Endoplasmic Reticulum Sorting and Kinesin-1 Command the Targeting of Axonal GABA(B) Receptors

In neuronal cells the intracellular trafficking machinery controls the availability of neurotransmitter receptors at the plasma membrane, which is a critical determinant of synaptic strength. Metabotropic γ amino-butyric acid (GABA) type B receptors (GABA(B)Rs) are neurotransmitter receptors that modulate synaptic transmission by mediating the slow and prolonged responses to GABA. GABA(B)Rs are obligatory heteromers constituted by two subunits, GABA(B)R1 and GABA(B)R2. GABA(B)R1a and GABA(B)R1b are the most abundant subunit variants. GABA(B)R1b is located in the somatodendritic domain whereas GABA(B)R1a is additionally targeted to the axon. Sushi domains located at the N-terminus of GABA(B)R1a constitute the only difference between both variants and are necessary and sufficient for axonal targeting. The precise targeting machinery and the organelles involved in sorting and transport have not been described. Here we demonstrate that GABA(B)Rs require the Golgi apparatus for plasma membrane delivery but that axonal sorting and targeting of GABA(B)R1a operate in a pre-Golgi compartment. In the axon GABA(B)R1a subunits are enriched in the endoplasmic reticulum (ER), and their dynamic behavior and colocalization with other secretory organelles like the ER-to-Golgi intermediate compartment (ERGIC) suggest that they employ a local secretory route. The transport of axonal GABA(B)R1a is microtubule-dependent and kinesin-1, a molecular motor of the kinesin family, determines axonal localization. Considering that progression of GABA(B)Rs through the secretory pathway is regulated by an ER retention motif our data contribute to understand the role of the axonal ER in non-canonical sorting and targeting of neurotransmitter receptors.     

Pharmacological analysis of the activation and receptor properties of the tonic GABA(C)R current in retinal bipolar cell terminals

GABAergic inhibition in the central nervous system (CNS) can occur via rapid, transient postsynaptic currents and via a tonic increase in membrane conductance, mediated by synaptic and extrasynaptic GABA(A) receptors (GABA(A)Rs) respectively. Retinal bipolar cells (BCs) exhibit a tonic current mediated by GABA(C)Rs in their axon terminal, in addition to synaptic GABA(A)R and GABA(C)R currents, which strongly regulate BC output. The tonic GABA(C)R current in BC terminals (BCTs) is not dependent on vesicular GABA release, but properties such as the alternative source of GABA and the identity of the GABA(C)Rs remain unknown. Following a recent report that tonic GABA release from cerebellar glial cells is mediated by Bestrophin 1 anion channels, we have investigated their role in non-vesicular GABA release in the retina. Using patch-clamp recordings from BCTs in goldfish retinal slices, we find that the tonic GABA(C)R current is not reduced by the anion channel inhibitors NPPB or flufenamic acid but is reduced by DIDS, which decreases the tonic current without directly affecting GABA(C)Rs. All three drugs also exhibit non-specific effects including inhibition of GABA transporters. GABA(C)R ρ subunits can form homomeric and heteromeric receptors that differ in their properties, but BC GABA(C)Rs are thought to be ρ1-ρ2 heteromers. To investigate whether GABA(C)Rs mediating tonic and synaptic currents may differ in their subunit composition, as is the case for GABA(A)Rs, we have examined the effects of two antagonists that show partial ρ subunit selectivity: picrotoxin and cyclothiazide. Tonic and synaptic GABA(C)R currents were differentially affected by both drugs, suggesting that a population of homomeric ρ1 receptors contributes to the tonic current. These results extend our understanding of the multiple forms of GABAergic inhibition that exist in the CNS and contribute to visual signal processing in the retina.     

The ubiquitin-like protein Plic-1 enhances the membrane insertion of GABAA receptors by increasing their stability within the endoplasmic reticulum

