Identification of an amino acid sequence within GABA(A) receptor beta3 subunits that is important for receptor assembly

GABA(A) receptors are chloride ion channels that can be opened by GABA, the most important inhibitory transmitter in the CNS. In the mammalian brain the majority of these pentameric receptors is composed of two alpha, two beta and one gamma subunit. To achieve the correct order of subunits around the pore, each subunit must form specific contacts via its plus (+) and minus (-) side. To identify a sequence on the beta3 subunit important for assembly, we generated various full-length or truncated chimeric beta3 constructs and investigated their ability to assemble with alpha1 and gamma2 subunits. It was demonstrated that replacement of the sequence beta3(76-89) by the homologous alpha1 sequence impaired assembly with alpha1 but not with gamma2 subunits in alpha1beta3gamma2-GABA(A) receptors. Other experiments indicated that assembly was impaired via the beta3(-) side of the chimeric subunit. Within the sequence beta3(76-89) the sequence beta3(85-89) seemed to be of primary importance for assembly with alpha1 subunits. A comparison with the structure of the acetylcholine-binding protein supports the conclusion that the sequence beta3(85-89) is located at the beta3(-) side and indicates that it contains amino acid residues that might directly interact with the (+) side of the neighbouring alpha1 subunit.     

GABA(A) receptor assembly. Identification and structure of gamma(2) sequences forming the intersubunit contacts with alpha(1) and beta(3) subunits

GABA(A) receptors are ligand-gated chloride channels composed of five homologous subunits that specifically recognize one another and assemble around an aqueous pore. To identify domains responsible for the specificity of subunit association, we constructed C-terminal truncated gamma(2) subunits, as well as mutated and chimeric fragments. From their ability to interfere with alpha(1)beta(3)gamma(2) receptor assembly and to associate with full-length subunits, we concluded that amino acid sequences gamma(2)-(91-104) and gamma(2)-(83-90) form the sites mediating assembly with alpha(1) and beta(3) subunits, respectively. Neural network-based secondary structure prediction, Monte Carlo optimization, and hydrophobicity analysis led to the conclusion that these sites also form the intersubunit contacts in the completely assembled receptor and provided important information on the benzodiazepine-binding site and structure of GABA(A) receptors.     

Molecular determinants of desensitization and assembly of the chimeric GABA(A) receptor subunits (alpha1/gamma2) and (gamma2/alpha1) in combinations with beta2 and gamma2

Two gamma-aminobutyric acid(A) (GABA(A)) receptor chimeras were designed in order to elucidate the structural requirements for GABA(A) receptor desensitization and assembly. The (alpha1/gamma2) and (gamma2/alpha1) chimeric subunits representing the extracellular N-terminal domain of alpha1 or gamma2 and the remainder of the gamma2 or alpha1 subunits, respectively, were expressed with beta2 and beta2gamma2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (alpha1/gamma2)beta2 and (alpha1/gamma2)beta2gamma2 but not the (gamma2/alpha1)beta2 and (gamma2/alpha1)beta2gamma2 subunit combinations formed functional receptor complexes as shown by whole-cell patch-clamp recordings and [3H]muscimol and [3H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (alpha1/gamma2)-containing receptors was pronounced, as opposed to the staining of the (gamma2/alpha1)-containing receptors, which was only slightly higher than background. To explain this, the (alpha1/gamma2) and (gamma2/alpha1) chimeras may act like alpha1 and gamma2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (alpha1/gamma2) chimeric subunit had characteristics different from the alpha1 subunit, since the (alpha1/gamma2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch-clamp recordings, which was independent of whether the chimera was expressed in combination with beta2 or beta2gamma2. Surprisingly, the (alpha1/gamma2)(gamma2/alpha1)beta2 subunit combination did desensitize, indicating that the C-terminal segment of the alpha1 subunit may be important for desensitization. Moreover, desensitization was observed for the (alpha1/gamma2)beta2gamma2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.     

