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.     

The availability of surface GABA B receptors is independent of gamma-aminobutyric acid but controlled by glutamate in central neurons

The efficacy of synaptic transmission depends on the availability of ionotropic and metabotropic neurotransmitter receptors at the plasma membrane, but the contribution of the endocytic and recycling pathways in the regulation of gamma-aminobutyric acid type B (GABA(B)) receptors remains controversial. To understand the mechanisms that regulate the abundance of GABA(B) receptors, we have studied their turnover combining surface biotin labeling and a microscopic immunoendocytosis assay in hippocampal and cortical neurons. We report that internalization of GABA(B) receptors is agonist-independent. We also demonstrate that receptors endocytose in the cell body and dendrites but not in axons. Additionally, we show that GABA(B) receptors endocytose as heterodimers via clathrin- and dynamin-1-dependent mechanisms and that they recycle to the plasma membrane after endocytosis. More importantly, we show that glutamate decreases the levels of cell surface receptors in a manner dependent on an intact proteasome pathway. These observations indicate that glutamate and not GABA controls the abundance of surface GABA(B) receptors in central neurons, consistent with their enrichment at glutamatergic synapses.     

Identification and characterization of a novel splice variant of beta3 subunit of GABA(A) receptor in rat testis and spermatozoa

Gamma-aminobutyric acid type A (GABA(A)) receptors are the major sites of inhibitory action of fast synaptic neurotransmission in the brain. Their receptors are also widely distributed in peripheral and endocrine tissues. A full-length cDNA encoding a novel splice variant of beta3 subunit of GABA(A) receptor, designated as beta3t, was identified in rat testis. This isoform contains a segment, having identical amino acid sequence as the beta3 subunit of neuronal GABA(A) receptors except for a section composed of 25 different amino acid sequence in the N-terminus. Northern blot shows that this isoform is found in rat testis. The beta3t isoform mRNA was detected in germ cells in the late step of spermatogenesis by in situ hybridization assay. Results of immunohistochemical and immunocytochemical assays indicate that the beta3t isoform is expressed in rat testis and spermatozoa. To determine a possible function of the N-terminal 25 amino acid segment, a recombinant plasmid of beta3t-EGFPC was constructed by fusing green fluorescent protein to the C-terminus of the beta3t isoform. The chimera product failed to be translocated unto the cell surface when expressed in HEK 293 cells; whereas, the beta3 subunit of rat brain is incorporated into the plasma membrane. In conclusion, the present results show that one variant of beta3 subunit of GABA(A) receptor, designated as beta3t, is found in germ cells of rat testis and sperm. The inability of the beta3t variant to target into the plasma membrane maybe a consequence of the unique 25 amino acid segment in the N-terminus.     

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.     

Activity-dependent phosphorylation of GABAA receptors regulates receptor insertion and tonic current

The expression of GABA(A) receptors and the efficacy of GABAergic neurotransmission are subject to adaptive compensatory regulation as a result of changes in neuronal activity. Here, we show that activation of L-type voltage-gated Ca(2+) channels (VGCCs) leads to Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of S383 within the β3 subunit of the GABA(A) receptor. Consequently, this results in rapid insertion of GABA(A) receptors at the cell surface and enhanced tonic current. Furthermore, we demonstrate that acute changes in neuronal activity leads to the rapid modulation of cell surface numbers of GABA(A) receptors and tonic current, which are critically dependent on Ca(2+) influx through L-type VGCCs and CaMKII phosphorylation of β3S383. These data provide a mechanistic link between activity-dependent changes in Ca(2+) influx through L-type channels and the rapid modulation of GABA(A) receptor cell surface numbers and tonic current, suggesting a homeostatic pathway involved in regulating neuronal intrinsic excitability in response to changes in activity.     

