Modulation of the expression of GABA(A) receptors in rat cerebellar granule cells by protein tyrosine kinases and protein kinase C

The expression of GABA(A) receptors in rat cerebellar granules in culture has been studied by beta(2/3) subunit immunocytochemistry and fluorescence confocal microscopy. These cells show labeling all over the cell bodies' plasma membrane and dendrites. Treatment with the protein tyrosine kinase (PTK) inhibitor genistein results in a decrease of the labeling associated with the beta(2/3) subunit in both cell bodies and dendrites. No effect was found with an inactive genistein analogue, daidzein. A similar effect was found with a protein kinase C (PKC) activator, phorbol myristate acetate (PMA). The effects of genistein and PMA are additive.The interpretation of the results is that PTK inhibition blocks exocytotic deposit of newly synthesized GABA(A) receptors onto the neuronal plasma membrane. On the other hand, PKC activation speeds up endocytotic removal of GABA(A) receptors.     

Effects of taurine on cell morphology and expression of low-affinity GABA receptors in cultured cerebellar granule cells

Effects of taurine and THIP were studied on the development of cultured cerebellar granule cells with regard to GABA receptor expression and morphological development. Culturing in the presence of taurine or THIP led to the formation of low affinity GABA receptors as revealed from Scatchard analysis of [³H]GABA binding. This formation of receptors was susceptible to inhibition upon culturing in the simultaneous presence of taurine and bicuculline demonstrating the involvement of the high affinity GABA receptors which are present on the cells regardless of the culture condition. Superfusion experiments on cells cultured under the different conditions demonstrated that the low affinity GABA receptors expressed after culturing in the presence of THIP or taurine mediated an inhibition by GABA of evoked transmitter release from the granule cells. Cells cultured in either plain culture media or in the presence of taurine were indistinguishable with respect to the number of neurite extending cells observed after 4 days in culture. In contrast, culturing in the presence of THIP increased the number of neurite extending cells by 8% relative to the controls.Special issue dedicated to Dr. Paola S. Timiras     

The effects of zolpidem treatment on GABA(A) receptors in cultured cerebellar granule cells: changes in functional coupling

AimsHypnotic zolpidem is a positive allosteric modulator of γ-aminobutyric acid (GABA) action, with preferential although not exclusive binding for α1 subunit-containing GABA_A receptors. The pharmacological profile of this drug is different from that of classical benzodiazepines, although it acts through benzodiazepine binding sites at GABA_A receptors. The aim of this study was to further explore the molecular mechanisms of GABA_A receptor induction by zolpidem.Main methodsIn the present study, we explored the effects of two-day zolpidem (10 μM) treatment on GABA_A receptors on the membranes of rat cerebellar granule cells (CGCs) using [³H]flunitrazepam binding and semi-quantitative PCR analysis.Key findingsTwo-day zolpidem treatment of CGCs did not significantly affect the maximum number (B_max) of [³H]flunitrazepam binding sites or the expression of α1 subunit mRNA. However, as shown by decreased GABA [³H]flunitrazepam binding, two-day exposure of CGCs to zolpidem caused functional uncoupling of GABA and benzodiazepine binding sites at GABA_A receptor complexes.SignificanceIf functional uncoupling of GABA and benzodiazepine binding sites at GABA_A receptors is the mechanism responsible for the development of tolerance following long-term administration of classical benzodiazepines, chronic zolpidem treatment may induce tolerance.     

AMPA and kainate receptors mediate mutually exclusive effects on GABA(A) receptor expression in cultured mouse cerebellar granule neurones