Gamma-aminobutyric acid receptors (GABA(A)R) are the major sites of fast inhibitory neurotransmission in the brain, and a critical determinant for the efficacy of neuronal inhibition is the number of these receptors that are expressed on the neuronal cell surface. GABA(A)Rs are heteropentamers that can be constructed from seven subunit classes with multiple members; alpha, beta, gamma(1-3), delta, epsilon(1-3), theta, and pi. Receptor assembly occurs within the endoplasmic reticulum, and it is evident that transport-competent combinations exiting this organelle can access the cell surface, whereas unassembled subunits are ubiquitinated and subject to proteasomal degradation. In a previous report the ubiquitin-like protein Plic-1 was shown to directly interact with GABA(A)Rs and promote their accumulation at the cell surface. In this study we explore the mechanisms by which Plic-1 regulates the membrane trafficking of GABA(A)Rs. Using both recombinant and neuronal preparations it was apparent that Plic-1 increased the stability of endoplasmic reticulum resident GABA(A)Rs together with an increase in the abundance of poly-ubiquitinated receptor subunits. Furthermore, Plic-1 elevated cell surface expression levels by selectively increasing their rates of membrane insertion. Thus, Plic-1 may play a significant role in regulating the strength of synaptic inhibition by increasing the stability of GABA(A)Rs within the secretory pathway and thereby promoting their insertion into the neuronal plasma membrane.     

GABAA receptor trafficking-mediated plasticity of inhibitory synapses

Proper developmental, neural cell-type-specific, and activity-dependent regulation of GABAergic transmission is essential for virtually all aspects of CNS function. The number of GABA(A) receptors in the postsynaptic membrane directly controls the efficacy of GABAergic synaptic transmission. Thus, regulated trafficking of GABA(A) receptors is essential for understanding brain function in both health and disease. Here we summarize recent progress in the understanding of mechanisms that allow dynamic adaptation of cell surface expression and postsynaptic accumulation and function of GABA(A) receptors. This includes activity-dependent and cell-type-specific changes in subunit gene expression, assembly of subunits into receptors, as well as exocytosis, endocytic recycling, diffusion dynamics, and degradation of GABA(A) receptors. In particular, we focus on the roles of receptor-interacting proteins, scaffold proteins, synaptic adhesion proteins, and enzymes that regulate the trafficking and function of receptors and associated proteins. In addition, we review neuropeptide signaling pathways that affect neural excitability through changes in GABA(A)R trafficking.     

GISP: a novel brain-specific protein that promotes surface expression and function of GABA(B) receptors

Synaptic transmission depends on the regulated surface expression of neurotransmitter receptors, but many of the cellular processes required to achieve this remain poorly understood. To better define specific mechanisms for the GABA(B) receptor (GABA(B)R) trafficking, we screened for proteins that bind to the carboxy-terminus of the GABA(B1) subunit. We report the identification and characterization of a novel 130-kDa protein, GPCR interacting scaffolding protein (GISP), that interacts directly with the GABA(B1) subunit via a coiled-coil domain. GISP co-fractionates with GABA(B)R and with the postsynaptic density and co-immunoprecipitates with GABA(B1) and GABA(B2) from rat brain. In cultured hippocampal neurons, GISP displays a punctate dendritic distribution and has an overlapping localization with GABA(B)Rs. When co-expressed with GABA(B)Rs in human embryonic kidney cells, GISP promotes GABA(B)R surface expression and enhances both baclofen-evoked extracellular signal-regulated kinase (ERK) phosphorylation and G-protein inwardly rectifying potassium channel (GIRK) currents. These results suggest that GISP is involved in the forward trafficking and stabilization of functional GABA(B)Rs.     

Two pools of Triton X-100-insoluble GABA(A) receptors are present in the brain, one associated to lipid rafts and another one to the post-synaptic GABAergic complex

Rat forebrain synaptosomes were extracted with Triton X-100 at 4 degrees C and the insoluble material, which is enriched in post-synaptic densities (PSDs), was subjected to sedimentation on a continuous sucrose gradient. Two pools of Triton X-100-insoluble gamma-aminobutyric acid type-A receptors (GABA(A)Rs) were identified: (i) a higher-density pool (rho = 1.10-1.15 mg/mL) of GABA(A)Rs that contains the gamma2 subunit (plus alpha and beta subunits) and that is associated to gephyrin and the GABAergic post-synaptic complex and (ii) a lower-density pool (rho = 1.06-1.09 mg/mL) of GABA(A)Rs associated to detergent-resistant membranes (DRMs) that contain alpha and beta subunits but not the gamma2 subunit. Some of these GABA(A)Rs contain the delta subunit. Two pools of GABA(A)Rs insoluble in Triton X-100 at 4 degrees C were also identified in cultured hippocampal neurons: (i) a GABA(A)R pool that forms clusters that co-localize with gephyrin and remains Triton X-100-insoluble after cholesterol depletion and (ii) a GABA(A)R pool that is diffusely distributed at the neuronal surface that can be induced to form GABA(A)R clusters by capping with an anti-alpha1 GABA(A)R subunit antibody and that becomes solubilized in Triton X-100 at 4 degrees C after cholesterol depletion. Thus, there is a pool of GABA(A)Rs associated to lipid rafts that is non-synaptic and that has a subunit composition different from that of the synaptic GABA(A)Rs. Some of the lipid raft-associated GABA(A)Rs might be involved in tonic inhibition.     