Phosphorylation and chronic agonist treatment atypically modulate GABAB receptor cell surface stability

GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. The dynamic control of the cell surface stability of GABA(B) receptors is likely to be of fundamental importance in the modulation of receptor signaling. Presently, however, this process is poorly understood. Here we demonstrate that GABA(B) receptors are remarkably stable at the plasma membrane showing little basal endocytosis in cultured cortical and hippocampal neurons. In addition, we show that exposure to baclofen, a well characterized GABA(B) receptor agonist, fails to enhance GABA(B) receptor endocytosis. Lack of receptor internalization in neurons correlates with an absence of agonist-induced phosphorylation and lack of arrestin recruitment in heterologous systems. We also demonstrate that chronic exposure to baclofen selectively promotes endocytosis-independent GABA(B) receptor degradation. The effect of baclofen can be attenuated by activation of cAMP-dependent protein kinase or co-stimulation of beta-adrenergic receptors. Furthermore, we show that increased degradation rates are correlated with reduced receptor phosphorylation at serine 892 in GABA(B)R2. Our results support a model in which GABA(B)R2 phosphorylation specifically stabilizes surface GABA(B) receptors in neurons. We propose that signaling pathways that regulate cAMP levels in neurons may have profound effects on the tonic synaptic inhibition by modulating the availability of GABA(B) receptors.     

Positive feedback regulation between gamma-aminobutyric acid type A (GABA(A)) receptor signaling and brain-derived neurotrophic factor (BDNF) release in developing neurons

During the early development of the nervous system, γ-aminobutyric acid (GABA) type A receptor (GABA(A)R)-mediated signaling parallels the neurotrophin/tropomyosin-related kinase (Trk)-dependent signaling in controlling a number of processes from cell proliferation and migration, via dendritic and axonal outgrowth, to synapse formation and plasticity. Here we present the first evidence that these two signaling systems regulate each other through a complex positive feedback mechanism. We first demonstrate that GABA(A)R activation leads to an increase in the cell surface expression of these receptors in cultured embryonic cerebrocortical neurons, specifically at the stage when this activity causes depolarization of the plasma membrane and Ca(2+) influx through L-type voltage-gated Ca(2+) channels. We further demonstrate that GABA(A)R activity triggers release of the brain-derived neurotrophic factor (BDNF), which, in turn by activating TrkB receptors, mediates the observed increase in cell surface expression of GABA(A)Rs. This BDNF/TrkB-dependent increase in surface levels of GABA(A)Rs requires the activity of phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC) and does not involve the extracellular signal-regulated kinase (ERK) 1/2 activity. The increase in GABA(A)R surface levels occurs due to an inhibition of the receptor endocytosis by BDNF, whereas the receptor reinsertion into the plasma membrane remains unaltered. Thus, GABA(A)R activity is a potent regulator of the BDNF release during neuronal development, and at the same time, it is strongly enhanced by the activity of the BDNF/TrkB/PI3K/PKC signaling pathway.     

Pharmacologic and therapeutic aspects of the developmentally regulated expression of GABA(A) and GABA(B) receptors: cerebellar granule cells as a model system.

Cerebellar granule neurons can be conveniently kept in culture. They constitute a useful model to study regulation of glutamatergic activity, in particular the inhibitory action of GABA (7-aminobutyrate). GABA exerts an inhibitory action on evoked transmitter release acting on both GABA(A) and GABA(B) receptors. The functional properties of these receptors are dependent upon the environment of the neurons during early development in culture as the expression of both receptor subtypes is enhanced by exposure of the neurons to GABA(A) receptor agonists. Thus, the inducible GABA(A) receptors are of low affinity and lack benzodiazepine sensitivity, and the G-protein coupling differs among the native and the inducible GABA(B) receptors. Moreover, the GABA(A) and the GABA(B) receptors are functionally coupled, leading to a disinhibitory action of GABA. Therefore drugs exhibiting selective agonist or antagonist action on subclasses of GABA(A) and GABA(B) may be of potential use as regulators of glutamatergic excitatory activity.     

