Functional properties of recombinant GABA(A) receptors composed of single or multiple beta subunit subtypes

GABA(A) receptor (GABAR) isoforms in the central nervous system are composed of combinations of alpha(1-6), beta(1-4), gamma(1-4), delta(1) and epsilon(1) subunit subtypes arranged in a pentamer. Many regions of the brain express high levels of mRNA encoding several different subunits and even multiple subunit subtypes. The stoichiometry of GABAR isoforms is unclear, and the number and identity of individual subunit subtypes that are coassembled remain uncertain. To examine the role of beta subunit subtypes in the functional properties of GABARS and to determine whether multiple beta subtypes can be coassembled in functional GABARs, plasmids containing cDNAs encoding rat beta1 and/or beta3, alpha5 and gamma2L subtypes were cotransfected into L929 fibroblasts. The properties of the expressed receptor populations were determined using whole-cell and single-channel recording techniques. The alpha5beta1gamma2L isoform was less sensitive to GABA than the alpha5beta3gamma2L isoform. alpha5beta1gamma2L isoform currents were also insensitive to the allosteric modulator loreclezole, while alpha5beta3gamma2L isoform currents were strongly potentiated by loreclezole. Fibroblasts transfected with plasmids containing cDNAs for both beta1 and beta3 subtypes along with alpha5 and gamma2L subtypes produced a receptor population with an intermediate sensitivity to GABA which was insensitive to loreclezole. These results suggest that functional GABARs can be formed that contain two different beta1 subunit subtypes with properties different from receptors that contain only a single beta1 subtype and that the beta1 subunit subtypes influence the response of GABARs to GABA and to the allosteric modulator loreclezole.     

Expression of GABA(A) receptor subunits in the hippocampus of the rat after kainic acid-induced seizures

The GABA(A) receptor is a ligand gated chloride channel consisting of five membrane spanning proteins for which 13 different genes have been identified in the mammalian brain. The present review summarizes recent work from our laboratory on the characterization of the immunocytochemical distribution of these GABA(A) receptor subunits in the rat brain and changes in immunoreactivity and mRNA expression after kainic acid-induced status epilepticus. A heterogeneous distribution of immunoreactive GABA(A) receptor subunits was observed. The most abundant ones were: alpha1, alpha2, alpha4, alpha5, beta2, beta3, gamma2, and delta. Alpha1, beta2, and gamma2 were about equally distributed in all subfields of the hippocampus; alpha4- and delta-subunits were preferentially found in the dentate molecular layer and in CA1; alpha2 was localized to the dentate molecular layer and CA3; alpha5 was found in the dendritic areas of CA1 to CA3; and beta1 was preferentially seen in CA2. Alpha1, beta2, gamma2 and delta were highly concentrated in interneurons. Kainic acid-induced seizures caused acute and chronic changes in the expression of mRNAs and immunoreactive proteins. Acute changes included decreases in alpha2, alpha5, beta1, beta3, gamma2 and delta mRNA levels (by about 25-50%), accompanied by increases (by about 50%) in alpha1, alpha4, and beta2 messages in granule cells (after 6-12 h). Chronic changes, characterized by losses in mRNA and immunoreactive proteins in CA1 and CA3, are undoubtedly due to seizure-related cell damage. However, compensatory expression of alpha2 and beta3 subunits, especially in CA3b/c, was observed. Furthermore, increases in mRNAs and immunoreactive proteins were seen for alpha1, alpha2 alpha4, beta1, beta2, beta3 and gamma2 in granule cells and in the molecular layer of the dentate gyrus at 7-30 days after kainic acid injection. The changes in the expression of GABA(A) receptor subunits, observed in practically all hippocampal subfields, may reflect altered GABA-ergic transmission during development of the epileptic syndrome. Increased expression of GABA(A) receptor subunits in the dendritic field of granule cells and CA3 suggest that GABA-ergic inhibition may be augmented at these levels. However, the lasting preservation of alpha1-, beta2-, and gamma2-subunits in interneurons could provide a basis for augmented inhibition of GABA-ergic interneurons, leading to net disinhibition.     