GABA(A) receptor composition is determined by distinct assembly signals within alpha and beta subunits

Key to understanding how receptor diversity is achieved and controlled is the identification of selective assembly signals capable of distinguishing between other subunit partners. We have identified that the beta1-3 subunits exhibit distinct assembly capabilities with the gamma2L subunit. Similarly, analysis of an assembly box in alpha1-(57-68) has revealed an absolute requirement for this region in the assembly of alphabeta receptors. Furthermore, a selective requirement for a single amino acid (Arg-66), previously shown to be essential for the formation of the low affinity GABA binding site, is observed. This residue is critical for the assembly of alpha1beta2 but not alpha1beta1 or alpha1beta3 receptors. We have confirmed the ability of the previously identified GKER signal in beta3 to direct the assembly of betagamma receptors. The GKER signal is also involved in driving assembly with the alpha1 subunit, conferring the ability to assemble with alpha1(R66A) on the beta2 subunit. Although this signal is sufficient to permit the formation of beta2gamma2 receptors, it is not necessary for beta3gamma2 receptor formation, suggesting the existence of alternative assembly signals. These findings support the belief that GABA(A) receptor assembly occurs via defined pathways to limit the receptor diversity.     

Identification of amino acid residues important for assembly of GABA receptor alpha1 and gamma2 subunits

Comparative models of GABA(A) receptors composed of alpha1 beta3 gamma2 subunits were generated using the acetylcholine-binding protein (AChBP) as a template and were used for predicting putative engineered cross-link sites between the alpha1 and the gamma2 subunit. The respective amino acid residues were substituted by cysteines and disulfide bond formation between subunits was investigated on co-transfection into human embryonic kidney (HEK) cells. Although disulfide bond formation between subunits could not be observed, results indicated that mutations studied influenced assembly of GABA(A) receptors. Whereas residue alpha1A108 was important for the formation of assembly intermediates with beta3 and gamma2 subunits consistent with its proposed location at the alpha1(+) side of GABA(A) receptors, residues gamma2T125 and gamma2P127 were important for assembly with beta3 subunits. Mutation of each of these residues also caused an impaired expression of receptors at the cell surface. In contrast, mutated residues alpha1F99C, alpha1S106C or gamma2T126C only impaired the formation of receptors at the cell surface when co-expressed with subunits in which their predicted interaction partner was also mutated. These data are consistent with the prediction that the mutated residue pairs are located close to each other.     

Assembly of GABA(A) receptors (Review)

GABA(A) receptors are the major inhibitory transmitter receptors in the central nervous system. They are chloride ion channels that can be opened by gamma-aminobutyric acid (GABA) and are the targets of action of a variety of pharmacologically and clinically important drugs. GABA(A) receptors are composed of five subunits that can belong to different subunit classes. The existence of 19 different subunits gives rise to the formation of a large variety of distinct GABA(A) receptor subtypes in the brain. The majority of GABA(A) receptors seems to be composed of two alpha, two beta and one gamma subunit and the occurrence of a defined subunit stoichiometry and arrangement in alphabetagamma receptors strongly indicates that assembly of GABA(A) receptors proceeds via defined pathways. Based on the differential ability of subunits to interact with each other, a variety of studies have been performed to identify amino acid sequences or residues important for assembly. Such residues might be involved in direct protein-protein interactions, or in stabilizing direct contact sites in other regions of the subunit. Several homo-oligomeric or hetero-oligomeric assembly intermediates could be the starting point of GABA(A) receptor assembly but so far no unequivocal assembly mechanism has been identified. Possible mechanisms of assembly of GABA(A) receptors are discussed in the light of recent publications.     

Forced subunit assembly in alpha1beta2gamma2 GABAA receptors. Insight into the absolute arrangement

The major isoform of the gamma-aminobutyric acid type A (GABA(A)) receptor is thought to be composed of 2alpha(1), 2beta(2), and 1gamma(2) subunit(s), which surround the ion pore. Definite evidence for the subunit arrangement is lacking. We show here that GABA(A) receptor subunits can be concatenated to a trimer that can be functionally expressed upon combination with a dimer. Many combinations did not result in the functional expression. In contrast, four different combinations of triple subunits with dual subunit constructs, all resulting in the identical pentameric receptor gamma(2)beta(2)alpha(1)beta(2)alpha(1), could be successfully expressed in Xenopus oocytes. We characterized the functional properties of these receptors in respect to agonist, competitive antagonist, and diazepam sensitivity. All properties were similar to those of wild type alpha(1)beta(2)gamma(2) GABA(A) receptors. Thus, together with information on the crystal structure of the homologous acetylcholine-binding protein (Brejc, K., van Dijk, W. J., Klaassen, R. V., Schuurmans, M., van Der Oost, J., Smit, A. B., and Sixma, T. K., (2001) Nature 411, 269-276, we provide evidence for an arrangement gamma(2)beta(2)alpha(1)beta(2)alpha(1), counterclockwise when viewed from the synaptic cleft. Forced subunit assembly will also allow receptors containing different subunit isoforms or mutant subunits to be expressed, each in a desired position. The methods established here should be applicable to the entire ion channel family comprising nicotinic acetylcholine, glycine, and 5HT(3) receptors.     