Protein kinase C phosphorylation regulates membrane insertion of GABAA receptor subtypes that mediate tonic inhibition

Tonic inhibition in the brain is mediated largely by specialized populations of extrasynaptic receptors, γ-aminobutyric acid receptors (GABA(A)Rs). In the dentate gyrus region of the hippocampus, tonic inhibition is mediated primarily by GABA(A)R subtypes assembled from α4β2/3 with or without the δ subunit. Although the gating of these receptors is subject to dynamic modulation by agents such as anesthetics, barbiturates, and neurosteroids, the cellular mechanisms neurons use to regulate their accumulation on the neuronal plasma membrane remain to be determined. Using immunoprecipitation coupled with metabolic labeling, we demonstrate that the α4 subunit is phosphorylated at Ser(443) by protein kinase C (PKC) in expression systems and hippocampal slices. In addition, the β3 subunit is phosphorylated on serine residues 408/409 by PKC activity, whereas the δ subunit did not appear to be a PKC substrate. We further demonstrate that the PKC-dependent increase of the cell surface expression of α4 subunit-containing GABA(A)Rs is dependent on Ser(443). Mechanistically, phosphorylation of Ser(443) acts to increase the stability of the α4 subunit within the endoplasmic reticulum, thereby increasing the rate of receptor insertion into the plasma membrane. Finally, we show that phosphorylation of Ser(443) increases the activity of α4 subunit-containing GABA(A)Rs by preventing current run-down. These results suggest that PKC-dependent phosphorylation of the α4 subunit plays a significant role in enhancing the cell surface stability and activity of GABA(A)R subtypes that mediate tonic inhibition.     

The sushi domains of secreted GABA(B1) isoforms selectively impair GABA(B) heteroreceptor function

GABA(B) receptors are the G-protein-coupled receptors for gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. GABA(B) receptors are promising drug targets for a wide spectrum of psychiatric and neurological disorders. Receptor subtypes exhibit no pharmacological differences and are based on the subunit isoforms GABA(B1a) and GABA(B1b). GABA(B1a) differs from GABA(B1b) in its ectodomain by the presence of a pair of conserved protein binding motifs, the sushi domains (SDs). Previous work showed that selectively GABA(B1a) contributes to heteroreceptors at glutamatergic terminals, whereas both GABA(B1a) and GABA(B1b) contribute to autoreceptors at GABAergic terminals or to postsynaptic receptors. Here, we describe GABA(B1j), a secreted GABA(B1) isoform comprising the two SDs. We show that the two SDs, when expressed as a soluble protein, bind to neuronal membranes with low nanomolar affinity. Soluble SD protein, when added at nanomolar concentrations to dissociated hippocampal neurons or to acute hippocampal slices, impairs the inhibitory effect of GABA(B) heteroreceptors on evoked and spontaneous glutamate release. In contrast, soluble SD protein neither impairs the activity of GABA(B) autoreceptors nor impairs the activity of postsynaptic GABA(B) receptors. We propose that soluble SD protein scavenges an extracellular binding partner that retains GABA(B1a)-containing heteroreceptors in proximity of the presynaptic release machinery. Soluble GABA(B1) isoforms like GABA(B1j) may therefore act as dominant-negative inhibitors of heteroreceptors and control the level of GABA(B)-mediated inhibition at glutamatergic terminals. Of importance for drug discovery, our data also demonstrate that it is possible to selectively impair GABA(B) heteroreceptors by targeting their SDs.     

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.     

A trafficking checkpoint controls GABA(B) receptor heterodimerization

Surface expression of GABA(B) receptors requires heterodimerization of GB1 and GB2 subunits, but little is known about mechanisms that ensure efficient heterodimer assembly. We found that expression of the GB1 subunit on the cell surface is prevented through a C-terminal retention motif RXR(R); this sequence is reminiscent of the ER retention/retrieval motif RKR identified in subunits of the ATP-sensitive K+ channel. Interaction of GB1 and GB2 through their C-terminal coiled-coil alpha helices masks the retention signal in GB1, allowing the plasma membrane expression of the assembled complexes. Because individual GABA(B) receptor subunits and improperly assembled receptor complexes are not functional even if expressed on the cell surface, we conclude that a trafficking checkpoint ensures efficient assembly of functional GABA(B) receptors.     