The role of an alpha subtype M2-M3 His in regulating inhibition of GABAA receptor current by zinc and other divalent cations

Sensitivity of GABAA receptors (GABARs) to inhibition by zinc and other divalent cations is influenced by the alpha subunit subtype composition of the receptor. For example, alpha6beta3gamma2L receptors are more sensitive to inhibition by zinc than alpha1beta3gamma2L receptors. We examined the role of a His residue located in the M2-M3 extracellular domain (rat alpha6 H273) in the enhanced zinc sensitivity conferred by the alpha6 subtype. The alpha1 subtype contains an Asn (N274) residue in the equivalent location. GABA-activated whole-cell currents were obtained from L929 fibroblasts after transient transfection with expression vectors containing GABAA receptor cDNAs. Mutation of alpha1 (alpha1(N274H)) or alpha6 (alpha6(H273N)) subtypes did not alter the GABA EC50 of alphabeta3gamma2L receptors. alpha1(N274H)beta3gamma2L receptor currents were as sensitive to zinc as alpha6beta3gamma2L receptor currents, although alpha6(H273N)beta3gamma2L receptor currents had the reduced zinc sensitivity of alpha1beta3gamma2L receptor currents. We also examined the activity of other inhibitory divalent cations with varying alpha subtype dependence: nickel, cadmium, and copper. alpha6beta3gamma2L receptor currents were more sensitive to nickel, equally sensitive to cadmium, and less sensitive to copper than alpha1beta3gamma2L receptor currents. Studies with alpha1 and alpha6 chimeric subunits indicated that the structural dependencies of the activity of some of these cations were different from zinc. Compared with alpha6beta3gamma2L receptor currents, alpha6(H273N)beta3gamma2L receptor currents had reduced sensitivity to cadmium and nickel, but the sensitivity to copper was unchanged. Compared with alpha1beta3gamma2L receptor currents, alpha1(N274H)beta3gamma2L receptor currents had increased sensitivity to nickel, but the sensitivity to cadmium and copper was unchanged. These findings indicate that H273 of the alpha6 subtype plays an important role in determining the sensitivity of recombinant GABARs to the divalent cations zinc, cadmium, and nickel, but not to copper. Our results also suggest that the extracellular N-terminal domain of the alpha1 subunit contributes to a regulatory site(s) for divalent cations, conferring high sensitivity to inhibition by copper and cadmium.     

Purification of four forms of the beta gamma subunit complex of G proteins containing different gamma subunits

To investigate the physiological significance of the diversity of gamma subunits of G proteins, we purified four forms of beta gamma of G proteins from bovine brain (beta gamma-B1, beta gamma-B2, beta gamma-B3), and spleen (beta gamma-S1) by the sequential chromatography on columns of DEAE-Sephacel, Ultrogel AcA 34, heptylamine-Sepharose, phenyl-5PW, and DEAE-5PW. Electrophoretic analyses showed that each beta gamma mainly contained the 36-kDa beta and a distinct but homogeneous gamma. These beta gamma complexes were subjected directly to proteolytic digestion and subsequent amino acid sequence analyses of their fragments. It was revealed that beta gamma-B1, -B2, and -B3 were identical to beta 1 gamma 7 (with a low level of beta 2 gamma 7), beta 1 gamma 2 and beta 1 gamma 3, respectively, while beta gamma-S1 was composed of beta 1 and an unidentified form of gamma. Then we examined the functional differences among these beta gamma complexes and the beta gamma of transducin (beta gamma-T, beta 1 gamma 1). Few differences were observed among all beta gamma complexes to enhance pertussis toxin-catalyzed ADP-ribosylation of the alpha subunits of G(o) and Gt. The four forms of beta gamma complexes purified from brain and spleen showed indistinguishable inhibitory effects on the release of GDP from G(o) alpha, but beta gamma-T was much less effective. Brain and spleen beta gamma complexes were equally effective in inhibiting calmodulin-stimulated adenylyl-cyclase activity, but beta gamma-T had a very weak inhibitory effect. Five forms of beta gamma facilitated metarhodopsin II-catalyzed binding of GTP gamma S to Gt alpha in a concentration-dependent manner with the following rank order of effectiveness: beta gamma-S1 > beta gamma-T > beta gamma-B1 > beta gamma-B2 > beta gamma-B3. Because the beta gamma complexes used in this study mostly contained the same beta subunit, the functional differences must be dependent on the gamma subunits. Thus, it seems likely that the receptor, the alpha subunits, and the effector are able to distinguish between the various gamma subunits.     