Gephyrin-independent GABA(A)R mobility and clustering during plasticity

The activity-dependent modulation of GABA-A receptor (GABA(A)R) clustering at synapses controls inhibitory synaptic transmission. Several lines of evidence suggest that gephyrin, an inhibitory synaptic scaffold protein, is a critical factor in the regulation of GABA(A)R clustering during inhibitory synaptic plasticity induced by neuronal excitation. In this study, we tested this hypothesis by studying relative gephyrin dynamics and GABA(A)R declustering during excitatory activity. Surprisingly, we found that gephyrin dispersal is not essential for GABA(A)R declustering during excitatory activity. In cultured hippocampal neurons, quantitative immunocytochemistry showed that the dispersal of synaptic GABA(A)Rs accompanied with neuronal excitation evoked by 4-aminopyridine (4AP) or N-methyl-D-aspartic acid (NMDA) precedes that of gephyrin. Single-particle tracking of quantum dot labeled-GABA(A)Rs revealed that excitation-induced enhancement of GABA(A)R lateral mobility also occurred before the shrinkage of gephyrin clusters. Physical inhibition of GABA(A)R lateral diffusion on the cell surface and inhibition of a Ca(2+) dependent phosphatase, calcineurin, completely eliminated the 4AP-induced decrease in gephyrin cluster size, but not the NMDA-induced decrease in cluster size, suggesting the existence of two different mechanisms of gephyrin declustering during activity-dependent plasticity, a GABA(A)R-dependent regulatory mechanism and a GABA(A)R-independent one. Our results also indicate that GABA(A)R mobility and clustering after sustained excitatory activity is independent of gephyrin.     

GABA spillover activates postsynaptic GABA(B) receptors to control rhythmic hippocampal activity

In the hippocampus, interneurons provide synaptic inhibition via the transmitter GABA, which can activate GABA(A) and GABA(B) receptors (GABA(A)Rs and GABA(B)Rs). Generally, however, GABA released by a single interneuron activates only GABA(A)Rs on its targets, despite the abundance of GABA(B)RS. Here, I show that during hippocampal rhythmic activity, simultaneous release of GABA from several interneurons activates postsynaptic GABA(B)Rs and that block of GABA(B)Rs increases oscillation frequency. Furthermore, if GABA uptake is inhibited, even GABA released by a single interneuron is enough to activate GABA(B)Rs. This occurs also on cells not directly contacted by that interneuron, indicating that GABA has to overcome uptake and exit the synaptic cleft to reach GABA(B)RS. Thus, activation of extrasynaptic GABA(B)Rs by pooling of GABA is an important mechanism regulating hippocampal network activity.     

Muskelin regulates actin filament- and microtubule-based GABA(A) receptor transport in neurons

Intracellular transport regulates protein turnover including endocytosis. Because of the spatial segregation of F-actin and microtubules, internalized cargo vesicles need to employ myosin and dynein motors to traverse both cytoskeletal compartments. Factors specifying cargo delivery across both tracks remain unknown. We identified muskelin to interconnect retrograde F-actin- and microtubule-dependent GABA(A) receptor (GABA(A)R) trafficking. GABA(A)Rs regulate synaptic transmission, plasticity, and network oscillations. GABA(A)R α1 and muskelin interact directly, undergo neuronal cotransport, and associate with myosin VI or dynein motor complexes in?subsequent steps of GABA(A)R endocytosis. Inhibition of either transport route selectively interferes with receptor internalization or degradation. Newly generated muskelin KO mice display depletion of both transport steps and a high-frequency ripple oscillation phenotype. A diluted coat color of muskelin KOs further suggests muskelin transport functions beyond neurons. Our data suggest the concept that specific trafficking factors help cargoes to traverse both F-actin and microtubule compartments, thereby regulating their fate.     

Regulation of GABA(A) receptor dynamics by interaction with purinergic P2X(2) receptors

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.     