GABA maintains the proliferation of progenitors in the developing chick ciliary marginal zone and non-pigmented ciliary epithelium

GABA is more than the main inhibitory neurotransmitter found in the adult CNS. Several studies have shown that GABA regulates the proliferation of progenitor and stem cells. This work examined the effects of the GABA(A) receptor system on the proliferation of retinal progenitors and non-pigmented ciliary epithelial (NPE) cells. qRT-PCR and whole-cell patch-clamp electrophysiology were used to characterize the GABA(A) receptor system. To quantify the effects on proliferation by GABA(A) receptor agonists and antagonists, incorporation of thymidine analogues was used. The results showed that the NPE cells express functional extrasynaptic GABA(A) receptors with tonic properties and that low concentration of GABA is required for a baseline level of proliferation. Antagonists of the GABA(A) receptors decreased the proliferation of dissociated E12 NPE cells. Bicuculline also had effects on progenitor cell proliferation in intact E8 and E12 developing retina. The NPE cells had low levels of the Cl-transporter KCC2 compared to the mature retina, suggesting a depolarising role for the GABA(A) receptors. Treatment with KCl, which is known to depolarise membranes, prevented some of the decreased proliferation caused by inhibition of the GABA(A) receptors. This supported the depolarising role for the GABA(A) receptors. Inhibition of L-type voltage-gated Ca(2+) channels (VGCCs) reduced the proliferation in the same way as inhibition of the GABA(A) receptors. Inhibition of the channels increased the expression of the cyclin-dependent kinase inhibitor p27(KIP1), along with the reduced proliferation. These results are consistent with that when the membrane potential indirectly regulates cell proliferation with hyperpolarisation of the membrane potential resulting in decreased cell division. The increased expression of p27(KIP1) after inhibition of either the GABA(A) receptors or the L-type VGCCs suggests a link between the GABA(A) receptors, membrane potential, and intracellular Ca(2+) in regulating the cell cycle.     

Hetero-oligomerization between GABAA and GABAB receptors regulates GABAB receptor trafficking

The neurotransmitter gamma-aminobutyric acid (GABA) mediates inhibitory signaling in the brain via stimulation of both GABA(A) receptors (GABA(A)R), which are chloride-permeant ion channels, and GABA(B) receptors (GABA(B)R), which signal through coupling to G proteins. Here we report physical interactions between these two different classes of GABA receptor. Association of the GABA(B) receptor 1 (GABA(B)R1) with the GABA(A) receptor gamma2S subunit robustly promotes cell surface expression of GABA(B)R1 in the absence of GABA(B)R2, a closely related GABA(B) receptor that is usually required for efficient trafficking of GABA(B)R1 to the cell surface. The GABA(B)R1/gamma2S complex is not detectably functional when expressed alone, as assessed in both ERK activation assays and physiological analyses in oocytes. However, the gamma2S subunit associates not only with GABA(B)R1 alone but also with the functional GABA(B)R1/GABA(B)R2 heterodimer to markedly enhance GABA(B) receptor internalization in response to agonist stimulation. These findings reveal that the GABA(B)R1/gamma2S interaction results in the regulation of multiple aspects of GABA(B) receptor trafficking, allowing for cross-talk between these two distinct classes of GABA receptor.     