Functional characterization of human gamma-aminobutyric acidA receptors containing the alpha 4 subunit

The alpha subunits are an important determinant of the pharmacology of gamma-aminobutyric acidA (GABAA) receptors with respect to agonists, antagonists, and modulatory compounds, particularly the benzodiazepines. The alpha 4 subunit is the least abundant subunit in the brain and the most similar in deduced primary amino acid sequence to the alpha 6 subunit. We demonstrate that the human alpha 4 subunit forms a functional receptor when expressed with beta gamma 2, demonstrating some properties similar to alpha 6 beta gamma 2 and some properties more akin to alpha 1 beta gamma 2. It also exhibited some properties that were unlike any other alpha subunit-containing receptor. GABA affinity seemed to be identical to that of the alpha 1 beta 1 gamma 2 receptor; however, the partial agonists 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol and piperidine-4-sulfonic acid showed lower efficacy than at either alpha 1 beta 1 gamma 2 or alpha 6 beta 1 gamma 2. Benzodiazepine pharmacology of alpha 4-containing receptors was similar to that of alpha 6-containing receptors with the exception of dimethoxy-4-ethyl-beta-carboline-3-carboxylate, which behaved as a partial inverse agonist. Pentobarbital potentiated alpha 4 beta 1 gamma 2 receptor GABA responses to a level comparable with alpha 6 beta 1 gamma 2 (approximately 700% of EC20); however, unlike alpha 6 beta 1 gamma 2 receptors, it did not elicit any direct activation of the receptor. Propofol also potentiated alpha 4 beta 1 gamma 2 GABA responses but to a level more comparable to that of alpha 1 beta 1 gamma 2, suggesting that these compounds act via different sites. Unlike other subunit combinations, propofol did not elicit a direct activation of the receptor. These results suggest that the mechanism for direct activation of the GABAA receptor by pentobarbital and propofol is absent on alpha 4-containing receptors. Furosemide, which non-competitively inhibits the GABAA receptor, showed 700-fold selectivity for alpha 6 beta 3 gamma 2 receptors over alpha 1-, alpha 2-, alpha 3-, and alpha 5-containing receptors and exhibited selectivity for alpha 4 beta 3 gamma 2 receptors (> 50-fold). These experiments reveal a unique pharmacology for alpha 4-containing receptors with some similarities to both alpha 6- and alpha 1-containing receptors.     

Rat and human hippocampal alpha5 subunit-containing gamma-aminobutyric AcidA receptors have alpha5 beta3 gamma2 pharmacological characteristics

The gamma-aminobutyric acid (GABA)A receptor is a hetero-oligomer consisting of five subunits, the combination of which confers unique pharmacological properties to the receptor. To understand the physiological role of native GABAA receptors, it is critical to determine their subunit compositions. The pharmacological characteristics of human alpha5 beta3 gamma2 and alpha5beta3gamma3 GABAA receptors stably expressed in L(tk-) cells were characterized with the alpha5-selective ligand [3H]L-655,708 and compared with the pharmacological characteristics of [3H]L-655,708 binding sites from rat and human hippocampus. Saturation analyses revealed a 9-fold selective affinity of [3H]L-655,708 for alpha5 beta3 gamma2 receptors (Kd = 1.7 +/- 0.4 nM), compared with alpha5 beta3 gamma3 receptors (Kd = 15 +/- 3 nM). Rat and human hippocampal [3H]L-655,708 binding sites had affinities of 2.2 +/- 0.6 and 1.0 +/- 0.2 nM, respectively, comparable to the affinity of alpha5 beta3 gamma2 receptors. Pharmacological analysis of [3H]L-655,708 binding sites in rat and human hippocampi revealed a strong correlation with the affinities of seven benzodiazepine site ligands for alpha5 beta3 gamma2 but not alpha5 beta3 gamma3 receptors. Immunoprecipitation of [3H]L-655,708 binding sites from rat hippocampus with a gamma2-selective antibody yielded 19 +/- 4% of total benzodiazepine binding sites measured using [3H]Ro15-1788, whereas no specific binding was measured after immunoprecipitation with an anti-gamma3 antibody. Combinatorial immunoprecipitations of [3H]muscimol binding sites with anti-alpha5 and anti-gamma2 or anti-alpha5 and anti-gamma3 antibodies established the preferential expression of alpha5 gamma2 receptors, accounting for 22 +/- 2% of total rat hippocampal GABAA receptors. These observations provide pharmacological and structural evidence for the prevalence of alpha5 beta3 gamma2 GABAA receptors in rat hippocampus, despite the clustering of alpha5 and gamma3 loci on the same chromosome.     

