Surface Expression Of GABAA Receptors Research Articles

Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum.

Protein homeostasis (proteostasis) deficiency is an important contributing factor to neurological and metabolic diseases. However, how the proteostasis network orchestrates the folding and assembly of multi-subunit membrane proteins is poorly understood. Previous proteomics studies identified Hsp47 (Gene: SERPINH1), a heat shock protein in the endoplasmic reticulum lumen, as the most enriched interacting chaperone for gamma-aminobutyric acid type A (GABAA) receptors. Here, we show that Hsp47 enhances the functional surface expression of GABAA receptors in rat neurons and human HEK293T cells. Furthermore, molecular mechanism study demonstrates that Hsp47 acts after BiP (Gene: HSPA5) and preferentially binds the folded conformation of GABAA receptors without inducing the unfolded protein response in HEK293T cells. Therefore, Hsp47 promotes the subunit-subunit interaction, the receptor assembly process, and the anterograde trafficking of GABAA receptors. Overexpressing Hsp47 is sufficient to correct the surface expression and function of epilepsy-associated GABAA receptor variants in HEK293T cells. Hsp47 also promotes the surface trafficking of other Cys-loop receptors, including nicotinic acetylcholine receptors and serotonin type 3 receptors in HEK293T cells. Therefore, in addition to its known function as a collagen chaperone, this work establishes that Hsp47 plays a critical and general role in the maturation of multi-subunit Cys-loop neuroreceptors.

Control of cell surface expression of GABAA receptors by a conserved region at the end of the N-terminal extracellular domain of receptor subunits

Type A γ-aminobutyric acid receptors (GABAARs) represent a family of pentameric GABA-gated Cl-/HCO3- ion channels which mediate inhibitory transmission in the central nervous system. Cell surface expression of GABAARs, a prerequisite for their function, is dependent on the appropriate assembly of the receptor subunits and their transient interactions with molecular chaperones within the endoplasmic reticulum (ER) and Golgi apparatus. Here, we describe a highly conserved amino acid sequence within the extracellular N-terminal domain of the receptor subunits adjoining the first transmembrane domain as a region important for GABAAR processing within the ER. Modifications of this region in the α1, β3, and γ2 subunits using insertion or site-directed mutagenesis impaired GABAAR trafficking to the cell surface in heterologous cell systems although they had no effect on the subunit assembly. We found that mutated receptors accumulated in the ER where they were shown to associate with chaperones calnexin, BiP, and Grp94. However, their surface expression was increased when ER-associated degradation or proteosome function was inhibited, while modulation of ER calcium stores had little effect. When compared to the wt, mutated receptors showed decreased interaction with calnexin, similar binding to BiP, and increased association with Grp94. Structural modeling of calnexin interaction with the wt or mutated GABAAR revealed that disruption in structure caused by mutations in the conserved region adjoining the first transmembrane domain may impair calnexin binding. Thus, this previously uncharacterized region plays an important role in intracellular processing of GABAARs at least in part by stabilizing their interaction with calnexin.

Control of cell surface expression of GABAA receptors by a conserved region at the end of the N-terminal extracellular domain of receptor subunits

Control of cell surface expression of GABAA receptors by a conserved region at the end of the N-terminal extracellular domain of receptor subunits

Metformin produces anxiolytic-like effects in rats by facilitating GABAA receptor trafficking to membrane.

Altered function or expression of GABAA receptors contributes to anxiety disorders. Benzodiazepines are widely prescribed for the treatment of anxiety. However, the long-term use of benzodiazepines increases the risk of developing drug dependence and tolerance. Thus, it is urgent to explore new therapeutic approaches. Metformin is widely used to treat Type 2 diabetes and other metabolic syndromes. However, the role of metformin in psychiatric disorders, especially anxiety, remains largely unknown. We examined the effects of metformin on anxiety-like behaviour of rats in open field test and elevated plus maze test. We also observed the effect of metformin (10μM, in vitro; 100mg·kg-1 , in vivo) on the trafficking of GABAA receptors, as mechanisms underlying the anxiolytic effects of metformin. Metformin (100mg·kg-1 , i.p. 30min) displayed a robust and rapid anxiolytic effect, without tolerance. Metformin up-regulated the surface expression of GABAA receptors and increased miniature inhibitory postsynaptic currents (mIPSCs). AMP-activated protein kinase (AMPK) activated by metformin-induced stimulation of forkhead box O3a (FoxO3a) transcriptional activity, followed by increased expression of GABAA receptor-associated protein (GABARAP) and its binding to GABAA receptors finally resulted in the membrane insertion of GABAA receptors. Metformin increased mIPSCs by up-regulating the membrane insertion of GABAA receptors, via a pathway involving AMPK, FoxO3a, and the GABAA receptor-associated protein. Thus metformin has a potential new use in the treatment of anxiety disorders.

