GABAAR isoform and subunit structural motifs determine synaptic and extrasynaptic receptor localisation (original) (raw)
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Dynamic mobility of functional GABAA receptors at inhibitory synapses
Nature Neuroscience, 2005
Importing functional GABA A receptors into synapses is fundamental for establishing and maintaining inhibitory transmission and for controlling neuronal excitability. By introducing a binding site for an irreversible inhibitor into the GABA A receptor a1 subunit channel lining region that can be accessed only when the receptor is activated, we have determined the dynamics of receptor mobility between synaptic and extrasynaptic locations in hippocampal pyramidal neurons. We demonstrate that the cell surface GABA A receptor population shows no fast recovery after irreversible inhibition. In contrast, after selective inhibition, the synaptic receptor population rapidly recovers by the import of new functional entities within minutes. The trafficking pathways that promote rapid importation of synaptic receptors do not involve insertion from intracellular pools, but reflect receptor diffusion within the plane of the membrane. This process offers the synapse a rapid mechanism to replenish functional GABA A receptors at inhibitory synapses and a means to control synaptic efficacy.
GABAA receptor subunit composition and competition at synapses are tuned by GABAB receptor activity
Molecular and Cellular Neuroscience, 2014
GABA B Rs have a well-established role in controlling neuronal excitability and presynaptic neurotransmitter release. We examined the role of GABA B R activity in modulating the number and lateral diffusion of GABA A Rs at inhibitory synapses. Changes in diffusion of GABA A Rs at synapses were observed when subunit heterogeneity was taken into account. While α1-GABA A Rs were unaffected, α2and α5-GABA A Rs showed inverse changes in enrichment and diffusion. The intracellular TM3-4 loop of α2 was sufficient to observe the changes in diffusion by GABA B R activity, whereas the loop of α5 was not. The opposing effect on α2and α5-GABA A Rs was caused by a competition between GABA A Rs for binding slots at synapses. Receptor immobilization by cross-linking revealed that α5-GABA A R trapping at synapses is regulated by modulation of α2-GABA A R mobility. Finally, PKC activity was determined to be part of the signaling pathway through which GABA B R activity modulates α2-GABA A R diffusion at synapses. These results outline a novel mechanism for tuning inhibitory transmission in a subunit-specific manner, and for the first time describe competition between GABA A Rs with different subunit compositions for binding slots at synapses.
Plasticity of GABA transporters: an unconventional route to shape inhibitory synaptic transmission
2014
The brain relies on GABAergic neurons to control the ongoing activity of neuronal networks. GABAergic neurons control the firing pattern of excitatory cells, the temporal structure of membrane potential oscillations and the time window for integration of synaptic inputs. These actions require a fine control of the timing of GABA receptor activation which, in turn, depends on the precise timing of GABA release from pre-synaptic terminals and GABA clearance from the extracellular space. Extracellular GABA is not subject to enzymatic breakdown, and its clearance relies entirely on diffusion and uptake by specific transporters. In contrast to glutamate transporters, GABA transporters are abundantly expressed in neuronal pre-synaptic terminals. GABA transporters move laterally within the plasma membrane and are continuously trafficked to/from intracellular compartments. It is hypothesized that due to their proximity to GABA release sites, changes in the concentration and lateral mobility of GABA transporters may have a significant effect on the time course of the GABA concentration profile in and out of the synaptic cleft. To date, this hypothesis remains to be tested. Here we use 3D Monte Carlo reaction-diffusion simulations to analyze how changes in the density of expression and lateral mobility of GABA transporters in the cell membrane affect the extracellular GABA concentration profile and the activation of GABA receptors. Our results indicate that these manipulations mainly alter the GABA concentration profile away from the synaptic cleft. These findings provide novel insights into how the ability of GABA transporters to undergo plastic changes may alter the strength of GABAergic signals and the activity of neuronal networks in the brain.
