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.     

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.     

A novel site on gamma 3 subunits important for assembly of GABA(A) receptors

Gamma-aminobutyric acid, type A (GABA(A)) receptors are ligand-gated chloride channels and are the major inhibitory transmitter receptors in the central nervous system. The majority of these receptors is composed of two alpha, two beta, and one gamma subunits. To identify sequences important for subunit assembly, we generated C-terminally truncated and chimeric gamma(3) constructs. From their ability to associate with full-length alpha(1) and beta(3) subunits, we concluded that amino acid sequence gamma(3)(70-84) either directly interacts with alpha(1) or beta(3) subunits or stabilizes a contact site elsewhere in the protein. The observation that this sequence contains amino acid residues homologous to gamma(2) residues contributing to the benzodiazepine-binding site at the alpha(1)/gamma(2) interface suggested that in alpha(1)beta(3)gamma(3) receptors the sequence gamma(3)(70-84) is located at the alpha(1)/gamma(3) interface. In the absence of alpha(1) subunits this sequence might allow assembly of beta(3) with gamma(3) subunits. Other experiments indicated that sequences gamma(3)(86-95) and gamma(3)(94-107), which are homologous to previously identified sequences important for assembly of gamma(2) subunits, are also important for assembly of gamma(3) subunits. This indicates that during assembly of the GABA(A) receptor, more than one N-terminal sequence is important for binding to the same neighboring subunit. Whether the three sequences investigated are involved in direct interaction or stabilize other regions involved in intersubunit contacts has to be further studied.     

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.     

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.     

Two invariant tryptophans on the alpha1 subunit define domains necessary for GABA(A) receptor assembly.

Two invariant tryptophan residues on the N-terminal extracellular region of the rat alpha1 subunit, Trp-69 and Trp-94, are critical for the assembly of the GABA(A) (gamma-aminobutyric acid, type A) receptor into a pentamer. These tryptophans are common not only to all GABA(A) receptor subunits, but also to all ligand-gated ion channel subunits. Converting each Trp residue to Phe and Gly by site-directed mutagenesis allowed us to study the role of these invariant tryptophan residues. Mutant alpha1 subunits, coexpressed with beta2 subunits in baculovirus-infected Sf9 cells, displayed high affinity binding to [(3)H]muscimol, a GABA site ligand, but no binding to [(35)S]t-butyl bicyclophosphorothionate, a ligand for the receptor-associated ion channel. Neither [(3)H]muscimol binding to intact cells nor immunostaining of nonpermeabilized cells gave evidence of surface expression of the receptor. When expressed with beta2 and gamma2 polypeptides, the mutant alpha1 polypeptides did not form [(3)H]flunitrazepam binding sites though wild-type alpha1 polypeptides did. The distribution of the mutant receptors on sucrose gradients suggests that the effects on ligand binding result from the inability of the mutant alpha1 subunits to form pentamers. We conclude that Trp-69 and Trp-94 participate in the formation of the interface between alpha and beta subunits, but not of the GABA binding site.     

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.     

Role of the conserved lysine residue in the middle of the predicted extracellular loop between M2 and M3 in the GABA(A) receptor.

In alpha1, beta2, and gamma2 subunits of the gamma-aminobutyric acid A (GABA(A)) receptor, a conserved lysine residue occupies the position in the middle of the predicted extracellular loop between the transmembrane M2 and M3 regions. In all three subunits, this residue was mutated to alanine. Whereas the mutation in alpha1 and beta2 subunits resulted each in about a sixfold shift of the concentration-response curve for GABA to higher concentrations, no significant effect by mutation in the gamma subunit was detected. The affinity for the competitive inhibitor bicuculline methiodide was not affected by the mutations in either the alpha1 subunit or the beta2 subunit. Concentration-response curves for channel activation by pentobarbital were also shifted to higher concentrations by the mutation in the alpha and beta subunits. Binding of [3H]Ro 15-1788 was unaffected by the mutation in the alpha subunit, whereas the binding of [3H]muscimol was shifted to lower affinity. Mutation of the residue in the alpha1 subunit to E, Q, or R resulted in an about eight-, 10-, or fivefold shift, respectively, to higher concentrations of the concentration-response curve for GABA. From these observations, it is concluded that the corresponding residues on the alpha1 and beta2 subunits are involved more likely in the gating of the channel by GABA than in the binding of GABA or benzodiazepines.     