Studies on animal models of epilepsy and cerebellar ataxia, e.g., stargazer mice (stg) have identified changes in the GABAergic properties of neurones associated with the affected brain loci. Whether these changes contribute to or constitute homeostatic adaptations to a state of altered neuronal excitability is as yet unknown. Using cultured cerebellar granule neurones from control [+/+; alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptor (AMPAR)-competent, Kainate receptor (KAR)-competent] and stg (AMPAR-incompetent, KAR-competent), we investigated whether non-NMDA receptor (NMDAR) activity regulates GABA(A) receptor (GABAR) expression. Neurones were maintained in 5 mmol/L KCl-containing basal media or depolarizing media containing either 25 mmol/L KCl or the non-NMDAR agonist kainic acid (KA) (100 micromol/L). KCl- and KA-mediated depolarization down-regulated GABAR alpha1, alpha6 and beta2, but up-regulated alpha4, beta3 and delta subunits in +/+ neurones. The KCl-evoked but not KA-evoked effects were reciprocated in stg neurones compatible with AMPAR-regulation of GABAR expression. Conversely, GABAR gamma2 expression was insensitive to KCl-mediated depolarization, but was down-regulated by KA-treatment in a 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)-reversible manner in +/+ and stg neurones compatible with a KAR-mediated response. KA-mediated up-regulation of GABAR alpha4, beta3 and delta was inhibited by L-type voltage-gated calcium channel (L-VGCC) blockers and the Ca2+/calmodulin-dependent protein kinase inhibitor, 4-[(2S)-2-[(5-isoquinolinylsulfonyl)methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl] phenyl isoquinoline sulfonic acid ester (KN-62). Up-regulation of GABAR alpha4 and beta3 was also prevented by calcineurin (CaN) inhibitors, FK506 and cyclosporin A. Down-regulation of GABAR alpha1, alpha6 and beta2 was independent of L-VGCC activity, but was prevented by inhibitors of CaN. Thus, we provide evidence that a KAR-mediated and at least three mutually exclusive AMPAR-mediated signalling mechanisms regulate neuronal GABAR expression.     

A GABA(B) mystery: the search for pharmacologically distinct GABA(B) receptors

The classification of neurotransmitter receptors into distinct pharmacological subtypes is of major importance in drug discovery. This quest is particularly important for neurotransmitter systems that are widely distributed. Because gamma-aminobutyric acid (GABA) receptors, both GABA(A) and GABA(B), are found throughout the neuroaxis, they are likely involved in all central nervous system functions. Accordingly, the therapeutic promise of GABA(B) receptor manipulation depends upon the identification of subtypes than can be specifically targeted.     

A chondroitin sulfate proteoglycan that is developmentally regulated in the cerebellar mossy fiber system.

It is known that the mammalian brain contains many kinds of proteoglycans, but almost all of them remain to be characterized. In this study, we prepared a monoclonal antibody against a phosphate-buffered saline-soluble brain proteoglycan (MAb 6B4). MAb 6B4 recognized a 600- to 1000-kDa chondroitin sulfate proteoglycan with a 250-kDa core protein (6B4 proteoglycan). The core protein of 6B4 proteoglycan carried the HNK-1 epitope. Immunohistochemical analysis of the adult rat brain indicated that this proteoglycan was expressed on the cell surfaces of a subset of neurons. In the hindbrain, 6B4 proteoglycan was highly expressed on the cerebellar Purkinje cells and Golgi cells, and at particular nuclei including the pontine nuclei and lateral reticular nucleus. Almost all of these nuclei were connected to the cerebellum through the mossy fiber system. A developmental study indicated that the expression of this proteoglycan changed dramatically during the formation of the cerebellar mossy fiber system. The mossy fibers from the pontine nuclei expressed 6B4 proteoglycan transiently from Embryonic Day 20 (E20) to Postnatal Day 30 (P30), during which time the axonal outgrowth and glomerular synapse formation occurred. The Purkinje cells, glomeruli, and Golgi cells began to be stained with MAb 6B4 from P10, P16, and P20, respectively. These expression stages correspond with the onset of their synapse formation. These results suggest that 6B4 proteoglycan is closely involved in the development of the cerebellar mossy fiber system.     