Detection and binding properties of GABA(A) receptor assembly intermediates

Density gradient centrifugation of native and recombinant gamma-aminobutyric acid, type A (GABA(A)) receptors was used to detect assembly intermediates. No such intermediates could be identified in extracts from adult rat brain or from human embryonic kidney (HEK) 293 cells transfected with alpha(1), beta(3), and gamma(2) subunits and cultured at 37 degrees C. However, subunit dimers, trimers, tetramers, and pentamers were found in extracts from the brain of 8-10-day-old rats and from alpha(1)beta(3)gamma(2) transfected HEK cells cultured at 25 degrees C. In both systems, alpha(1), beta(3), and gamma(2) subunits could be identified in subunit dimers, indicating that different subunit dimers are formed during GABA(A) receptor assembly. Co-transfection of HEK cells with various combinations of full-length and C-terminally truncated alpha(1) and beta(3) or alpha(1) and gamma(2) subunits and co-immunoprecipitation with subunit-specific antibodies indicated that even subunits containing no transmembrane domain can assemble with each other. Whereas alpha(1)gamma(2), alpha(1)Ngamma(2), alpha(1)gamma(2)N, and alpha(1)Ngamma(2)N, combinations exhibited specific [(3)H]Ro 15-1788 binding, specific [(3)H]muscimol binding could only be found in alpha(1)beta(3) and alpha(1)beta(3)N, but not in alpha(1)Nbeta(3) or alpha(1)Nbeta(3)N combinations. This seems to indicate that a full-length alpha(1) subunit is necessary for the formation of the muscimol-binding site and for the transduction of agonist binding into channel gating.     

Arg-274 and Leu-277 of the gamma-aminobutyric acid type A receptor alpha 2 subunit define agonist efficacy and potency

Alanine-scanning mutagenesis and the whole cell voltage clamp technique were used to investigate the function of the extracellular loop between the second and third transmembrane domains (TM2-TM3) of the gamma-aminobutyric acid type A receptor (GABA(A)-R). A conserved arginine residue in the TM2-TM3 loop of the GABA(A)-R alpha(2) subunit was mutated to alanine, and the mutant alpha(2)(R274A) was co-expressed with wild-type beta(1) and gamma(2S) subunits in human embryonic kidney (HEK) 293 cells. The GABA EC(50) was increased by about 27-fold in the mutant receptor relative to receptors containing a wild-type alpha(2) subunit. Similarly, the GABA EC(50) at alpha(2)(L277A)beta(1)gamma(2S) and alpha(2)(K279A)beta(1)gamma(2S) GABA(A)-R combinations was increased by 51- and 4-fold, respectively. The alpha(2)(R274A) or alpha(2)(L277A) mutations also reduced the maximal response of piperidine-4-sulfonic acid relative to GABA by converting piperidine-4-sulfonic acid into a weak partial agonist at the GABA(A)-R. Based on these results, we propose that alpha(2)(Arg-274) and alpha(2)(Leu-277) are crucial to the efficient transduction of agonist binding into channel gating at the GABA(A)-R.     

Unique assignment of inter-subunit association in GABA(A) alpha 1 beta 3 gamma 2 receptors determined by molecular modeling