Functional expression of metabotropic GABAB receptors in primary cultures of astrocytes from rat cerebral cortex

GABA(B) receptor subunits are widely expressed on neurons throughout the central nervous system (CNS), at both pre- and postsynaptic sites, where they mediate the late and slow component of the inhibitory response to the major inhibitory neurotransmitter GABA. Recently, GABA(B) receptors have been reported to be expressed in astrocytes and microglia in the rat CNS by immunocytochemistry. However, there are few reports available for the functional characterization of GABA(B) receptors on astrocytes. In the present study, we therefore investigated the functional expression and characteristics of GABA(B) receptors in primary cultures of astrocytes from rat cerebral cortex. In the presence of 10 microM GTP, forskolin concentration-dependently increased adenylylcyclase (AC) activity in membranes prepared from rat astrocytes. The selective GABA(B) agonist (R)-baclofen concentration-dependently reduced forskolin-stimulated AC activity in the presence of 10 microM GTP. This effect was reversed by the selective GABA(B) antagonists, CGP-55845 and CGP-54626, and was completely abolished by treatment of astrocytic membranes with pertussis toxin. In addition, RT-PCR, Western blotting, and immunocytochemistry clearly showed that metabotropic GABA(B) receptor isoforms (GABA(B)R1 and GABA(B)R2) are expressed in rat cerebrocortical astrocytes. Taken collectively, these results demonstrate that functionally active metabotropic GABA(B) receptors are expressed in rat cerebrocortical astrocytes.     

Blocking L-type Voltage-gated Ca2+ Channels with Dihydropyridines Reduces ��-Aminobutyric Acid Type A Receptor Expression and Synaptic Inhibition

γ-Aminobutyric acid type A receptors (GABA_ARs) are the major sites of fast inhibitory neurotransmission in the brain, and the numbers of these receptors at the cell surface can determine the strength of GABAergic neurotransmission. Chronic changes in neuronal activity lead to an adaptive modulation in the efficacy of GABAergic synaptic inhibition, brought about in part by changes in the number of synaptic GABA_ARs, a mechanism known as homeostatic synaptic plasticity. Reduction in the number of GABA_ARs in response to prolonged neuronal activity blockade is dependent on the ubiquitin-proteasome system. The underlying biochemical pathways linking chronic activity blockade to proteasome-dependent degradation of GABA_ARs are unknown. Here, we show that chronic blockade of L-type voltage-gated calcium channels (VGCCs) with nifedipine decreases the number of GABA_ARs at synaptic sites but not the overall number of inhibitory synapses. In parallel, blockade of L-type VGCCs decreases the amplitude but not the frequency of miniature inhibitory postsynaptic currents or expression of the glutamic acid decarboxylase GAD65. We further reveal that the activation of L-type VGCCs regulates the turnover of newly translated GABA_AR subunits in a mechanism dependent upon the activity of the proteasome and thus regulates GABA_AR insertion into the plasma membrane. Together, these observations suggest that activation of L-type VGCCs can regulate the abundance of synaptic GABA_ARs and the efficacy of synaptic inhibition, revealing a potential mechanism underlying the homeostatic adaptation of fast GABAergic inhibition to prolonged changes in activity.     

GABA and neuroligin signaling: linking synaptic activity and adhesion in inhibitory synapse development

GABA-mediated synaptic inhibition is crucial in neural circuit operations. In mammalian brains, the development of inhibitory synapses and innervation patterns is often a prolonged postnatal process, regulated by neural activity. Emerging evidence indicates that gamma-aminobutyric acid (GABA) acts beyond inhibitory transmission and regulates inhibitory synapse development. Indeed, GABA(A) receptors not only function as chloride channels that regulate membrane voltage and conductance but also play structural roles in synapse maturation and stabilization. The link from GABA(A) receptors to postsynaptic and presynaptic adhesion is probably mediated, partly by neuroligin-reurexin interactions, which are potent in promoting GABAergic synapse formation. Therefore, similar to glutamate signaling at excitatory synapse, GABA signaling may coordinate maturation of presynaptic and postsynaptic sites at inhibitory synapses. Defining the many steps from GABA signaling to receptor trafficking/stability and neuroligin function will provide further mechanistic insights into activity-dependent development and possibly plasticity of inhibitory synapses.