Targeted disruption of the GABA(A) receptor delta subunit gene leads to an up-regulation of gamma 2 subunit-containing receptors in cerebellar granule cells

GABA(A) receptors are chloride channels composed of five subunits. Cerebellar granule cells express abundantly six subunits belonging to four subunit classes. These are assembled into a number of distinct receptors, but the regulation of their relative proportions is yet unknown. Here, we studied the composition of cerebellar GABA(A) receptors after targeted disruption of the delta subunit gene. In membranes and extracts of delta-/- cerebellum, [(3)H]muscimol binding was not significantly changed, whereas [(3)H]Ro15-4513 binding was increased by 52% due to an increase in diazepam-insensitive binding. Immunocytochemical and Western blot analysis revealed no change in alpha(6) subunits but an increased expression of gamma(2) subunits in delta-/- cerebellum. Immunoaffinity chromatography of cerebellar extracts indicated there was an increased coassembly of alpha(6) and gamma(2) subunits and that 24% of all receptors in delta-/- cerebellum did not contain a gamma subunit. Because 97% of delta subunits are coassembled with alpha(6) subunits in the cerebellum of wild-type mice, these results indicated that, in delta-/- mice, alpha(6)betagamma(2) and alphabeta receptors replaced delta subunit-containing receptors. The availability of the delta subunit, thus, influences the level of expression or the extent of assembly of the gamma(2) subunit, although these two subunits do not occur in the same receptor.     

Distinct patterns of expression and regulation of GABA receptors containing the delta subunit in cerebellar granule and hippocampal neurons

Neuronal plasticity is achieved by regulation of the expression of genes for neurotransmitter receptors such as the type A receptor (GABA(A)R) for gamma-aminobutyric acid. We now show that two different rat neuronal populations in culture manifest distinct patterns of GABA(A)R plasticity in response to identical stimuli. Whereas prolonged exposure to ethanol had no effect on expression of the delta subunit of GABA(A)Rs at the mRNA or protein level in cerebellar granule neurons, it increased the abundance of delta subunit mRNA and protein in hippocampal neurons. Subsequent ethanol withdrawal transiently down-regulated delta subunit expression in cerebellar granule neurons and gradually normalized that in hippocampal neurons. These effects of ethanol exposure and withdrawal were accompanied by corresponding functional changes in GABA(A)Rs. GABA(A)Rs containing the delta subunit were also distributed differentially in the cerebellar and hippocampal neurons. These findings reveal complex and distinct mechanisms of regulation of the expression of GABA(A)Rs that contain the delta subunit in different neuronal types.     

Regulation of the expression of low affinity GABAA receptors in rat cerebellar granule cells

Summary. GABA_A receptors of cerebellar granule cells obtained from neonatal rats and kept in culture were studied by labelled muscimol binding. The data show that, according to the maturational state of those cells in vivo, one or two binding components appear. The low affinity component seems to be the one appearing later. The expression of this component seems to be regulated by protein tyrosine phosphorylation. In fact, its expression is down regulated by the protein tyrosine kinase (PTK) inhibitor, genistein. Viceversa, its expression is upregulated by insulin like growth factor I (IGF-I), most probably via PTK activation. A possible interpretation of the data is that in vivo IGF-I is one of the endogenous messages leading to the expression of this component during development. Another endogenous factor involved may be GABA itself. Low affinity GABA_A receptors appear to be the ones involved in inhibitory synaptic transmission at glomeruli. Whereas the high affinity ones probably correspond to extrasynaptic GABA_A receptors mediating the tonic form of inhibition in cerebellar granules. Received December 12, 2000 Accepted February 12, 2001     

GABA-agonists induce the formation of low-affinity GABA-receptors on cultured cerebellar granule cells via preexisting high affinity GABA receptors