Synaptic GABAA receptors are directly recruited from their extrasynaptic counterparts
The EMBO Journal, 2006
GABA A receptors mediate the majority of fast synaptic inhibition in the brain. The accumulation of these ligand-gated ion channels at synaptic sites is a prerequisite for neuronal inhibition, but the molecular mechanisms underlying this phenomenon remain obscure. To further understand these processes, we have examined the cellular origins of synaptic GABA A receptors. To do so, we have created fluorescent GABA A receptors that are capable of binding a-bungarotoxin (Bgt), facilitating the visualization of receptor endocytosis, exocytosis and delivery to synaptic sites. Imaging with Bgt in hippocampal neurons revealed that GABA A receptor endocytosis occurred exclusively at extrasynaptic sites, consistent with the preferential colocalization of extrasynaptic receptors with the AP2 adaptin. Receptor insertion into the plasma membrane was also predominantly extrasynaptic, and pulse-chase analysis revealed that these newly inserted receptors were then able to access directly synaptic sites. Therefore, our results demonstrate that synaptic GABA A receptors are directly recruited from their extrasynaptic counterparts. Moreover, they illustrate a dynamic mechanism for neurons to modulate GABA A receptor number at inhibitory synapses by controlling the stability of extrasynaptic receptors.
Activity-Dependent Tuning of Inhibitory Neurotransmission Based on GABAAR Diffusion Dynamics
Neuron, 2009
An activity-dependent change in synaptic efficacy is a central tenet in learning, memory, and pathological states of neuronal excitability. The lateral diffusion dynamics of neurotransmitter receptors are one of the important parameters regulating synaptic efficacy. We report here that neuronal activity modifies diffusion properties of type-A GABA receptors (GABA A R) in cultured hippocampal neurons: enhanced excitatory synaptic activity decreases the cluster size of GABA A Rs and reduces GABAergic mIPSC. Single-particle tracking of the GABA A R g2 subunit labeled with quantum dots reveals that the diffusion coefficient and the synaptic confinement domain size of GABA A R increases in parallel with neuronal activity, depending on Ca 2+ influx and calcineurin activity. These results indicate that GABA A R diffusion dynamics are directly linked to rapid and plastic modifications of inhibitory synaptic transmission in response to changes in intracellular Ca 2+ concentration. This transient activity-dependent reduction of inhibition would favor the onset of LTP during conditioning.
Journal of Biological Chemistry, 2012
Background: GABA A receptor ␥2 and ␦ subunits are thought to be responsible for synaptic and extrasynaptic targeting. Results: We demonstrate here that ␣2 and ␣6 subunits can target ␦/␥2 chimeras to synaptic and extrasynaptic sites. Conclusion: The ␣ subunits play a direct role in GABA A receptor targeting. Significance: Different subunits of GABA A receptors encode intrinsic signals to control subcellular targeting. GABA A receptors (GABA A-Rs) are localized at both synaptic and extrasynaptic sites, mediating phasic and tonic inhibition, respectively. Previous studies suggest an important role of ␥2 and ␦ subunits in synaptic versus extrasynaptic targeting of GABA A-Rs. Here, we demonstrate differential function of ␣2 and ␣6 subunits in guiding the localization of GABA A-Rs. To study the targeting of specific subtypes of GABA A-Rs, we used a molecularly engineered GABAergic synapse model to precisely control the GABA A-R subunit composition. We found that in neuron-HEK cell heterosynapses, GABAergic events mediated by ␣23␥2 receptors were very fast (rise time ϳ2 ms), whereas events mediated by ␣63␦ receptors were very slow (rise time ϳ20 ms). Such an order of magnitude difference in rise time could not be attributed to the minute differences in receptor kinetics. Interestingly, synaptic events mediated by ␣63 or ␣63␥2 