Posttranslational modifications of the 5'-AMP-activated protein kinase beta1 subunit

The AMP-activated protein kinase (AMPK) consists of catalytic alpha and noncatalytic beta and gamma subunits and is responsible for acting as a metabolic sensor for AMP levels. There are multiple genes for each subunit and the rat liver AMPK alpha1 and alpha2 catalytic subunits are associated with beta1 and gamma1 noncatalytic subunits. We find that the isolated gamma1 subunit is N-terminally acetylated with no other posttranslational modification. The isolated beta1 subunit is N-terminally myristoylated. Transfection of COS cells with AMPK subunit cDNAs containing a nonmyristoylatable beta1 reduces, but does not eliminate, membrane binding of AMPK heterotrimer. The isolated beta1 subunit is partially phosphorylated at three sites, Ser24/25, Ser182, and Ser108. The Ser24/25 and Ser108 sites are substoichiometrically phosphorylated and can be autophosphorylated in vitro. The Ser-Pro site in the sequence LSSS182PPGP is stoichiometrically phosphorylated, and no additional phosphate is incorporated into this site with autophosphorylation. Based on labeling studies in transfected cells, we conclude that alpha1 Thr172 is a major, although not exclusive, site of both basal and stimulated alpha1 phosphorylation by an upstream AMPK kinase.     

U-89843A is a novel allosteric modulator of gamma-aminobutyric acidA receptors

A group of pyrrolopyrimidine derivatives were examined for their interaction with rat recombinant gamma-aminobutyric acid (GABA)A receptors using the whole cell patch clamp and equilibrium binding techniques. In the alpha 1 beta 2 gamma 2 subtype of GABAA receptors expressed in human embryonic kidney cells, a prototype pyrrolopyrimidine, U-89843A (7H-pyrrol[2,3-d]pyrimidine,6,7-methyl-2,4-di- 1-pyrrolidinyl,hydrochloride), dose-dependently enhanced 5 microM GABA-induced Cl- currents with a maximal enhancement of 362 +/- 91%, a half-maximal concentration of 2 +/- 0.4 microM and a slope factor of 1.1 +/- 0.4. The drug also inhibited [35S]t-butylbicyclophosphorothionate binding in rat cerebrocortical membranes with a similar half-maximal inhibitory concentration. The enhancement of Cl- currents by U-89843A was insensitive to Ro 15-1788 (a benzodiazepine antagonist), was also observed in the alpha 3 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 subtypes (no selectivity to different alpha-isoforms unlike many benzodiazepines), but was absent in the receptor subtypes consisting of two subunits (alpha 1 beta 2, alpha 1 gamma 2 and beta 2 gamma 2). It has been known that neurosteroids and barbiturates are uniformly active in both the two subunit receptors, substituted pyrazinones are only active in the alpha 1 beta 2 subtype and loreclezole is active in the subtypes containing beta 2. We propose that U-89843A interacts with an allosteric site on GABAA receptors distinct from the sites for benzodiazepines, barbiturates, neurosteroids, substituted pyrazinones or loreclezole.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interferon-gamma potentiates interleukin (IL)-6 and tumor necrosis factor-alpha but not IL-1beta induced by endotoxin in the brain