The brefeldin A-inhibited GDP/GTP exchange factor 2, a protein involved in vesicular trafficking, interacts with the beta subunits of the GABA receptors

We have found that the brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2) interacts with the beta subunits of the gamma-aminobutyric acid type-A receptor (GABA(A)R). BIG2 is a Sec7 domain-containing guanine nucleotide exchange factor known to be involved in vesicular and protein trafficking. The interaction between the 110 amino acid C-terminal fragment of BIG2 and the large intracellular loop of the GABA(A)R beta subunits was revealed with a yeast two-hybrid assay. The native BIG2 and GABA(A)Rs interact in the brain since both coprecipitated from detergent extracts with either anti-GABA(A)R or anti-BIG2 antibodies. In transfected human embryonic kidney cell line 293 cells, BIG2 promotes the exit of GABA(A)Rs from endoplasmic reticulum. Double label immunofluorescence of cultured hippocampal neurons and electron microscopy immunocytochemistry of rat brain tissue show that BIG2 concentrates in the trans-Golgi network. BIG2 is also present in vesicle-like structures in the dendritic cytoplasm, sometimes colocalizing with GABA(A)Rs. BIG2 is present in both inhibitory GABAergic synapses that contain GABA(A)Rs and in asymmetric excitatory synapses. The results are consistent with the hypotheses that the interaction of BIG2 with the GABA(A)R beta subunits plays a role in the exocytosis and trafficking of assembled GABA(A)R to the cell surface.     

Tracking cell surface GABAB receptors using an alpha-bungarotoxin tag

GABA(B) receptors mediate slow synaptic inhibition in the central nervous system and are important for synaptic plasticity as well as being implicated in disease. Located at pre- and postsynaptic sites, GABA(B) receptors will influence cell excitability, but their effectiveness in doing so will be dependent, in part, on their trafficking to, and stability on, the cell surface membrane. To examine the dynamic behavior of GABA(B) receptors in GIRK cells and neurons, we have devised a method that is based on tagging the receptor with the binding site components for the neurotoxin, alpha-bungarotoxin. By using the alpha-bungarotoxin binding site-tagged GABA(B) R1a subunit (R1a(BBS)), co-expressed with the R2 subunit, we can track receptor mobility using the small reporter, alpha-bungarotoxin-conjugated rhodamine. In this way, the rates of internalization and membrane insertion for these receptors could be measured with fixed and live cells. The results indicate that GABA(B) receptors rapidly turnover in the cell membrane, with the rate of internalization affected by the state of receptor activation. The bungarotoxin-based method of receptor-tagging seems ideally suited to follow the dynamic regulation of other G-protein-coupled receptors.     

Interleukin-1beta enhances GABAA receptor cell-surface expression by a phosphatidylinositol 3-kinase/Akt pathway: relevance to sepsis-associated encephalopathy

Sepsis-associated encephalopathy (SAE) is a frequent but poorly understood neurological complication in sepsis that negatively influences survival. Here we present clinical and experimental evidence that this brain dysfunction may be related to altered neurotransmission produced by inflammatory mediators. Compared with septic patients, SAE patients had higher interleukin-1beta (IL-1beta) plasma levels; interestingly, these levels decreased once the encephalopathy was resolved. A putative IL-1beta effect on type A gamma-aminobutyric acid receptors (GABA(A)Rs), which mediate fast synaptic transmission in most cerebral inhibitory synapses in mammals, was investigated in cultured hippocampal neurons and in Xenopus oocytes expressing native or foreign rat brain GABA(A)Rs, respectively. Confocal images in both cell types revealed that IL-1beta increases recruitment of GABA(A)Rs to the cell surface. Moreover, brief applications of IL-1beta to voltage-clamped oocytes yielded a delayed potentiation of the GABA-elicited chloride currents (I(GABA)); this effect was suppressed by IL-1ra, the natural IL-1 receptor (IL-1RI) antagonist. Western blot analysis combined with I(GABA) recording and confocal images of GABA(A) Rs in oocytes showed that IL-1beta stimulates the IL-1RI-dependent phosphatidylinositol 3-kinase activation and the consequent facilitation of phospho-Akt-mediated insertion of GABA(A)Rs into the cell surface. The interruption of this signaling pathway by specific phosphatidylinositol 3-kinase or Akt inhibitors suppresses the cytokine-mediated effects on GABA(A)R, whereas activation of the conditionally active form of Akt1 (myr-Akt1.ER*) with 4-hydroxytamoxifen reproduces the effects. These findings point to a previously unrecognized signaling pathway that connects IL-1beta with increased "GABAergic tone." We propose that through this mechanism IL-1beta might alter synaptic strength at central GABAergic synapses and so contribute to the cognitive dysfunction observed in SAE.     