receptors were significantly slower than those mediated by ␣23 or ␣23␥2 receptors, suggesting a differential role of ␣ subunit in receptor targeting. This was confirmed by differential targeting of the same ␦-␥2 chimeric subunits to synaptic or extrasynaptic sites, depending on whether it was co-assembled with the ␣2 or ␣6 subunit. In addition, insertion of a gephyrin-binding site into the intracellular domain of ␣6 and ␦ subunits brought ␣63␦ receptors closer to synaptic sites. Therefore, the ␣ subunits, together with the ␥2 and ␦ subunits, play a critical role in governing synaptic versus extrasynaptic targeting of GABA A-Rs, possibly through differential interactions with gephyrin. Neural inhibition in the brain is mostly mediated by GABA A receptors (GABA A-Rs). 2 To date, 19 isoforms of GABA A-R subunits have been identified as follows: ␣1-6, 1-3, ␥1-3, ␦, ⑀, , , and 1-3 (1, 2). Most GABA A-Rs expressed in the brain are composed of two ␣, two , and one ␥ subunits, of which the ␥ subunit can be substituted by ␦, ⑀, , or (3, 4). There are two forms of GABAergic inhibition, phasic and tonic (5, 6). Phasic inhibition is mediated by postsynaptically clustered GABA A-Rs composed of ␣1-3, 2-3, and ␥2 subunits. Tonic inhibition is mediated by extrasynaptic GABA A-Rs typically composed of ␣4/6 (and possibly ␣1),  and ␦ subunits (7-9), as well as ␣5␥2 subunits (10-12). Blocking tonic inhibition significantly enhanced neuronal excitability (5, 13-15). Malfunction of tonic inhibition is implicated in epilepsy, abnormal cognition and memory, sleep disorders, anxiety, depression, schizophrenia, and alcohol addiction (16-23). Although the mechanisms for synaptic receptor targeting have been extensively studied, little is known about the molecular mechanisms specifying extrasynaptic targeting of ␦ subunit-containing GABA A-Rs. Neurons deficient in the ␣1 or ␣2 or ␣3 subunits showed diminished postsynaptic GABA A-R clusters in different subcellular localizations (24-27). The ␥2 subunit, and particularly its intracellular loop (IL) and the fourth transmembrane domain (TM4), plays a critical role in synaptic clustering of GABA A-Rs (28-31). In contrast, the ␦ subunit-containing GABA A-Rs are mainly localized at extrasynaptic membranes (7, 8). Thus, the ␥2 and ␦ subunits have been thought to be involved in the synaptic versus extrasynaptic targeting of GABA A-Rs. However, the mostly extrasynaptic ␣5␥2 and punctated ␣1␦ GABA A-Rs suggest that ␥2 and ␦ subunits cannot be solely responsible for guiding GABA A-R targeting (9, 10, 12). Here, we employed a molecularly engineered synapse model to investigate the mechanism of ␦-GABA A-R targeting. We
Diffusion Barriers Constrain Receptors at Synapses
PLoS ONE, 2012
The flux of neurotransmitter receptors in and out of synapses depends on receptor interaction with scaffolding molecules. However, the crowd of transmembrane proteins and the rich cytoskeletal environment may constitute obstacles to the diffusion of receptors within the synapse. To address this question, we studied the membrane diffusion of the caminobutyric acid type A receptor (GABA A R) subunits clustered (c2) or not (a5) at inhibitory synapses in rat hippocampal dissociated neurons. Relative to the extrasynaptic region, c2 and a5 showed reduced diffusion and increased confinement at both inhibitory and excitatory synapses but they dwelled for a short time at excitatory synapses. In contrast, c2 was ,3fold more confined and dwelled ,3-fold longer in inhibitory synapses than a5, indicating faster synaptic escape of a5. Furthermore, using a gephyrin dominant-negative approach, we showed that the increased residency time of c2 at inhibitory synapses was due to receptor-scaffold interactions. As shown for GABA A R, the excitatory glutamate receptor 2 subunit (GluA2) of the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) had lower mobility in both excitatory and inhibitory synapses but a higher residency time at excitatory synapses.