Recent publications defined requirements for inter-subunit contacts in a benzodiazepine-sensitive GABA(A) receptor (GABA(A)R alpha 1 beta 3 gamma 2). There is strong evidence that the heteropentameric receptor contains two alpha 1, two beta 3, and one gamma 2 subunit. However, the available data do not distinguish two possibilities: When viewed clockwise from an extracellular viewpoint the subunits could be arranged in either gamma 2 beta 3 alpha 1 beta 3 alpha 1 or gamma 2 alpha 1 beta 3 alpha 1 beta 3 configurations. Here we use molecular modeling to thread the relevant GABA(A)R subunit sequences onto a template of homopentameric subunits in the crystal structure of the acetylcholine binding protein (AChBP). The GABA(A) sequences are known to have 15-18% identity with the acetylcholine binding protein and nearly all residues that are conserved within the nAChR family are present in AChBP. The correctly aligned GABA(A) sequences were threaded onto the AChBP template in the gamma 2 beta 3 alpha 1 beta 3 alpha 1 or gamma 2 alpha 1 beta 3 alpha 1 beta 3 arrangements. Only the gamma 2 alpha 1 beta 3 alpha 1 beta 3 arrangement satisfied three known criteria: (1) alpha 1 His(102) binds at the gamma 2 subunit interface in proximity to gamma 2 residues Thr(142), Phe(77), and Met(130); (2) alpha 1 residues 80-100 bind near gamma 2 residues 91-104; and (3) alpha 1 residues 58-67 bind near the beta 3 subunit interface. In addition to predicting the most likely inter-subunit arrangement, the model predicts which residues form the GABA and benzodiazepine binding sites.     

Endoplasmic reticulum retention and associated degradation of a GABAA receptor epilepsy mutation that inserts an aspartate in the M3 transmembrane segment of the alpha1 subunit

A GABA(A) receptor alpha1 subunit epilepsy mutation (alpha1(A322D)) introduces a negatively charged aspartate residue into the hydrophobic M3 transmembrane domain of the alpha1 subunit. We reported previously that heterologous expression of alpha1(A322D)beta2gamma2 receptors in mammalian cells resulted in reduced total and surface alpha1 subunit protein. Here we demonstrate the mechanism of this reduction. Total alpha1(A322D) subunit protein was reduced relative to wild type protein by a similar amount when expressed alone (86 +/- 6%) or when coexpressed with beta2 and gamma2S subunits (78 +/- 6%), indicating an expression reduction prior to subunit oligomerization. In alpha1beta2gamma2S receptors, endoglycosidase H deglycosylated only 26 +/- 5% of alpha1 subunits, consistent with substantial protein maturation, but in alpha1(A322D)beta2gamma2S receptors, endoglycosidase H deglycosylated 91 +/- 4% of alpha1(A322D) subunits, consistent with failure of protein maturation. To determine the cellular localization of wild type and mutant subunits, the alpha1 subunit was tagged with yellow (alpha1-YFP) or cyan (alpha1-CFP) fluorescent protein. Confocal microscopic imaging demonstrated that 36 +/- 4% of alpha1-YFPbeta2gamma2 but only 5 +/- 1% alpha1(A322D)-YFPbeta2gamma2 colocalized with the plasma membrane, whereas the majority of the remaining receptors colocalized with the endoplasmic reticulum (55 +/- 4% alpha1-YFPbeta2gamma2S, 86 +/- 3% alpha1(A322D)-YFP). Heterozygous expression of alpha1-CFPbeta2gamma2S and alpha1(A322D)-YFPbeta2gamma2S or alpha1-YFPbeta2gamma2S and alpha1(A322D)-CFPbeta2gamma2S receptors showed that membrane GABA(A) receptors contained primarily wild type alpha1 subunits. These data demonstrate that the A322D mutation reduces alpha1 subunit expression after translation, but before assembly, resulting in endoplasmic reticulum-associated degradation and membrane alpha1 subunits that are almost exclusively wild type subunits.     

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.     

Spontaneous cross-link of mutated alpha1 subunits during GABA(A) receptor assembly