The female epilepsy protein PCDH19 is a new GABAAR-binding partner that regulates GABAergic transmission as well as migration and morphological maturation of hippocampal neurons.

The PCDH19 gene (Xp22.1) encodes the cell-adhesion protein protocadherin-19 (PCDH19) and is responsible for a neurodevelopmental pathology characterized by female-limited epilepsy, cognitive impairment and autistic features, the pathogenic mechanisms of which remain to be elucidated. Here, we identified a new interaction between PCDH19 and GABAA receptor (GABAAR) alpha subunits in the rat brain. PCDH19 shRNA-mediated downregulation reduces GABAAR surface expression and affects the frequency and kinetics of miniature inhibitory postsynaptic currents (mIPSCs) in cultured hippocampal neurons. In vivo, PCDH19 downregulation impairs migration, orientation and dendritic arborization of CA1 hippocampal neurons and increases rat seizure susceptibility. In sum, these data indicate a role for PCDH19 in GABAergic transmission as well as migration and morphological maturation of neurons.

The Influence of Regional Distribution and Pharmacologic Specificity of GABAAR Subtype Expression on Anesthesia and Emergence.

Anesthetics produce unconsciousness by modulating ion channels that control neuronal excitability. Research has shown that specific GABAA receptor (GABAAR) subtypes in particular regions of the central nervous system contribute to different hyperpolarizing conductances, and behaviorally to distinct components of the anesthetized state. The expression of these receptors on the neuron cell surface, and thus the strength of inhibitory neurotransmission, is dynamically regulated by intracellular trafficking mechanisms. Pharmacologic or activity-based perturbations to these regulatory systems have been implicated in pathology of several neurological conditions, and can alter the individual response to anesthesia. Furthermore, studies are beginning to uncover how anesthetic exposure itself elicits enduring changes in subcellular physiology, including the processes that regulate ion channel trafficking. Here, we review the mechanisms that determine GABAAR surface expression, and elaborate on influences germane to anesthesia and emergence. We address known trafficking differences between the intrasynaptic receptors that mediate phasic current and the extra-synaptic receptors mediating tonic current. We also describe neurophysiologic consequences and network-level abnormalities in brain function that result from receptor trafficking aberrations. We hypothesize that the relationship between commonly used anesthetic agents and GABAAR surface expression has direct consequences on mature functioning neural networks and by extension ultimately influence the outcome of patients that undergo general anesthesia. Rational design of new anesthetics, anesthetic techniques, EEG-based monitoring strategies, or emergence treatments will need to take these effects into consideration.

Surface expression of GABAA receptors in the rat nucleus accumbens is increased in early but not late withdrawal from extended-access cocaine self-administration

It is well established that cocaine-induced changes in glutamate receptor expression in the nucleus accumbens (NAc) play a significant role in animal models of cocaine addiction. Far less is known about cocaine-induced changes in GABA transmission, despite its importance in regulating NAc output via local interneurons and medium spiny neuron (MSN) axon collaterals (GABA ‘microcircuit’). Here we investigated whether GABAA receptor surface or total expression is altered following an extended-access cocaine self-administration regimen that produces a time-dependent intensification (incubation) of cue-induced cocaine craving in association with strengthening of AMPA receptor (AMPAR) transmission onto MSN. Rats self-administered cocaine or saline (control condition) 6h/day for 10 days. NAc tissue was obtained and surface proteins biotinylated on three withdrawal days (WD) chosen to span incubation of craving and associated AMPAR plasticity: WD2, WD25 and WD48. Immunoblotting was used to measure total and surface expression of three GABAA receptor subunits (α1, α2, and α4) that are strongly expressed in the NAc. We found a transient increase in surface, but not total, expression of the α2 subunit on WD2 from cocaine self-administration, an effect that was no longer observed by WD25. The expression of α1 and α4 subunits was not altered at these withdrawal times. On WD48, when AMPAR transmission is significantly potentiated, we did not find any alteration in GABAA receptor surface or total expression. Our findings suggest that the strengthening of AMPAR-mediated glutamate transmission in the NAc is not accompanied by compensatory strengthening of GABAergic transmission through insertion of additional GABAA receptors.

