Cloning and Characterization of a GABAA Receptor γ2 Subunit Variant

Pei, Jin; Zhang, Juan; Rowe-Teeter, Courtney; Yang, Junming; Stuve, Laura L.; Fu, Glenn K.

doi:10.1074/jbc.m308656200

Cited by 16 publications

(17 citation statements)

References 42 publications

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“…This Ser lies in loop B of the current structurally based 6-loop model of the ligand binding domain, first found in the nAChRs (68) and extended also (69) to vertebrate GABRs. The essentiality of this Ser in the human GABR ␥2 subunit has been demonstrated very recently by its mutation, which prevents active receptor formation with wild-type ␣ and ␤ subunits (58). We note that all of the evidence cited here supports the concept of a constant and essential role for the framework (consensus E) that we have used in the analysis.…”

Section: Common Features Of the Gabr Genessupporting

confidence: 61%

“…Our analyses of Protein product cannot reach cell surface (58) a The total number of exons, coding and non-coding, as determined or confirmed here on the genome. b The ␤2 gene has 11 exons: exon 1 has 74 bp of 5Ј-utr (unreported), exon 2 has 152 bp of 5Ј-utr plus 77 bp coding for the signal peptide, exon 10 is expressed only in the "␤2L" form.…”

Section: Common Features Of the Gabr Genesmentioning

confidence: 99%

“…In this form, an additional 120-bp exon is created by a weak splice site inside intron 5 by an Alu repetitive element residing there (58). The protein product may affect the trafficking of active GABA A receptors (58).…”

Section: Six Genomic Mechanisms For Creation Of Multiple Forms Of Gabmentioning

confidence: 99%

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Analysis of the Set of GABAA Receptor Genes in the Human Genome

Simon

Wakimoto

Fujita

et al. 2004

Journal of Biological Chemistry

237

196

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The genes of the ionotropic ␥-aminobutyric acid receptor (GABR) subunits have shown an unusual chromosomal clustering, but only now can this be fully specified by analyses of the human genome. We have characterized the genes encoding the 18 known human GABR subunits, plus one now located here, for their precise locations, sizes, and exon/intron structures. Clusters of 17 of the 19, distributed between five chromosomes, are specified in detail, and their possible significance is considered. By applying search algorithms designed to recognize sequences of all known GABRtype subunits in species from man down to nematodes, we found no new GABR subunit is detectable in the human genome. However, the sequence of the human orthologue of the rat GABR 3 receptor subunit was uncovered by these algorithms, and its gene could be analyzed. Consistent with those search results, orthologues of the ␤4 and ␥4 subunits from the chicken, not cloned from mammals, were not detectable in the human genome by specific searches for them. The relationships are consistent with the mammalian subunit being derived from the ␤ line and ⑀ from the ␥ line, with mammalian loss of ␤4 and ␥4. In their structures the human GABR genes show a basic pattern of nine coding exons, with six different genomic mechanisms for the alternative splicing found in various subunits. Additional noncoding exons occur for certain subunits, which can be regulatory. A dicysteine loop and its exon show remarkable constancy between all GABR subunits and species, of deduced functional significance.

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Section: Common Features Of the Gabr Genessupporting

confidence: 61%

Section: Common Features Of the Gabr Genesmentioning

confidence: 99%

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Analysis of the Set of GABAA Receptor Genes in the Human Genome

Simon

Wakimoto

Fujita

et al. 2004

Journal of Biological Chemistry

237

196

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“…These two residues flank a novel alternative exon (16,17). When the residues are mutated, oligomerization of ␥2 with ␣2 and ␤1 is prevented, and cell surface access of ␥2 is abolished.…”

Section: ␥-Aminobutyric Acid Type a (Gaba A )mentioning

confidence: 99%

Serine 171, a Conserved Residue in the γ-Aminobutyric Acid Type A (GABAA) Receptor γ2 Subunit, Mediates Subunit Interaction and Cell Surface Localization