gamma-Aminobutyric acid, type A (GABA(A)) receptor alpha1 subunits containing a cysteine mutation at a position in the channel mouth (H109C) surprisingly formed a spontaneous cross-link with each other in receptors composed of alpha1H109C, beta3, and gamma2 subunits. Cross-linking of two alpha1H109C subunits did not significantly change the affinity of [(3)H]muscimol or [(3)H]Ro15-1788 binding in alpha1H109Cbeta3gamma2 receptors, but GABA displayed a reduced potency for activating chloride currents. On reduction of the disulfide bond, however, GABA activation as well as diazepam modulation was similar in mutated and wild-type receptors, suggesting that these receptors exhibited the same subunit stoichiometry and arrangement. Disulfide bonds could not be reoxidized by copper phenanthroline after having been reduced in completely assembled receptors, suggesting that cross-linking can only occur at an early stage of assembly. The cross-link of alpha1H109C subunits and the subsequent transport of the resulting homodimers to the cell surface caused a reduction of the intracellular pool of alpha1H109C subunits and a reduced formation of completely assembled receptors. The formation of alpha1H109C homodimers as well as of correctly assembled GABA(A) receptors containing cross-linked alpha1H109C subunits could indicate that homodimerization of alpha1 subunits via contacts located in the channel mouth might be one starting point of GABA(A) receptor assembly. Alternatively the assembly mechanism might have started with the formation of heterodimers followed by a cross-link of mutated alpha1 subunits at the heterotrimeric stage. The formation of cross-linked alpha1H109C homodimers would then have occurred independently in a separate pathway.     

Effects of treatment with GABA(A) receptor subunit antisense oligodeoxynucleotides on GABA-stimulated 36Cl- influx in the rat cerebral cortex

GABA(A) receptor function was studied in cerebral cortical vesicles prepared from rats after intracerebroventricular microinjections of antisense oligodeoxynucleotides (aODNs) for alpha1, gamma2, beta1, beta2 subunits. GABA(A) receptor alpha1 subunit aODNs decreased alpha1 subunit mRNA by 59+/-10%. Specific [3H]GABA binding was decreased by alpha1 or beta2 subunit aODNs (to 63+/-3% and 64+/-9%, respectively) but not changed by gamma2 subunit aODNs (94+/-5%). Specific [3H]flunitrazepam binding was increased by alpha1 or beta2 subunit aODNs (122+/-8% and 126+/-11%, respectively) and decreased by gamma2 subunit aODNs (50+/-13%). The "knockdown" of specific subunits of the GABA(A )receptor significantly influenced GABA-stimulated 36Cl- influx. Injection of alpha1 subunit aODNs decreased basal 36Cl- influx and the GABA Emax; enhanced GABA modulation by diazepam; and decreased antagonism of GABA activity by bicuculline. Injection of gamma2 subunit aODNs increased the GABA Emax; reversed the modulatory efficacy of diazepam from enhancement to inhibition of GABA-stimulation; and reduced the antagonist effect of bicuculline. Injection of beta2 subunit aODNs reduced the effect of diazepam whereas treatment with beta1 subunit aODNs had no effect on the drugs studied. Conclusions from our studies are: (1) alpha1 subunits promote, beta2 subunits maintain, and gamma2 subunits suppress GABA stimulation of 36Cl- influx; (2) alpha1 subunits suppress, whereas beta2, and gamma2 subunits promote allosteric modulation by benzodiazepines; (3) diazepam can act as an agonist or inverse agonist depending on the relative composition of the receptor subunits: and (4) the mixed competitive/non-competitive effects of bicuculline result from activity at alpha1 and gamma2 subunits and the lack of activity at beta1 and beta2 subunits.     

Intracellular trafficking of GABA(A) receptors

Some of the mechanisms that control the intracellular trafficking of GABA(A) receptors have recently been described. Following the synthesis of alpha, beta, and gamma subunits in the endoplasmic reticulum, ternary receptor complexes assemble slowly and are inefficiently inserted into surface membranes of heterologous cells. While beta3, beta4, and gamma2S subunits appear to contain polypeptide sequences that alone are sufficient for surface targeting, these sequences are neither conserved nor essential for surface expression of heteromeric GABA(A) receptors formed from alpha1beta or alpha1betagamma subunits. At the neuronal surface, native GABA(A) receptor clustering and synaptic targeting require a gamma2 subunit and the participation of gephyrin, a clustering protein for glycine receptors. A linker protein, such as the GABA(A) receptor associated protein (GABARAP), may be necessary for the formation of GABA(A) receptor aggregates containing gephyrin. A substantial fraction of surface receptors are sequestered by endocytosis, another process which apparently requires a GABA(A) receptor gamma2 subunit. In heterologous cells, constitutive endocytosis seems to predominate while, in cortical neurons, internalization is evoked when receptors are occupied by GABA(A) agonists. After constitutive endocytosis, receptors are relatively stable and can be rapidly recycled to the cell surface, a process that may be regulated by protein kinase C. On the other hand, a portion of the intracellular GABA(A) receptors derived from ligand-dependent endocytosis is apparently degraded. The clustering of GABA(A) receptors at synapses and at coated pits are two mechanisms that may compete for a pool of diffusable receptors, providing a model for plasticity at inhibitory synapses.     