GABA Potency at GABAA Receptors Found in Synaptic and Extrasynaptic Zones
Frontiers in Cellular Neuroscience, 2012
The potency of GABA is vitally important for its primary role in activating GABA A receptors and acting as an inhibitory neurotransmitter. Although numerous laboratories have presented information, directly or indirectly, on GABA potency, it is often difficult to compare across such studies given the inevitable variations in the methods used, the cell types studied, whether native or recombinant receptors are examined, and their relevance to native synaptic and extrasynaptic GABA A receptors. In this review, we list the most relevant isoforms of synaptic and extrasynaptic GABA A receptors that are thought to assemble in surface membranes of neurons in the central nervous system. Using consistent methodology in one cell type, the potencies of the endogenous neurotransmitter GABA are compared across a spectrum of GABA A receptors. The highest potency for GABA is measured when activating extrasynaptic-type α6 subunit-containing receptors, whereas synaptic-type α2β3γ2 and α3β3γ2 receptors exhibited the lowest potency, and other GABA A receptor subtypes that are found both in synaptic and extrasynaptic compartments, showed intermediate sensitivities to GABA. The relatively simple potency relationship between GABA and its target receptors is important as it serves as one of the major determinants of GABA A receptor activation, with consequences for the development of inhibition, either by tonic or phasic mechanisms.
Journal of Neuroscience, 2011
The majority of fast synaptic inhibition in the brain is mediated by benzodiazepine-sensitive ␣1-subunit-containing GABA type A receptors (GABA A Rs); however, our knowledge of the mechanisms neurons use to regulate their synaptic accumulation is rudimentary. Using immunoprecipitation, we demonstrate that GABA A Rs and gephyrin are intimately associated at inhibitory synapses in cultured rat neurons. In vitro we reveal that the E-domain of gephyrin directly binds to the ␣1 subunit with an affinity of ϳ20 M, mediated by residues 360 -375 within the intracellular domain of this receptor subunit. Mutating residues 360 -375 decreases both the accumulation of ␣1-containing GABA A Rs at gephyrin-positive inhibitory synapses in hippocampal neurons and the amplitude of mIPSCs. We also demonstrate that the affinity of gephyrin for the ␣1 subunit is modulated by Thr375, a putative phosphorylation site. Mutation of Thr375 to a phosphomimetic, negatively charged amino acid decreases both the affinity of the ␣1 subunit for gephyrin, and therefore receptor accumulation at synapses, and the amplitude of mIPSCs. Finally, single-particle tracking reveals that gephyrin reduces the diffusion of ␣1-subunit-containing GABA A Rs specifically at inhibitory synapses, thereby increasing their confinement at these structures. Our results suggest that the direct binding of gephyrin to residues 360 -375 of the ␣1 subunit and its modulation are likely to be important determinants for the stabilization of GABA A Rs at synaptic sites, thereby modulating the strength of synaptic inhibition.
Expression of Distinct Subunits of GABAA Receptor Regulates Inhibitory Synaptic Strength
Journal of Neurophysiology, 2004
Expression of distinct ␣ subunits of GABA A receptor regulates inhibitory synaptic strength. . Distinct ␣ subunit subtypes in the molecular assembly of GABA A receptors are a critical determinant of the functional properties of inhibitory synapses and their modulation by a range of pharmacological agents. We investigated the contribution of these subunits to the developmental changes of inhibitory synapses in cerebellar granule neurons in primary cultures from wild-type and ␣1 subunit Ϫ/Ϫ mice. The decay time of miniature inhibitory postsynaptic currents (mIPSCs) halved between 6 days in vitro (DIV6) and DIV12. This was paralleled by the decrease of ␣2 and ␣3 subunits, the increase of ␣1 and ␣6 subunits expression at synapses, and changes in the action of selective ␣ subunit modulators. A small but significant shortening