Because interferon-gamma (IFN gamma) is present in the central nervous system during neurologic diseases associated with inflammation, its effect on endotoxin-induced cytokines was studied. Cerebrospinal fluid (CSF) and serum levels of interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha (TNF alpha), their messenger RNA expression in brain areas (hypothalamus, hippocampus, and striatum) and in spleen were evaluated 2 and 8 h after endotoxin [lipopolysaccharide (LPS), 25 microg/rat i.c.v.], IFN gamma (2.5 microg/rat i.c.v.) or after their coadministration in rats. CSF and serum IL-1beta levels were increased by LPS alone and IFN gamma coadministration did not furtherly increase them. IFN gamma potentiated LPS effect on IL-6 and TNF alpha levels in both CSF and serum. LPS and IFN-gamma coadministration did not alter IL-1beta messenger RNA expression induced by LPS in brain areas and in spleen, but it potentiated that of IL-6 and TNF alpha. The present in vivo data show that i.c.v. coadministration of LPS and IFN gamma results in a potentiation of cytokine production (IL-6 and TNF alpha) which may trigger a cascade of events relevant to neurodegenerative processes. This action is independent of IL-1beta because the production of this cytokine is not altered by IFN gamma treatment.     

The gamma 2 subunit of the GABAA receptor is concentrated in synaptic junctions containing the alpha 1 and beta 2/3 subunits in hippocampus, cerebellum and globus pallidus

The gamma 2 subunit is necessary for the expression of the full benzodiazepine pharmacology of GABAA receptors and is one of the major subunits in the brain. In order to determine the location of channels containing the gamma 2 subunit in relation to GABA-releasing terminals on the surface of neurons, a new polyclonal antipeptide antiserum was developed to the gamma 2 subunit and used in high resolution, postembedding, immunoelectron-microscopic procedures. Dual immunogold labelling of the same section for two subunits, and up to three sections of the same synapse reacted for different subunits, were used to characterize the subunit composition of synaptic receptors. The gamma 2 subunit was present in type 2, "symmetrical" synapses in each of the brain areas studied, with the exception of the granule cell layer of the cerebellum. The gamma 2 subunit was frequently co-localized in the same synaptic junction with the alpha 1 and beta 2/3 subunits. The immunolabelling of synapses was coincident with the junctional membrane specialization of the active zone. Immunolabelling for the receptor often occurred in multiple clusters in the synapses. In the hippocampus, the gamma 2 subunit was present in basket cell synapses on the somata and proximal dendrites and in axo-axonic cell synapses on the axon initial segment of pyramidal and granule cells. Some synapses on the dendrites of GABAergic interneurones were densely labelled for the gamma 2, alpha 1 and beta 2/3 subunits. In the cerebellum, the gamma 2 subunit was present in both distal and proximal Purkinje cell dendritic synapses established by stellate and basket cell, respectively. On the soma of Purkinje cells, basket cell synapses were only weakly labelled. Synapses on interneuron dendrites were more densely labelled for the gamma 2, alpha 1 and beta 2/3 subunits than synapses on Purkinje or granule cells. Although immunoperoxidase and immunofluorescence methods show an abundance of the gamma 2 subunit in granule cells, the labelling of Golgi synapses was much weaker with the immunogold method than that of the other cell types. In the globus pallidus, many type 2 synapses were labelled for the gamma 2 subunit together with alpha 1 and beta 2/3 subunits. The results show that gamma 2 and beta 2/3 subunits receptor channels are highly concentrated in GABAergic synapses that also contain the alpha 1 and beta 2/3 subunits. Channels containing the gamma 2 subunit are expressed in synapses on functionally distinct domains of the same neuron receiving GABA from different presynaptic sources. There are quantitative differences in the density of GABAA receptors at synapses on different cell types in the same brain area.     

Coexistence of two beta subunit isoforms in the same gamma-aminobutyric acid type A receptor