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.     

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.     

Differential protein mobility of the gamma-aminobutyric acid, type A, receptor alpha and beta subunit channel-lining segments

The gamma-aminobutyric acid, type A (GABAA), receptor ion channel is lined by the second membrane-spanning (M2) segments from each of five homologous subunits that assemble to form the receptor. Gating presumably involves movement of the M2 segments. We assayed protein mobility near the M2 segment extracellular ends by measuring the ability of engineered cysteines to form disulfide bonds and high affinity Zn(2+)-binding sites. Disulfide bonds formed in alpha1beta1E270Cgamma2 but not in alpha1N275Cbeta1gamma2 or alpha1beta1gamma2K285C. Diazepam potentiation and Zn2+ inhibition demonstrated that expressed receptors contained a gamma subunit. Therefore, the disulfide bond in alpha1beta1E270Cgamma2 formed between non-adjacent subunits. In the homologous acetylcholine receptor 4-A resolution structure, the distance between alpha carbon atoms of 20' aligned positions in non-adjacent subunits is approximately 19 A. Because disulfide trapping involves covalent bond formation, it indicates the extent of movement but does not provide an indication of the energetics of protein deformation. Pairs of cysteines can form high affinity Zn(2+)-binding sites whose affinity depends on the energetics of forming a bidentate-binding site. The Zn2+ inhibition IC50 for alpha1beta1E270Cgamma2 was 34 nm. In contrast, it was greater than 100 microM in alpha1N275Cbeta1gamma2 and alpha1beta1gamma2K285C receptors. The high Zn2+ affinity in alpha1beta1E270Cgamma2 implies that this region in the beta subunit has a high protein mobility with a low energy barrier to translational motions that bring the positions into close proximity. The differential mobility of the extracellular ends of the beta and alpha M2 segments may have important implications for GABA-induced conformational changes during channel gating.     

Mutational analysis of roles for extracellular cysteine residues in the assembly and function of human alpha 7-nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChR) containing alpha7 subunits self-assemble into simple, homopentameric complexes. However, successful heterologous expression of functional alpha7-nAChR has only been achieved in a few host cell types, such as the SH-EP1 human epithelial cell line. All ionotropic glycine receptor, GABA(A) receptor, 5-HT(3) receptor, and nAChR subunits contain a pair of highly conserved cysteine residues (C150 and C164 for alpha7 subunits) in their N-terminal extracellular domain. These residues are thought to be involved in the formation of a conserved cystine loop that is critical to the proper folding and assembly of subunits. However, nAChR alpha7 (and alpha8) subunits also contain a third cysteine residue, C138, N-terminal to the conserved cysteine pair. Using SH-EP1 cells as a host for heterologous expression, we evaluated the roles of C138, C150, and C164 in subunit folding, assembly, and cell surface expression and function of alpha7-nAChR. Results indicate that mutation of C138, but not of C150 or C164, yields an nAChR that can assemble to form (125)I-labeled alpha-bungarotoxin binding sites expressed on the cell surface. Further, whole-cell patch clamp recordings demonstrate that mutation of C138 to alanine does not alter the function of the fully assembled alpha7-nAChR. These results indicate that C150 and C164 are required for surface expression, but that C138 is neither necessary for nor inhibitory toward the surface expression and function of human alpha7-nAChR. These results suggest that disulfide bond formation between C138 and either C150 or C164, if it occurs, has no significant effect on alpha7-nAChR assembly or function.     

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.     

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.     