Propofol Regulates the Surface Expression of GABAA Receptors: Implications in Synaptic Inhibition.

The anesthetic propofol is thought to induce rapid hypnotic sedation by potentiating γ-aminobutyric acid receptor (GABAAR) activity. Little is known about the molecular mechanisms of propofol in modulating inhibitory synaptic transmission. We aimed to investigate the role of propofol in modulating surface expression of GABAARs. C57BL/6 mice received an intraperitoneal injection of propofol. Hippocampal pyramidal neurons were prepared from embryonic day-18 mice and were treated with propofol. Proteins on the plasma membrane were analyzed using cell surface biotinylation, immunoblotting and enzyme-linked immunosorbent assay. Electrophysiological activities were recorded from hippocampal cells in acute brain slices of mice. The interaction between GABAARs and clathrin adaptor protein 2 was assessed by immunoprecipitation. Phosphorylation of GABAARs was shown by in vitro kinase assay. Propofol facilitated membrane accumulation of GABAARβ3 subunits. Propofol mediated phosphorylation of GABAARβ3 by protein kinase Cε which blocked the interaction between GABAARβ3 and the β-adaptin subunit of adaptor protein 2, resulting in an inhibition of the receptor endocytosis in hippocampal pyramidal neurons. Coincident with increased GABAARs surface level, propofol enhanced evoked and miniature synaptic GABA receptor currents. This study offers new insight on the regulatory mechanism of propofol in inhibiting neuronal excitability.

BIG1, a Brefeldin A-Inhibited Guanine Nucleotide-Exchange Factor, Is Required for GABA-Gated Cl– Influx Through Regulation of GABAA Receptor Trafficking

GABAA receptors (GABAARs) mediate the majority of fast synaptic inhibition. Trafficking regulation and protein-protein interactions that maintain the appropriate number of GABAARs at the cell surface are considered to be important mechanisms for controlling the strength of synaptic inhibition. Here, we report that BIG1, a brefeldin A (BFA)-inhibited guanine nucleotide-exchange factor (GEF) which has a known role in vesicle trafficking, is a new binding partner of GABAARs. Treatment of neurons with BFA, an uncompetitive inhibitor of BIG1 GEF activity, or depletion of BIG1 by small RNA interference (siRNA) significantly decreased GABAARs at the neuronal surface and suppressed GABA-gated influx of chloride ions. Over-expression of HA-tagged BIG1-E793K, a dominant-negative mutant, also significantly decreased GABAARs at the neuronal surface, but had no effect on the total amount of GABAARs. Inhibition of GABAAR endocytosis by muscimol increased both GABAARs and BIG1 at the neuronal surface in a time-dependent fashion, and this increase could be abolished by bicuculline. Finally, depletion of BIG1 by siRNA inhibited the muscimol-stimulated increase of GABAARs. Those data suggest an important function of BIG1 in trafficking of GABAARs to the cell surface through its GEF activity. Thus, we identify an important role of BIG1 in modulating GABA-gated Cl(-) influx through the regulation of cell surface expression of GABAARs.