Three novel subunit-specific antisera to the beta1, beta2, and beta3 subunits of rat gamma-aminobutyric acid type A (GABAA) receptors have been used to study the native receptor in the rat brain. Affinity-purified anti-beta1, anti-beta2, and anti-beta3 antibodies recognized in immunoblots protein bands of 57, 55, and 57 kDa, respectively. Quantitative immunoprecipitation of solubilized GABAA receptors from various rat brain regions showed that the beta2 subunit was the most abundant isoform in cerebellum (in 96% of the GABAA receptors) and cerebral cortex (64%) but that it was the least abundant isoform in hippocampus (44%). The beta3 subunit was found most abundant in hippocampus (64%) followed by cerebral cortex (48%) and cerebellum (33%). The beta1 subunit was present in a very small proportion of the cerebellar GABAA receptors (3%), but it was present in a high proportion of the GABAA receptors from the hippocampus (49%) and cerebral cortex (32%). Quantitative receptor immunoprecipitation or immunopurification followed by immunoblotting experiments have revealed the existence of colocalization of two different beta subunit isoforms in a significant proportion of the brain GABAA receptors. Thus, in the rat cerebral cortex 33% of the GABAA receptors have both beta2 and beta3 subunits, and 19% of the receptors have both beta1 and beta3 subunits. The extent of colocalization of beta subunit isoforms varied among brain regions, being highest in hippocampus and lowest in cerebellum. These and other results taken together suggest that the number of alpha, beta, and gamma subunits (stoichiometry) in the brain GABAA receptor pentamers might not be unique. It might vary depending on receptor type.     

Creating an education model for cardiac patients

gamma-Aminobutyric acidA (GABA(A)) gated chloride ion channels were expressed from human recombinant cDNA using the baculovirus/Sf-9 insect cell expression system. The electrophysiological effects in whole-cell currents of 5-(4-piperidyl) isoxazol-3-ol (4-PIOL), a GABA(A) receptor partial agonist, were investigated on GABA(A) receptor complexes of alpha1beta2gamma2S subunits as well as a slightly modified construct of alpha1(valine 121)beta2gamma2S subunits. Here we report that (1)4-PIOL induces an inward whole-cell current in a concentration-dependent manner in both alpha1(val 121)beta2gamma2S and alpha1(ile 121)beta2gamma2S receptor subunit combinations. (2) The 4-PIOL induced whole-cell currents were more pronounced in alpha1(val 121)beta2gamma2S than in alpha1(ile 121)beta2gamma2S receptor subunit combinations. (3) 4-PIOL inhibited GABA-induced responses on alpha1(ile 121)beta2gamma2S and alpha1(val 121)beta2gamma2S receptor combinations with similar potency.     

Direct interaction of the alpha and gamma subunits of the G proteins. Purification and analysis by limited proteolysis

The heterotrimeric G proteins are often regarded functionally as a heterodimer, consisting of a guanine nucleotide-binding alpha subunit and a beta gamma subunit complex. Since the tightly associated beta gamma subunit complex can be separated only under denaturing conditions, studies aimed at determining the individual contributions of the beta and gamma subunits in terms of binding to the various alpha subunits, interacting with receptors, and regulating effectors, have not been possible. To circumvent this problem, we have used baculovirus-infected cells to direct the individual expression of the beta 1 and gamma 2 subunits. Application of extracts from baculovirus-infected cells to an alpha subunit of G protein (G(o) alpha)-affinity matrix resulted in the selective retention and AMF-specific elution of the expressed gamma 2 subunit, but not the expressed beta 1 subunit. Overall, these and other data provide the first evidence of a direct association between the gamma and alpha subunits, which is dependent on prenylation of gamma. The apparent direct association between the gamma and alpha subunits was further probed by limited trypsin proteolysis. Upon addition of trypsin, the G(o) alpha subunit was rapidly cleaved to a 24-kDa fragment. However, in the presence of the purified gamma 2 subunit, trypsin cleavage of the G(o) alpha subunit was completely prevented. This demonstration of a direct association between the gamma and alpha subunits is particularly intriguing in light of the increasingly large number of known alpha, beta, and gamma subunits, which raises important questions regarding the assembly of these subunits into functionally distinct G proteins. Thus, a direct association between the gamma and alpha subunits, which exhibit the greatest structural diversity, may provide the basis for the selective assembly of these subunits into G proteins with functional diversity.     