Gephyrin-mediated γ-aminobutyric acid type A and glycine receptor clustering relies on a common binding site

Gephyrin is the major protein determinant for the clustering of inhibitory neurotransmitter receptors. Earlier analyses revealed that gephyrin tightly binds to residues 398-410 of the glycine receptor β subunit (GlyR β) and, as demonstrated only recently, also interacts with GABA(A) receptors (GABA(A)Rs) containing the α1, α2, and α3 subunits. Here, we dissect the molecular basis underlying the interactions between gephyrin and GABA(A)Rs containing these α-subunits and compare them to the crystal structure of the gephyrin-GlyR β complex. Biophysical and biochemical assays revealed that, in contrast to its tight interaction with GlyR β, gephyrin only loosely interacts with GABA(A)R α2, whereas it has an intermediate affinity for the GABA(A)R α1 and α3 subunits. Despite the wide variation in affinities and the low overall sequence homology among the identified receptor subunits, competition assays confirmed the receptor-gephyrin interaction to be a mutually exclusive process. Selected gephyrin point mutants that critically weaken complex formation with GlyR β also abolished the GABA(A)R α1 and α3 interactions. Additionally, we identified a common binding motif with two conserved aromatic residues that are central for gephyrin binding. Consistent with the biochemical data, mutations of the corresponding residues within the cytoplasmic domain of α2 subunit-containing GABA(A)Rs attenuated clustering of these receptors at postsynaptic sites in hippocampal neurons. Taken together, our experiments provide key insights regarding similarities and differences in the complex formation between gephyrin and GABA(A)Rs compared with GlyRs and, hence, the accumulation of these receptors at postsynaptic sites.     

Modulation of GABAA receptor activity by phosphorylation and receptor trafficking: implications for the efficacy of synaptic inhibition

Fast synaptic inhibition in the brain is largely mediated by GABA(A) receptors. These ligand-gated ion channels are crucial in the control of cell and network activity. Therefore, modulating their function or cell surface stability will have major consequences for neuronal excitation. It has become clear that the stability and activity of GABA(A) receptors at synapses can be dynamically modulated by receptor trafficking and phosphorylation. Here, we discuss these regulatory mechanisms, and their consequences for the efficacy of GABA(A) receptor mediated synaptic inhibition.     

Trafficking and synaptic anchoring of ionotropic inhibitory neurotransmitter receptors

Neurotransmitter receptors are subject to microtubule-based transport between intracellular organelles and the neuronal plasma membrane. Receptors that arrive at plasma membrane compartments diffuse laterally within the plane of the cellular surface. To achieve immobilization at their sites of action, cytoplasmic receptor residues bind to submembrane proteins, which are coupled to the underlying cytoskeleton by multiprotein scaffolds. GABA(A)Rs (gamma-aminobutyric type A receptors) and GlyRs (glycine receptors) are the major inhibitory receptors in the central nervous system. At inhibitory postsynaptic sites, all GlyRs and the majority of GABA(A)Rs directly or indirectly couple to gephyrin, a multimeric PSD (postsynaptic density) component. In addition to cluster formations at axo-dendritic contacts, individual GABA(A)R subtypes also anchor and concentrate at extrasynaptic positions, either through association with gephyrin or direct interaction with the ERM (ezrin/radixin/moesin) family protein radixin. In addition to their role in diffusion trapping of surface receptors, scaffold components also undergo rapid exchange to/from and between postsynaptic specializations, leading to a dynamic equilibrium of receptor-scaffold complexes. Moreover, scaffold components serve as adaptor proteins that mediate specificity in intracellular transport complexes. In the present review, we discuss the dynamic delivery, stabilization and removal of inhibitory receptors at synaptic sites.     