The kinetics of specific GABA-binding to membranes isolated from cerebellar granule cells, cultured for 12 days from dissociated cerebella of 7-day-old rats was studied using [³H]GABA as the ligand. The granule cells were cultured in the presence of the specific GABA receptor agonist 4, 5, 6, 7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 150 M) or THIP plus the antagonist bicuculline methobromide (150 M of each) or in the absence of the agonist or antagonist. Membranes isolated from granule cells cultured in a medium without the GABA agonist revealed a single binding site for GABA with a binding constant (K _D) of 7.9±0.4 nM and aB _max of 3.42±0.08 pmol×mg^–1 protein. Membranes from cells cultured in the presence of THIP had two binding sites for GABA withK _D-values of 6.8±0.9 nM and 476±311 nM, respectively. The correspondingB _max values were 4.41±0.42 pmol×mg^–1 and 5.81±1.20 pmol×mg^–1. The effect of culturing the cells in THIP was antagonized by the simultaneous presence of bicuculline in the culture media, i.e. no significant low-affinity binding for GABA was found on the membranes from granule cells cultured in both THIP and bicuculline. TheK _D value (14.3±1.4 nM) for the high affinity binding site was, however, slightly increased compared to the non-treated cells. These findings suggest that the ability of THIP to induce formation of low-affinity GABA receptors is mediated by preexisting high-affinity GABA-receptors on the granule cells.     

Modulation of cell surface GABA(B) receptors by desensitization, trafficking and regulated degradation

Inhibitory neurotransmission ensures normal brain function by counteracting and integrating excitatory activity.-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system,and mediates its effects via two classes of receptors:the GABA A and GABA B receptors.GABA A receptors are heteropentameric GABA-gated chloride channels and responsible for fast inhibitory neurotransmission.GABA B receptors are heterodimeric G protein coupled receptors (GPCR) that mediate slow and prolonged inhibitory transmission.The extent of inhibitory neurotransmission is determined by a variety of factors,such as the degree of transmitter release and changes in receptor activity by posttranslational modifications (e.g.,phosphorylation),as well as by the number of receptors present in the plasma membrane available for signal transduction.The level of GABA B receptors at the cell surface critically depends on the residence time at the cell surface and finally the rates of endocytosis and degradation.In this review we focus primarily on recent advances in the understanding of trafficking mechanisms that determine the expression level of GABA B receptors in the plasma membrane,and thereby signaling strength.     

Sleep is differently modulated by basal forebrain GABA(A) and GABA(B) receptors

There is evidence that GABA plays a major role in sleep regulation. GABA(A) receptor agonists and different compounds interacting with the GABA(A) receptor complex, such as barbiturates and benzodiazepines, can interfere with the sleep/wake cycle. On the other hand, there is very little information about the possible role of GABA(B) receptors in sleep modulation. The nucleus basalis of Meynert (NBM), a cholinergic area in the basal forebrain, plays a pivotal role in the modulation of sleep and wakefulness, and both GABA(A) and GABA(B) receptors have been described within the NBM. This study used unilateral infusions in the NBM to determine the effects of 3-hydroxy-5-aminomethylisoxazole hydrobromide (muscimol hydrobromide, a GABA(A) receptor subtype agonist) and beta-(aminomethyl)-4-chlorobenzenepropanoic acid (baclofen, a GABA(B) receptor subtype agonist) on sleep parameters in freely moving rats by means of polygraphic recordings. Muscimol (0.5 nmol) and baclofen (0.7 nmol) induced an increase in slow-wave sleep and an inhibition of wakefulness. Muscimol, but not baclofen, also caused a decrease in desynchronized sleep parameters. The results reported here indicate that 1) the NBM activation of both GABA(A) and GABA(B) receptors influences the sleep/wake cycle, and 2) GABA(A) but not GABA(B) receptors are important for desynchronized sleep modulation, suggesting that the two GABAergic receptors play different roles in sleep modulation.     

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.     

Clathrin-independent endocytosis of GABA(A) receptors in HEK 293 cells.