Disrupted GABAAR trafficking and synaptic inhibition in a mouse model of Huntington's disease

Growing evidence suggests that Huntington's disease (HD), a neurodegenerative movement disorder caused by the mutant huntingtin (htt) with an expanded polyglutamine (polyQ) repeat, is associated with the altered intracellular trafficking and synaptic function. GABAA receptors, the key determinant of the strength of synaptic inhibition, have been found to bind to the huntingtin associated protein 1 (HAP1). HAP1 serves as an adaptor linking GABAA receptors to the kinesin family motor protein 5 (KIF5), controlling the transport of GABAA receptors along microtubules in dendrites. In this study, we found that GABAAR-mediated synaptic transmission is significantly impaired in a transgenic mouse model of HD expressing polyQ-htt, which is accompanied by the diminished surface expression of GABAA receptors. Moreover, the GABAAR/HAP1/KIF5 complex is disrupted and dissociated from microtubules in the HD mouse model. These results suggest that GABAAR trafficking and function is impaired in HD, presumably due to the interference of KIF5-mediated microtubule-based transport of GABAA receptors. The diminished inhibitory synaptic efficacy could contribute to the loss of the excitatory/inhibitory balance, leading to increased neuronal excitotoxicity in HD.

Surface Expression of GABAA Receptors

Surface Expression of GABAA Receptors

D1‐like dopamine receptors regulate GABAA receptor function to modulate hippocampal neural progenitor cell proliferation

The proliferation and differentiation of neural progenitor (NP) cells can be regulated by neurotransmitters including GABA and dopamine. The present study aimed to examine how these two neurotransmitter systems interact to affect post-natal hippocampal NP cell proliferation in vitro. Mouse hippocampal NP cells express functional GABAA receptors, which upon activation led to an increase in intracellular calcium levels via the opening of L-type calcium channels. Activation of these GABAA receptors also caused a significant decrease in proliferation; an effect that required the entry of calcium through L-type calcium channels. Furthermore, while activation of D1-like dopamine receptors had no effect on proliferation, it abrogated the suppressive effects of GABAA receptor activation on proliferation. The effects of D1-like dopamine receptors are associated with a decrease in the ability of GABAA receptors to increase intracellular calcium levels, and a reduction in the surface expression of GABAA receptors. In this way, D1-like dopamine receptor activation can increase the proliferation of NP cells by preventing GABAA receptor-mediated inhibition of proliferation. These results suggest that, in conditions where NP cell proliferation is under the tonic suppression of GABA, agonists which act through D1-like dopamine receptors may increase the proliferation of neural progenitors.

Selective Changes in Hippocampal GABAA Receptors during Status Epilepticus

Subunit-Specific Trafficking of >GABAA Receptors During Status Epilepticus. Goodkin HP, Joshi S, Mtchedlishvili Z, Brar J, Kapur J. J Neurosci 2008 5;28(10):2527–2538. It is proposed that a reduced surface expression of GABAA receptors (GABARs) contributes to the pathogenesis of status epilepticus (SE), a condition characterized by prolonged seizures. This hypothesis was based on the finding that prolonged epileptiform bursting (repetitive bursts of prolonged depolarizations with superimposed action potentials) in cultures of dissociated hippocampal pyramidal neurons (dissociated cultures) results in the increased intracellular accumulation of GABARs. However, it is not known whether this rapid modification in the surface-expressed GABAR pool results from selective, subunit-dependent or nonselective, subunit-independent internalization of GABARs. In hippocampal slices obtained from animals undergoing prolonged SE (SE-treated slices), we found that the surface expression of the GABAR β2/3 and γ2 subunits was reduced, whereas that of the δ subunit was not. Complementary electrophysiological recordings from dentate granule cells in SE-treated slices demonstrated a reduction in GABAR-mediated synaptic inhibition, but not tonic inhibition. A reduction in the surface expression of the γ2 subunit, but not the δ subunit was also observed in dissociated cultures and organotypic hippocampal slice cultures when incubated in an elevated KCl external medium or an elevated KCl external medium supplemented with NMDA, respectively. Additional studies demonstrated that the reduction in the surface expression of the γ2 subunit was independent of direct ligand binding of the GABAR. These findings demonstrate that the regulation of surface-expressed GABAR pool during SE is subunit-specific and occurs independent of ligand binding. The differential modulation of the surface expression of GABARs during SE has potential implications for the treatment of this neurological emergency.