Distribution and function of laminins in the neuromuscular system of developing, adult, and mutant mice

Laminins, heterotrimers of alpha, beta, and gamma chains, are prominent constituents of basal laminae (BLs) throughout the body. Previous studies have shown that laminins affect both myogenesis and synaptogenesis in skeletal muscle. Here we have studied the distribution of the 10 known laminin chains in muscle and peripheral nerve, and assayed the ability of several heterotrimers to affect the outgrowth of motor axons. We show that cultured muscle cells express four different alpha chains (alpha1, alpha2, alpha4, and alpha5), and that developing muscles incorporate all four into BLs. The portion of the muscle's BL that occupies the synaptic cleft contains at least three alpha chains and two beta chains, but each is regulated differently. Initially, the alpha2, alpha4, alpha5, and beta1 chains are present both extrasynaptically and synaptically, whereas beta2 is restricted to synaptic BL from its first appearance. As development proceeds, alpha2 remains broadly distributed, whereas alpha4 and alpha5 are lost from extrasynaptic BL and beta1 from synaptic BL. In adults, alpha4 is restricted to primary synaptic clefts whereas alpha5 is present in both primary and secondary clefts. Thus, adult extrasynaptic BL is rich in laminin 2 (alpha2beta1gamma1), and synaptic BL contains laminins 4 (alpha2beta2gamma1), 9 (alpha4beta2gamma1), and 11 (alpha5beta2gamma1). Likewise, in cultured muscle cells, alpha2 and beta1 are broadly distributed but alpha5 and beta2 are concentrated at acetylcholine receptor-rich "hot spots," even in the absence of nerves. The endoneurial and perineurial BLs of peripheral nerve also contain distinct laminin chains: alpha2, beta1, gamma1, and alpha4, alpha5, beta2, gamma1, respectively. Mutation of the laminin alpha2 or beta2 genes in mice not only leads to loss of the respective chains in both nerve and muscle, but also to coordinate loss and compensatory upregulation of other chains. Notably, loss of beta2 from synaptic BL in beta2(-/-) "knockout" mice is accompanied by loss of alpha5, and decreased levels of alpha2 in dystrophic alpha2(dy/dy) mice are accompanied by compensatory retention of alpha4. Finally, we show that motor axons respond in distinct ways to different laminin heterotrimers: they grow freely between laminin 1 (alpha1beta1gamma1) and laminin 2, fail to cross from laminin 4 to laminin 1, and stop upon contacting laminin 11. The ability of laminin 11 to serve as a stop signal for growing axons explains, in part, axonal behaviors observed at developing and regenerating synapses in vivo.     

The interaction of the general anesthetic etomidate with the gamma-aminobutyric acid type A receptor is influenced by a single amino acid

The gamma-aminobutyric acid type A (GABAA) receptor is a transmitter-gated ion channel mediating the majority of fast inhibitory synaptic transmission within the brain. The receptor is a pentameric assembly of subunits drawn from multiple classes (alpha1-6, beta1-3, gamma1-3, delta1, and epsilon1). Positive allosteric modulation of GABAA receptor activity by general anesthetics represents one logical mechanism for central nervous system depression. The ability of the intravenous general anesthetic etomidate to modulate and activate GABAA receptors is uniquely dependent upon the beta subunit subtype present within the receptor. Receptors containing beta2- or beta3-, but not beta1 subunits, are highly sensitive to the agent. Here, chimeric beta1/beta2 subunits coexpressed in Xenopus laevis oocytes with human alpha6 and gamma2 subunits identified a region distal to the extracellular N-terminal domain as a determinant of the selectivity of etomidate. The mutation of an amino acid (Asn-289) present within the channel domain of the beta3 subunit to Ser (the homologous residue in beta1), strongly suppressed the GABA-modulatory and GABA-mimetic effects of etomidate. The replacement of the beta1 subunit Ser-290 by Asn produced the converse effect. When applied intracellularly to mouse L(tk-) cells stably expressing the alpha6beta3gamma2 subunit combination, etomidate was inert. Hence, the effects of a clinically utilized general anesthetic upon a physiologically relevant target protein are dramatically influenced by a single amino acid. Together with the lack of effect of intracellular etomidate, the data argue against a unitary, lipid-based theory of anesthesia.     