Differential Regulation of GABAB Receptor Trafficking by Different Modes of N-methyl-d-aspartate (NMDA) Receptor Signaling

Inhibitory GABA_B receptors (GABA_BRs) can down-regulate most excitatory synapses in the CNS by reducing postsynaptic excitability. Functional GABA_BRs are heterodimers of GABA_B1 and GABA_B2 subunits and here we show that the trafficking and surface expression of GABA_BRs is differentially regulated by synaptic or pathophysiological activation of NMDA receptors (NMDARs). Activation of synaptic NMDARs using a chemLTP protocol increases GABA_BR recycling and surface expression. In contrast, excitotoxic global activation of synaptic and extrasynaptic NMDARs by bath application of NMDA causes the loss of surface GABA_BRs. Intriguingly, exposing neurons to extreme metabolic stress using oxygen/glucose deprivation (OGD) increases GABA_B1 but decreases GABA_B2 surface expression. The increase in surface GABA_B1 involves enhanced recycling and is blocked by the NMDAR antagonist AP5. The decrease in surface GABA_B2 is also blocked by AP5 and by inhibiting degradation pathways. These results indicate that NMDAR activity is critical in GABA_BR trafficking and function and that the individual subunits can be separately controlled to regulate neuronal responsiveness and survival.     

Changes in ventral respiratory column GABAaR ε- and δ-subunits during hibernation mediate resistance to depression by EtOH and pentobarbital

During hibernation in the 13-lined ground squirrel, Ictidomys tridecemlineatus, the cerebral cortex is electrically silent, yet the brainstem continues to regulate cardiorespiratory function. Previous work showed that neurons in slices through the medullary ventral respiratory column (VRC) but not the cortex are insensitive to high doses of pentobarbital during hibernation, leading to the hypothesis that GABA(A) receptors (GABA(A)R) in the VRC undergo a seasonal modification in subunit composition. To test whether alteration of GABA(A)R subunits are responsible for hibernation-associated pentobarbital insensitivity, we examined an array of subunits using RT-PCR and Western blots and identified changes in ε- and δ-subunits in the medulla but not the cortex. Using immunohistochemistry, we confirmed that during hibernation, the expression of ε-subunit-containing GABA(A)Rs nearly doubles in the VRC. We also identified a population of δ-subunit-containing GABA(A)Rs adjacent to the VRC that were differentially expressed during hibernation. As δ-subunit-containing GABA(A)Rs are particularly sensitive to ethanol (EtOH), multichannel electrodes were inserted in slices of medulla and cortex from hibernating squirrels and EtOH was applied. EtOH, which normally inhibits neuronal activity, excited VRC but not cortical neurons during hibernation. This excitation was prevented by bicuculline pretreatment, indicating the involvement of GABA(A)Rs. We propose that neuronal activity in the VRC during hibernation is unaffected by pentobarbital due to upregulation of ε-subunit-containing GABA(A)Rs on VRC neurons. Synaptic input from adjacent inhibitory interneurons that express δ-subunit-containing GABA(A)Rs is responsible for the excitatory effects of EtOH on VRC neurons during hibernation.     

GABA coordinates with insulin in regulating secretory function in pancreatic INS-1 β-cells

Pancreatic islet β-cells produce large amounts of γ-aminobutyric acid (GABA), which is co-released with insulin. GABA inhibits glucagon secretion by hyperpolarizing α-cells via type-A GABA receptors (GABA(A)Rs). We and others recently reported that islet β-cells also express GABA(A)Rs and that activation of GABA(A)Rs increases insulin release. Here we investigate the effects of insulin on the GABA-GABA(A)R system in the pancreatic INS-1 cells using perforated-patch recording. The results showed that GABA produces a rapid inward current and depolarizes INS-1 cells. However, pre-treatment of the cell with regular insulin (1 μM) suppressed the GABA-induced current (I(GABA)) by 43%. Zinc-free insulin also suppressed I(GABA) to the same extent of inhibition by regular insulin. The inhibition of I(GABA) occurs within 30 seconds after application of insulin. The insulin-induced inhibition of I(GABA) persisted in the presence of PI3-kinase inhibitor, but was abolished upon inhibition of ERK, indicating that insulin suppresses GABA(A)Rs through a mechanism that involves ERK activation. Radioimmunoassay revealed that the secretion of C-peptide was enhanced by GABA, which was blocked by pre-incubating the cells with picrotoxin (50 μM, p<0.01) and insulin (1 μM, p<0.01), respectively. Together, these data suggest that autocrine GABA, via activation of GABA(A)Rs, depolarizes the pancreatic β-cells and enhances insulin secretion. On the other hand, insulin down-regulates GABA-GABA(A)R signaling presenting a feedback mechanism for fine-tuning β-cell secretion.