of mIPSCs was observed with development in cells from Ϫ/Ϫ mice together with a decrease in the expression of ␣3 subunit. In contrast, the expression of ␣2 subunit at inhibitory synapses in Ϫ/Ϫ cells was significantly higher than in ϩ/ϩ cells at DIV11-12. ␣5 subunit was not detected, and increased sensitivity to a selective ␣4/␣6 subunit agonist suggests increased expression of extrasynaptic receptors in Ϫ/Ϫ mice. 2/3 subunit expression and loreclezole sensitivity increased with development in ϩ/ϩ but not in Ϫ/Ϫ cells, supporting the preferential association of the ␣1 with the 2 subunit. Synaptic charge transfer strongly decreased with development but was not different between cells in the ϩ/ϩ and Ϫ/Ϫ groups until DIV11-12. Our results uncover a pattern of sequential expression of ␣ subunits underlying the changes in functional efficacy of GABAergic networks with development. RA. Effects of ␥2S subunit incorporation on GABA A receptor macroscopic kinetics. Neuropharmacology 44: 1003-1012, 2003. Bollan K, King D, Robertson LA, Brown K, Taylor PM, Moss SJ, and Connolly CN. GABA A receptor composition is determined by distinct assembly signals within ␣ and  Subunits. J Biol Chem 278: 4747-4755, 2003. Brickley SG, Cull-Candy SG, and Farrant M. Development of a tonic form of synaptic inhibition in rat cerebellar granule cells resulting from persistent activation of GABA A receptors. J Physiol 497: 753-759, 1996. Brickley SG, Revilla V, Cull-Candy SG, Wisden W, and Farrant M. Adaptive regulation of neuronal excitability by a voltage-independent potassium conductance. Nature 409: 88 -92, 2001. Brown N, Kerby J, Bonnert TP, Whiting PJ, and Wafford KA. Pharmacological characterization of a novel cell line expressing human ␣43␦ GABA A receptors. Br J Pharmacol 136: 965-974, 2002. Brunig I, Scotti E, Sidler C, and Fritschy JM. Intact sorting, targeting, and clustering of GABA A receptor subtypes in hippocampal neurons in vitro. Wenthold RJ, Bredt DS, and Nicoll RA. Stargazing regulates synaptic targeting of AMPA receptors by two distinct mechanisms. Nature 408: 936 -943, 2000. Christie SB, Miralles CP, and De Blas AL GAB Aergic innervation organizes synaptic and extrasynaptic GABA A receptor clustering in cultured hippocampal neurons. J Neurosci 22: 684 -697, 2002. Ebert B, Thompson SA, Saounatsou K, McKernan R, Krogsgaard-Larsen P, and Wafford KA. Differences in agonist/antagonist binding affinity and receptor transduction using recombinant human GABA A receptors. Mol Pharmacol 52: 1150 -1156, 1997. Farrant M and Brickley SG. Properties of GABA A receptor-mediated transmission at newly formed Golgi-granule cell synapses in the cerebellum. Neuropharmacology 44: 181-189, 2003. Farrant M, Wisden W, Cull-Candy S, and Brickley SG. Absence of tonic GABA A mediated conductance in cerebellar granule cells of ␣6 Ϫ/Ϫ mice. Soc Neurosci Abstr 25: 246, 1999. Fisher JL, Hinkle DJ, and Macdonald RL. Loreclezole inhibition of recombinant ␣11␥2L GABA A receptor single channel currents. Neuropharmacology 39: 235-245, 2000. Fritschy JM, Paysan J, and Mohler H. Switch in the expression of rat GABA A receptor subtypes during postnatal development: an immunohistochemical study. J Neurosci 9: 5302-5324, 1994. Gallo V, Kingsbury A, Balazs R, and Jorgensen OS. The role of depolarization in the survival and differentiation of cerebellar granule cells in culture. J Neurosci 7: 2203-2213, 1987. Gao B and Fritschy JM. Cerebellar granule cells in vitro recapitulate the in vivo pattern of GABA A -receptor subunit expression. Brain Res Dev Brain Res 88: 1-16, 1995. Gingrich KJ, Roberts WA, and Kass RS. Dependence of the GABA A receptor gating kinetics on the ␣-subunit isoform: implications for structurefunction relations and synaptic transmission. J Physiol 489: 529 -543, 1995. Hamann M, Rossi DJ, and Attwell D. Tonic and spillover inhibition of granule cells control information flow through cerebellar cortex. Neuron 33: 625-633, 2002. Kaneda M, Farrant M, and Cull-Candy SG. Whole-cell and single-channel currents activated by GABA and glycine in granule cells of the rat cerebellum.