SB-205384: a GABA(A) receptor modulator with novel mechanism of action that shows subunit selectivity

1. SB-205384, and its (+) enantiomer (+)-SB-205384 were tested for their modulatory effects on human GABA(A) receptor subunit combinations expressed in Xenopus oocytes by electrophysiological methods. 2. The slowing of the decay rate induced by SB-205384 on native GABA-activated currents in rat neurones was also seen on GABA(A) currents in oocytes expressing human GABA(A) subunits. This temporal effect was observed for the alpha3beta2gamma2 subunit combination with little effect in subunit combinations containing either alpha1 or alpha2. 3. Potentiation of the peak amplitude of the GABA-activated currents by SB-205384 or (+)-SB-205384 was less specific for a particular subunit combination, although the greatest effect at 10 microM drug was seen on the alpha3beta2gamma2 subunit combination. 4. In contrast, zolpidem, a benzodiazepine site modulator, did not significantly slow decay rates of GABA(A) currents in oocytes expressing the alpha3beta2gamma2 subunit combination. Zolpidem, as expected, did selectively potentiate GABA-activated currents on oocytes expressing the gamma2 subunit compared to those containing the gamma1. 5. The results show that the novel kinetic modulatory profile of SB-205384 is selective for the alpha3beta2gamma2 subunit combination. This suggests that the compound is binding to a novel regulatory site on the subunit complex.     

The role of the GABA(A) receptor alpha1 subunit N-terminal extracellular domain in propofol potentiation of chloride current

Propofol (2,6-diisopropylphenol), an intravenous general anesthetic in active clinical use today, potentiates the action of gamma-aminobutyric acid (GABA) at the type-A receptor and also directly induces current in the absence of GABA. We expressed different combinations of murine GABA(A) receptor alpha1, beta3 and gamma2 subunits in Xenopus oocytes to investigate the subunit dependence of propofol potentiation of pentobarbital-induced current. Pentobarbital induces current in all beta3-subunit-containing receptors, whereas current gating by GABA requires the presence of both alpha1 and beta3 subunits. Therefore, pentobarbital rather than GABA was used to induce current in order to separate the subunit dependence of current gating from the subunit dependence of potentiating action of propofol. alpha1beta3gamma2, alpha1beta3, beta3gamma2, or beta3 subunit combinations all responded to pentobarbital in a dose-dependent manner. True potentiation was defined as the current magnitude to simultaneous application of pentobarbital and propofol exceeding the additive responses to individual drug applications. A dose-dependent propofol potentiation of pentobarbital-induced current was observed in oocytes injected with alpha1beta3 or alpha1beta3gamma2 but not in beta3gamma2 or beta3 subunits, suggesting that the alpha1 subunit was necessary for this modulatory action of propofol. Further examination of the propofol potentiation in chimeras between the alpha1 and beta3 subunits showed that the extracellular amino-terminal half of the alpha1 subunit was sufficient to support propofol potentiation. The different requirements of the receptor structure for the agonistic (gating) and the potentiating actions suggest that these two actions of propofol are distinct processes mediated through its action at distinct sites.     

Developmental cues modulate GABAA receptor subunit mRNA expression in cultured cerebellar granule neurons

The developmental expression of mRNAs encoding the GABAA receptor was analyzed in the rat cerebellar cortex and in cultured cerebellar granule neurons. Our studies in vivo reveal that the alpha 1-, beta 2-, and gamma 2-subunit mRNA levels in the cerebellar cortex rise dramatically during the second post-natal week, a period temporally correlated with extensive cerebellar maturation. To determine if these increases were preprogrammed or dependent on extrinsic factors, we examined subunit mRNA expression in granule cell cultures prepared at embryonic day 19 (E19) and postnatal day 10 (P10), immature and mature stages of cerebellar development, respectively. In E19 cultures, the alpha 1, beta 2, and gamma 2 GABAA receptor subunit mRNAs were present and their levels remained constant over the 21 d culture period. These results suggest that GABAA receptor gene expression is not intrinsic to the immature granule cells. A different pattern was found in P10 cultures where the three subunit mRNAs were initially present at levels approximately sixfold higher than those found at E19. The beta 2- and gamma 2-subunit mRNAs remained constant for 4 d and then increased sixfold between 4 and 7 d in culture. The magnitude and time course of these increases were similar to the developmental changes that occurred in vivo. Thus, our findings raise the possibility that signals encountered during development are necessary to induce GABAA receptor subunit mRNA expression. Moreover, these cues have been received by granule neurons prior to P10.(ABSTRACT TRUNCATED AT 250 WORDS)