Surface Expression of GABAA Receptors Is Transcriptionally Controlled by the Interplay of cAMP-response Element-binding Protein and Its Binding Partner Inducible cAMP Early Repressor

The regulated expression of type A gamma-aminobutyric acid (GABA) receptor (GABA(A)R) subunit genes plays a critical role in neuronal maturation and synaptogenesis. It is also associated with a variety of neurological diseases. Changes in GABA(A) receptor alpha1 subunit gene (GABRA1) expression have been reported in animal models of epilepsy, alcohol abuse, withdrawal, and stress. Understanding the genetic mechanism behind such changes in alpha subunit expression will lead to a better understanding of the role that signal transduction plays in control over GABA(A)R function and brings with it the promise of providing new therapeutic tools for the prevention or cure of a variety of neurological disorders. Here we show that activation of protein kinase C increases alpha1 subunit levels via phosphorylation of CREB (pCREB) that is bound to the GABRA1 promoter (GABRA1p). In contrast, activation of protein kinase A decreases levels of alpha1 even in the presence of pCREB. Decrease of alpha1 is dependent upon the inducible cAMP early repressor (ICER) as directly demonstrated by ICER-induced down-regulation of endogenous alpha1-containing GABA(A)Rs at the cell surface of cortical neurons. Taken together with the fact that there are less alpha1gamma2-containing GABA(A)Rs in neurons after protein kinase A stimulation and that activation of endogenous dopamine receptors down-regulates alpha1 subunit mRNA levels subsequent to induction of ICER, our studies identify a transcriptional mechanism for regulating the cell surface expression of alpha1-containing GABA(A)Rs that is dependent upon the formation of CREB heterodimers.

Impact of receptor changes on treatment of status epilepticus

Neurotransmitter receptors play a key role in the pathogenesis and current treatment strategies for status epilepticus (SE). Benzodiazepines are commonly used to treat SE exert their anticonvulsant action by stimulating γ-aminobutyric type A receptors (GABAA receptors). These drugs are quite effective in terminating SE in approximately 55–66% of patients, but there is a residual group of patients with seizures resistant to benzodiazepeines. The mechanisms underlying initiation and maintenance of prolonged SE were explored.

Regulation of Gephyrin and GABAAReceptor Binding within the Amygdala after Fear Acquisition and Extinction

Both the acquisition and extinction of conditioned fear appear to require the basolateral amygdala (BLA). Because these two forms of learning have opposing effects on the expression of conditioned fear, we hypothesized that they may modulate GABAergic tone differentially within the BLA. Previously, we reported that gene expression for the GABA(A) receptor clustering protein gephyrin was significantly downregulated in the BLA after fear acquisition (Ressler et al., 2002). Here we demonstrate an analogous decrease in BLA gephyrin protein levels, together with a decrease in the surface expression of GABA(A) receptors in the BLA after fear acquisition, as evidenced by decreased binding of H3-flunitrazepam. In marked contrast, gephyrin mRNA and protein levels in the BLA significantly increased after extinction training, as did H3-flunitrazepam binding. These results implicate the protein gephyrin in both fear acquisition and extinction and suggest that the modulation of gephyrin and GABA(A) receptor expression in the BLA may play a role in the experience-dependent plasticity underlying both of these types of learning. Furthermore, these results demonstrate that physiologically relevant, dynamic alterations of GABAergic synapses occur during the consolidation phase of BLA-dependent learning and may interact with previously described alterations in glutamatergic transmission to initiate and stabilize memory formation in vivo.

Activity-dependent regulation of GABAA receptors.

GABAA receptor heterogeneity is based on the combinatorial assembly of a large family of subunits into distinct receptor subtypes. A neuron-specific expression pattern of receptor subtypes has been demonstrated in adult rat brain, which can be reproduced in vitro in primary neuron cultures. This suggests that genetic programs established during ontogeny govern the expression of gamma-aminobutyric acid (GABAA) receptor subtypes. Activity-dependent mechanisms nevertheless modulate on a short-term basis the cell surface expression of GABAA receptors, as demonstrated in cultured hippocampal neurons upon blockade of synaptic transmission or application of brain-derived neurotrophic factor. Preliminary evidence points to changes in protein phosphorylation as a mechanism underlying short-term activity-dependent regulation of GABAA receptors. In vivo, chronic pharmacological modulation of neuronal activity during development, while having marked effects on the rate of cortical growth, failed to influence the expression of GABAA receptor subtypes, suggesting that additional factors are involved.