Multiple modes for conferring surface expression of homomeric beta1 GABAA receptors

The gamma-aminobutyric acid type A (GABA(A)) receptor assembles from individual subunits to form ligand-gated ion channels. Human (h) beta3 subunits assemble to form homomeric surface receptors in somatic cells, but hbeta1 subunits do not. We have identified three distinct sets of amino acid residues in the N-terminal extracellular domain of the hbeta1 subunit, which when mutated to the homologous residue in hbeta3 allow expression as a functional homomeric receptor. The three sets likely result in three modes of assembly. Mode 1 expression results from a single amino acid change at residue hbeta1 Asp-37. Mode 2 expression results from mutations of residues between positions 44 and 73 together with residues between positions 169 and 173. Finally, mode 3 results from the mutations A45V and K196R. Examination of homology-based structural models indicates that many of the residues are unlikely to be involved in physical inter-subunit interactions, suggesting that a major alteration is stabilization of an assembly competent form of the subunit. These mutations do not, however, have a major effect on the surface expression of heteromeric receptors which include the alpha1 subunit.     

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.     

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.     

Spontaneous thermal motion of the GABA(A) receptor M2 channel-lining segments

The gamma-aminobutyric acid type A (GABA(A)) receptor channel opening involves translational and rotational motions of the five channel-lining, M2 transmembrane segments. The M2 segment's extracellular half is loosely packed and undergoes significant thermal motion. To characterize the extent of the M2 segment's motion, we used disulfide trapping experiments between pairs of engineered cysteines. In alpha1beta1 gamma2S receptors the single gamma subunit is flanked by an alpha and beta subunit. The gamma2 M2-14' position is located in the alpha-gamma subunit interface. Gamma2 13' faces the channel lumen. We expressed either the gamma2 14' or the gamma2 13' cysteine substitution mutants with alpha1 cysteine substitution mutants between 12' and 16' and wild-type beta1. Disulfide bonds formed spontaneously between gamma2 14'C and both alpha1 15'C and alpha1 16'C and also between gamma2 13'C and alpha1 13'C. Oxidation by copper phenanthroline induced disulfide bond formation between gamma2 14'C and alpha1 13'C. Disulfide bond formation rates with gamma2 14'C were similar in the presence and absence of GABA, although the rate with alpha1 13'C was slower than with the other two positions. In a homology model based on the acetylcholine receptor structure, alphaM2 would need to rotate in opposite directions by approximately 80 degrees to bring alpha1 13' and alpha1 15' into close proximity with gamma2 14'. Alternatively, translational motion of alphaM2 would reduce the extent of rotational motion necessary to bring these two alpha subunit residues into close proximity with the gamma2 14' position. These experiments demonstrate that in the closed state the M2 segments undergo continuous spontaneous motion in the region near the extracellular end of the channel gate. Opening the gate may involve similar but concerted motions of the M2 segments.     

Consequence of the presence of two different beta subunit isoforms in a GABA(A) receptor

The major isoforms of GABA(A) receptors are thought to be composed of two alpha, two beta and one gamma subunit(s). GABA(A) receptors containing two beta1 subunits respond differently to the anticonvulsive compound loreclezole and the general anaesthetic etomidate than receptors containing two beta2 subunits. Receptors containing beta2 subunits show a much larger allosteric stimulation by these agents than those containing beta1 subunits. We were interested to know how receptors containing both beta1 and beta2 subunits, in different positions respond to loreclezole and etomidate. To answer this question, subunits were fused at the DNA level to form dimeric and trimeric subunits. Concatenated receptors (alpha1-beta1-alpha1/gamma2-beta1, alpha1-beta2-alpha1/gamma2-beta1, alpha1-beta1-alpha1/gamma2-beta2 and alpha1-beta2-alpha1/gamma2-beta2) were expressed in Xenopus ooctyes and functionally compared in their response to the agonist GABA and to the positive allosteric modulators, loreclezole and etomidate. We have shown that (I) in the presence of both beta1 and beta2 subunits in the same pentamer (mixed receptors) direct gating by etomidate is similar to exclusively beta1 containing receptors; (II) In mixed receptors, stimulation by etomidate assumed characteristics intermediate to exclusively beta1 or beta2 containing receptors, but the values for the concentrations < 10 microM were always much closer to those observed in alpha1-beta1-alpha1/gamma2-beta1 receptors; and (III) mixed receptors show no positional effects.