Lateral mobility and anchoring of recombinant GABAA receptors depend on subunit composition.

The clustering of type A gamma-aminobutyric acid receptors (GABA(A)R) at discrete and functionally significant domains on the nerve cell surface is an important determinant in the integration of synaptic inputs. To discern the role that the subunits of the GABA(A)R play in determining the receptor's cell surface topography and mobility, the alpha1, beta1, beta3, and gamma2s subunits were transfected into COS7, HEK293, and PC12 cells and the distribution and cell surface mobility of these recombinant receptors were examined. Our results show that alpha1 subunits are retained in the endoplasmic reticulum while beta1 and beta3 subunits are sorted to the plasma membrane where they form clusters. Co-expression and co-assembly of alpha1 and beta3 subunits result in the rescue of intracellular alpha1 subunits, which are transported as alphabeta subunit complexes to the cell surface where they formed clusters. Fluorescence photobleach recovery and single particle tracking of recombinant receptors show that, despite clustering, beta3 subunit homooligomers are mobile within a cell surface domain. Inclusion of alpha1 in beta3 or beta3gamma2s complexes, however, dramatically reduces the receptor's lateral mobility in COS 7 and PC12 cells and anchors GABA(A)Rs on the cell surface, suggesting the formation of a direct link to a component of the cytoskeleton. The mobility of recombinant receptors that include the alpha1 subunit mirrors the mobility of GABA(A)Rs on cell bodies and dendrites of cortical and spinal cord neurons. The results suggest that incorporation of alpha1 subunits give rise to a population of GABA(A)Rs that are immobilized on the cell surface.     

alpha4beta3delta GABA(A) receptors characterized by fluorescence resonance energy transfer-derived measurements of membrane potential

Selective modulators of gamma-aminobutyric acid, type A (GABA(A)) receptors containing alpha(4) subunits may provide new treatments for epilepsy and premenstrual syndrome. Using mouse L(-tk) cells, we stably expressed the native GABA(A) receptor subunit combinations alpha(3)beta(3)gamma(2,) alpha(4)beta(3)gamma(2), and, for the first time, alpha(4)beta(3)delta and characterized their properties using a novel fluorescence resonance energy transfer assay of GABA-evoked depolarizations. GABA evoked concentration-dependent decreases in fluorescence resonance energy transfer that were blocked by GABA(A) receptor antagonists and, for alpha(3)beta(3)gamma(2) and alpha(4)beta(3)gamma(2) receptors, modulated by benzodiazepines with the expected subtype specificity. When combined with alpha(4) and beta(3), delta subunits, compared with gamma(2), conferred greater sensitivity to the agonists GABA, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP), and muscimol and greater maximal efficacy to THIP. alpha(4)beta(3)delta responses were markedly modulated by steroids and anesthetics. Alphaxalone, pentobarbital, and pregnanolone were all 3-7-fold more efficacious at alpha(4)beta(3)delta compared with alpha(4)beta(3)gamma(2.) The fluorescence technique used in this study has proven valuable for extensive characterization of a novel GABA(A) receptor. For GABA(A) receptors containing alpha(4) subunits, our experiments reveal that inclusion of delta instead of gamma(2) subunits can increase the affinity and in some cases the efficacy of agonists and can increase the efficacy of allosteric modulators. Pregnanolone was a particularly efficacious modulator of alpha(4)beta(3)delta receptors, consistent with a central role for this subunit combination in premenstrual syndrome.     