The endocytosis of GABA(A) receptors was investigated in HEK 293 cells expressing receptor alpha1beta2- and alpha1beta2gamma2-subunit combinations. For assessment of internalized receptors by radioimmunoassay or immunofluorescence, a triple c-myc epitope was introduced into the amino terminus of the beta2 subunit. An assay based on biotin inaccessibility was used for alpha1 subunits. GABA(A) alpha1beta2- and alpha1beta2gamma2-subunit receptors were internalized with a t(1/2) of 5.5 min at 37 degrees C. With both subunit combinations, phorbol 12-myristate 3-acetate enhanced internalization by nearly 100%. Treatment of the cells with hypertonic sucrose prevented both the basal and phorbol ester-induced endocytosis of GABA(A) receptors. GF 109203X, an inhibitor of protein kinase C, blocked the stimulation by phorbol ester but had no detectable effect on basal receptor endocytosis. Coexpression with a dominant-negative mutant of dynamin (K44A) led to a 100% enhancement of GABA(A) receptor internalization, while the endocytosis of beta(2)-adrenergic receptors was completely prevented. The results indicate that the endocytosis of GABA(A) alpha1beta2-subunit receptors in HEK cells is constitutive, positively modulated by activation of protein kinase C, and occurs by a mechanism that requires neither the participation of a GABA(A) receptor gamma2 subunit nor a clathrin-mediated pathway.     

Evidence of two populations of GABA(A) receptors in cerebellar granule cells in culture: different desensitization kinetics, pharmacology, serine/threonine kinase sensitivity, and localization

GABA(A) receptors of rat cerebellar granule cells in culture have been studied by the whole cell patch clamp technique. The biphasic desensitization kinetic observed could be due either to different desensitization mechanisms of a single receptor population or to different receptor populations. The overall data indicate that the latter hypothesis is most probably the correct one. In fact, the fast desensitizing component was selectively potentiated by a benzodiazepine agonist and preferentially down-regulated by activation of the protein serine/threonine kinases A and G, as a consequence of the latter characteristic that receptor population was preferentially down-regulated by previous activation of N-methyl-d-aspartate glutamate receptors, via production of nitric oxide and PKG activation, most probably in dendrites. The other population is benzodiazepine insensitive and not influenced by activation of PKA or PKG. This slowly desensitizing population may correspond to the extrasynaptic delta subunit containing GABA(A) receptors described by other authors. Instead, the rapidly desensitizing population appears to represent dendritic synaptic GABA(A) receptors.     

Antisense suppression of GABA(A) receptor beta subunit levels in cultured cerebellar granule neurons demonstrates their importance in receptor expression

GABA(A) receptors in the CNS are pentameric molecules composed of alpha, beta, gamma, delta, epsilon and theta subunits. Studies on transfected cells have shown that GABA(A) receptor beta subunit isoforms can direct alpha1 subunit localization within the cell. To examine the role of selected subunits in governing GABA(A) receptor expression in neurons, cultures of rat cerebellar granule cells were grown with antisense or sense oligodeoxynucleotides (ODNs) specific for the alpha 1, beta 2 or gamma 2 subunits. These subunits are all expressed in granule neurons where they are thought to contribute to an abundant receptor type. Following ODN treatment, subunit expression and distribution were examined by western blotting, immunocytochemistry and RT-PCR. Treatment of the cultures with the antisense, but not the corresponding sense, ODNs reduced the levels of the targeted subunit polypeptides. In addition, the beta 2 antisense ODN reduced the level of the alpha1 subunit polypeptide without altering the level of its mRNA. In contrast, treatment with the beta 2 subunit antisense ODN did not alter gamma 2 subunit polypeptide expression, distribution or mRNA level. These findings suggest that the alpha1 subunit requires a beta subunit for assembly into GABA(A) receptors in cerebellar granule neurons.     

GABA(B) receptors: synaptic functions and mechanisms of diversity

GABA(B) receptors are the G-protein-coupled receptors for GABA, the main inhibitory neurotransmitter in the mammalian central nervous system. They are implicated in a variety of neurological and psychiatric disorders. With the cloning of GABA(B) receptors ten years ago, substantial progress was made in our understanding of this receptor system. Here, we review current concepts of synaptic GABA(B) functions and present the evidence that points to specific roles for receptor subtypes. We discuss ultrastructural studies revealing that most GABA(B) receptors are located remote from GABAergic terminals, which raises questions as to when such receptors become activated. Finally, we provide possible explanations for the perplexing situation that GABA(B) receptor subtypes that have indistinguishable properties in vitro generate distinct GABA(B) responses in vivo.