Pei

Walther

Zhang

et al. 2004

Journal of Biological Chemistry

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Serine 171 in the GABA A receptor ␥2 subunit is highly conserved in the ligand-gated ion channel superfamily. In this paper, we report that mutating serine 171 within ␥2 to glycine or cysteine prevents the interaction of ␥2 with ␣2 and ␤1 when these subunits are co-expressed in human embryo kidney 293 cells, resulting in intracellular retention of ␥2. Structure analysis based on a three-dimensional homology model of ␥2 (Ernst, M., Brauchart, D., Boresch, S., and Sieghart, W. (2003) Neuroscience 119, 933-943) reveals that serine 171 may play a critical role in the formation and stabilization of an exposed turn structure that is part of the subunit interaction site. Mutation of serine 171 in the ␥2 subunit could therefore result in alteration of the structure of the subunit interaction site, preventing correct subunit assembly. ␥-Aminobutyric acid type A (GABA A )1 receptors are members of the ligand-gated ion channel superfamily, which also includes nicotinic acetylcholine (ACh), glycine, glutamate, and 5-hydroxytryptamine type 3 (5HT3) receptors. Members of this superfamily share significant sequence similarity, implying a common evolutionary origin (2).GABA A receptors are the major inhibitory receptors in the brain and are the targets of clinically important drugs such as benzodiazepines, barbiturates, neurosteroids, and anesthetics (3). Several distinct classes of GABA A receptor subunits have been identified (3-8). Heterologous expression of recombinant GABA A receptor subunits in cultured cells demonstrates that the combination of ␣ and ␤ subunits is sufficient for creating the GABA binding sites to elicit GABA-gated Cl Ϫ currents (9). Benzodiazepines bind to a distinct site on the GABA A receptor and positively modulate GABA-gated C Ϫ current (10). Formation of the benzodiazepine binding site requires the presence of a third component, the ␥ subunit (9, 10). Typical native GABA A receptors have been proposed to be pentameric transmembrane proteins formed by two ␣, two ␤, and one ␥ subunits (9). Assembly of the hetero-pentamers occurs in the endoplasmic reticulum and requires the interaction between subunit-specific contact sites. Access to the cell surface is restricted to the correctly assembled GABA A receptors (11-15).We identified previously two highly conserved residues, serine 171 and tyrosine 172, in the ␥2 subunit. These two residues flank a novel alternative exon (16,17). When the residues are mutated, oligomerization of ␥2 with ␣2 and ␤1 is prevented, and cell surface access of ␥2 is abolished. In the present study, using single amino acid substitutions, we further demonstrate that serine 171 within ␥2 plays a critical role in mediating the subunit interaction and cell surface expression of ␥2, whereas tyrosine 172, as well as other residues surrounding Ser-171/ Tyr-172, exhibit little or no effect on the function of the ␥2 subunit. Mapping of the ␥2 protein sequence onto the threedimensional homology model of the extracellular component of the GABA A receptor generated from the ACh-binding prote...

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“…Additional diversity of receptor structure is accomplished by alternative splicing of some of the subunit mRNAs and by RNA editing that might affect kinetics, subunit assembly and cell-surface expression of GABAA receptors [27]. For example, the γ2 subunit appears in three alternatively spliced variants termed γ2L, γ2S and γ2XL [28], although physiological and pharmacological relevance of this diversity is still not completely resolved. Nova, a neuron-specific RNA binding protein, regulates alternative splicing of γ2 subunit by acting on an intronic splicing enhancer which lies far downstream of the regulated exon, and general dysregulation of Nova's splicing enhancer function may underlie the neurologic defects seen in patients with Nova's absence [29].…”

Section: Gabaa Receptors and Their Subunitsmentioning

confidence: 99%

GABA Receptors: Pharmacological Potential and Pitfalls

Jembrek¹,

Vlainić

2015

CPD

115

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Gamma-amino butyric acid (GABA), the major inhibitory neurotransmitter in the mammalian central nervous system, plays a key role in the regulation of neuronal transmission throughout the brain, affecting numerous physiological and psychological processes. Changes in GABA levels provoke disbalance between excitatory and inhibitory signals, and are involved in the development of numerous neuropsychiatric disorders. GABA exerts its effects via ionotropic (GABAA) and metabotropic (GABAB) receptors. Both types of receptors are targeted by many clinically important drugs that affect GABAergic function and are widely used in the treatment of anxiety disorder, epilepsy, insomnia, spasticity, aggressive behaviour, and other pathophysiological conditions and diseases. Of particular importance are drugs that modulate GABAA receptor complex, such as benzodiazepines, barbiturates, neuroactive steroids, intravenous and inhalational anesthetics, and ethanol. Molecular interactions and subsequent pharmacological effects induced by drugs acting at GABAA receptors are extremely complex due to structural heterogeneity of GABAA receptors and existence of numerous allosterically interconnected binding sites and various chemically distinct ligands that are able to bound to them. There is a growing interest in the development and application of subtype-selective drugs that will achieve specific therapeutic benefits without undesirable side effects. The aim of this review is to briefly summarize the key pharmacological properties of GABA receptors, and to present selected novel findings with the potential to open new perspectives in the development of more effective therapeutic strategies.

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Cloning and Characterization of a GABAA Receptor γ2 Subunit Variant

Cited by 16 publications

References 42 publications

Analysis of the Set of GABAA Receptor Genes in the Human Genome

Analysis of the Set of GABAA Receptor Genes in the Human Genome

Serine 171, a Conserved Residue in the γ-Aminobutyric Acid Type A (GABAA) Receptor γ2 Subunit, Mediates Subunit Interaction and Cell Surface Localization

GABA Receptors: Pharmacological Potential and Pitfalls

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