch Antagonisten induzierte Blockierung von P2X2 Rezeptoren führt zu einer Stabilisierung der GABAA/P2X2 Rezeptor-Komplexe an der Zelloberfläche. Diese Resultate zeigen einen neuen Mechanismus auf, bei dem die Assoziation von P2X2 Rezeptoren mit anderen Rezeptoren für einen spezifischen, zielgerichteten Transport zur neuronalen Zellmembran genutzt werden kann. Damit steht ein Reservepool an extrasynaptischen Rezeptoren zur Verfügung, der zur Regulation der neuronalen Erregbarkeit beitragen kann. Die vorliegenden Ergebnisse lassen auch den Schluss zu, dass P2X Rezeptor Antagonisten nicht nur die erregenden Aktivität von krankheits-bedingt im Überschuss freigesetztem ATP blockieren, sondern gleichzeitig auch zu einer verstärkten synaptischen Inhibierung durch GABAA Rezeptoren beitragen können.GABAARs in the spinal cord are evolving as an important target for drug development against pain. Purinergic P2X2Rs are also expressed in spinal cord neurons and are known to cross-talk with GABAARs. Here we investigated a possible "dynamic" interaction between GABAARs and P2X2Rs using co-immunoprecipitation and FRET studies in HEK 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 GABAARs inside the cell, thus stabilizing these receptors and using them for co-trafficking to the cell surface. P2X2Rs and GABAARs are then co-inserted into the cell membrane and are primarily located extra-synaptically. Furthermore, agonist induced activation of P2X2Rs results in disassembly of the receptor complex and destabilization of GABAARs 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 P2XRs with other receptors could be used for specific targeting to the neuronal membrane, 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 GABAARs.submitted by Amulya Nidhi ShrivastavaAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Med. Univ., Diss., 2010OeBB(VLID)171535

Cell biology and dynamics of Neuronal Na+/K+-ATPase in health and diseases

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Dynamic micro-organization of P2X7 receptors revealed by PALM based single particle tracking

Adenosine triphosphate (ATP)-gated P2X7 receptors (P2X7Rs) are members of the purinergic receptor family that are expressed in several cell types including neurons. A high concentration of ATP is required for the channel opening of P2X7Rs compared to other members of this receptor family. Recent work suggests that ATP binding to members of the P2X receptor family determines the diffusion and localization of these receptors on the plasma membrane of neurons. Here, we employed single particle tracking photoactivated localization microscopy (sptPALM) to study the diffusion and ATP-dependence of rat P2X7Rs. Dendra2-tagged P2X7Rs were transfected in hippocampal neurons and imaged on proximal dendrites. Our results suggest the presence of two populations of P2X7Rs within the extra-synaptic membrane: a population composed of rapidly diffusing receptors and one stabilized within nanoclusters (~100 nm diameter). P2X7R trajectories were rarely observed at synaptic sites. P2X7R mutations in the ATP-binding site (K64A) or the conserved phosphorylation site (K17A) resulted in faster- and slower-diffusing receptors, respectively. Furthermore, ATP differentially accelerated wild type and K17A-mutant receptors but not K64A-mutant receptors. Our results indicate that receptor conformation plays a critical role in regulating ATP-mediated changes in P2X7R diffusion and micro-organization

Differential Membrane Binding and Seeding of Distinct α\alpha-Synuclein Fibrillar Polymorphs

International audienceThe aggregation of the protein $\alpha$-Synuclein ($\alpha$-Syn) leads to different synucleinopathies. We recently showed that structurally distinct fibrillar $\alpha$-Synuclein polymorphs trigger either Parkinson’s Disease or Multiple System Atrophy hallmarks in vivo. Here, we establish structural-molecular basis for these observations. We show that distinct fibrillar $\alpha$-Syn polymorphs bind to and cluster differentially at the plasma membrane in both primary neuronal cultures and organotypic hippocampal slice cultures from wild-type mice. We demonstrate $\alpha$ polymorph-dependent and concentration-dependent seeding. We show a polymorph-dependent differential synaptic re-distribution of $\alpha$3-Na$^+$/K$^+$-ATPase, GluA2-AMPA and GluN2B-NMDA receptors but not GluA1-AMPA and mGluR5 receptors. We also demonstrate polymorph-dependent alteration in neuronal network activity upon seeded aggregation of $\alpha$-Syn. Our findings bring new insight into how distinct $\alpha$-Syn polymorphs differentially bind to and seed monomeric $\alpha$-Syn aggregation within neurons, thus affectingneuronal homeostasis through the redistribution of synaptic protein

Clustering of Tau fibrils impairs the synaptic composition of α3‐Na + /K + ‐ ATP ase and AMPA receptors

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Data in support of the identification of neuronal and astrocyte proteins interacting with extracellularly applied oligomeric and fibrillar α-synuclein assemblies by mass spectrometry.

International audienceα-Synuclein (α-syn) is the principal component of Lewy bodies, the pathophysiological hallmark of individuals affected by Parkinson disease (PD). This neuropathologic form of α-syn contributes to PD progression and propagation of α-syn assemblies between neurons. The data we present here support the proteomic analysis used to identify neuronal proteins that specifically interact with extracellularly applied oligomeric or fibrillar α-syn assemblies (conditions 1 and 2, respectively) (doi: 10.15252/embj.201591397[1]). α-syn assemblies and their cellular partner proteins were pulled down from neuronal cell lysed shortly after exposure to exogenous α-syn assemblies and the associated proteins were identified by mass spectrometry using a shotgun proteomic-based approach. We also performed experiments on pure cultures of astrocytes to identify astrocyte-specific proteins interacting with oligomeric or fibrillar α-syn (conditions 3 and 4, respectively). For each condition, proteins interacting selectively with α-syn assemblies were identified by comparison to proteins pulled-down from untreated cells used as controls. The mass spectrometry data, the database search and the peak lists have been deposited to the ProteomeXchange Consortium database via the PRIDE partner repository with the dataset identifiers PRIDE: PXD002256 to PRIDE: PXD002263 and doi: 10.6019/PXD002256 to 10.6019/PXD002263