GABAA receptor associated proteins: a key factor regulating GABAA receptor function

gamma-Aminobutyric acid (GABA), an important inhibitory neurotransmitter in both vertebrates and invertebrates, acts on GABA receptors that are ubiquitously expressed in the CNS. GABA(A) receptors also represent a major site of action of clinically relevant drugs, such as benzodiazepines, barbiturates, ethanol, and general anesthetics. It has been shown that the intracellular M3-M4 loop of GABA(A) receptors plays an important role in regulating GABA(A) receptor function. Therefore, studies of the function of receptor intracellular loop associated proteins become important for understanding mechanisms of regulating receptor activity. Recently, several labs have used the yeast two-hybrid assay to identify proteins interacting with GABA(A) receptors, for example, the interaction of GABA(A) receptor associated protein (GABARAP) and Golgi-specific DHHC zinc finger protein (GODZ) with gamma subunits, PRIP, phospholipase C-related, catalytically inactive proteins (PRIP-1) and (PRIP-2) with GABARAP and receptor gamma2 and beta subunits, Plic-1 with some alpha and beta subunits, radixin with the alpha5 subunit, HAP1 with the beta1 subunit, GABA(A) receptor interacting factor-1 (GRIF-1) with the beta2 subunit, and brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2) with the beta3 subunit. These proteins have been shown to play important roles in modulating the activities of GABA(A) receptors ranging from enhancing trafficking, to stabilizing surface and internalized receptors, to regulating modification of GABA(A) receptors. This article reviews the current studies of GABA(A) receptor intracellular loop-associated proteins.     

Subunit arrangement of gamma-aminobutyric acid type A receptors

The GABA(A) receptors are ligand-gated chloride channels. The subunit stoichiometry of the receptors is controversial; four, five, or six subunits per receptor molecule have been proposed for alphabeta receptors, whereas alphabetagamma receptors are assumed to be pentamers. In this study, alpha-beta and beta-alpha tandem cDNAs from the alpha1 and beta2 subunits of the GABA(A) receptor were constructed. We determined the minimal length of the linker that is required between the two subunits for functional channel expression for each of the tandem constructs. 10- and 23-amino acid residues are required for alpha-beta and beta-alpha, respectively. The tandem constructs either alone or in combination with each other failed to express functional channels in Xenopus oocytes. Therefore, we can exclude tetrameric or hexameric alphabeta GABA(A) receptors. We can also exclude proteolysis of the tandem constructs. In addition, the tandem constructs were combined with single alpha, beta, or gamma subunits to allow formation of pentameric arrangements. In contrast to the combination with alpha subunits, the combination with either beta or gamma subunits led to expression of functional channels. Therefore, a pentameric arrangement containing two alpha1 and three beta2 subunits is proposed for the receptor composed of alpha and beta subunits. Our findings also favor an arrangement betaalphagammabetaalpha for the receptor composed of alpha, beta, and gamma subunits.     

An asymmetric contribution to gamma-aminobutyric type A receptor function of a conserved lysine within TM2-3 of alpha1, beta2, and gamma2 subunits

Mutations that impair the expression and/or function of gamma-aminobutyric acid type A (GABAA) receptors can lead to epilepsy. The familial epilepsy gamma2(K289M) mutation affects a basic residue conserved in the TM2-3 linker of most GABAA subunits. We investigated the effect on expression and function of the Lys --> Met mutation in mouse alpha1(K278M), beta2(K274M), and gamma2(K289M) subunits. Compared with cells expressing wild-type and alpha1beta2gamma2(K289M) receptors, cells expressing alpha1(K278M)beta2gamma2 and alpha1beta2(K274M)gamma2 receptors exhibited reduced agonist-evoked current density and reduced GABA potency, with no change in single channel conductance. The low current density of alpha1beta2(K274M)gamma2 receptors coincided with reduced surface expression. By contrast the surface expression of alpha1(K278M)beta2gamma2 receptors was similar to wild-type and alpha1beta2gamma2(K289M) receptors suggesting that the alpha1(K278M) impairs function. In keeping with this interpretation GABA-activated channels mediated by alpha1(K278M)beta2gamma2 receptors had brief open times. To a lesser extent gamma2(K289M) also reduced mean open time, whereas beta2(K274M) had no effect. We used propofol as an alternative GABAA receptor agonist to test whether the functional deficits of mutant subunits were specific to GABA activation. Propofol was less potent as an activator of alpha1(K278M)beta2gamma2 receptors. By contrast, neither beta2(K274M) nor gamma2(K289M) affected the potency of propofol. The beta2(K274M) construct was unique in that it reduced the efficacy of propofol activation relative to GABA. These data suggest that the alpha1 subunit Lys-278 residue plays a pivotal role in channel gating that is not dependent on occupancy of the GABA binding site. Moreover, the conserved TM2-3 loop lysine has an asymmetric function in different GABAA subunits.