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Mutation of glutamate 155 of the GABAA receptor beta2 subunit produces a spontaneously open channel: a trigger for channel activation.

ABSTRACT: Protein movements underlying ligand-gated ion channel activation are poorly understood. The binding of agonist initiates a series of conformational movements that ultimately lead to the opening of the ion channel pore. Although little is known about local movements within the GABA-binding site, a recent structural model of the GABA(A) receptor (GABA(A)R) ligand-binding domain predicts that beta2Glu155 is a key residue for direct interactions with the neurotransmitter (Cromer et al., 2002). To elucidate the role of the beta2Ile154-Asp163 region in GABA(A)R activation, each residue was individually mutated to cysteine and coexpressed with wild-type alpha1 subunits in Xenopus laevis oocytes. Seven mutations increased the GABA EC50 value (8- to 3400-fold), whereas three mutations (E155C, S156C, and G158C) also significantly increased the 2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl) pyridazinium (SR-95531) K(I) value. GABA, SR-95531, and pentobarbital slowed N-biotinylaminoethyl methanethiosulfonate modification of T160C and D163C, indicating that beta2Thr160 and beta2Asp163 are located in or near the GABA-binding site and that this region undergoes structural rearrangements during channel gating. Cysteine substitution of beta2Glu155 resulted in spontaneously open GABA(A)Rs and differentially decreased the GABA, piperidine-4-sulfonic acid (partial agonist), and SR-95531 sensitivities, indicating that the mutation perturbs ligand binding as well as channel gating. Tethering thiol-reactive groups onto beta2E155C closed the spontaneously open channels, suggesting that beta2Glu155 is a control element involved in coupling ligand binding to channel gating. Structural modeling suggests that the beta2 Ile154-Asp163 region is a protein hinge that forms a network of interconnections that couples binding site movements to the cascade of events leading to channel opening.

SUBMITTER: Newell JG

PROVIDER: S-EPMC6730373 | biostudies-literature | 2004 Dec

REPOSITORIES: biostudies-literature

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Mutation of glutamate 155 of the GABAA receptor beta2 subunit produces a spontaneously open channel: a trigger for channel activation.

Newell J Glen JG McDevitt Ross A RA Czajkowski Cynthia C

The Journal of neuroscience : the official journal of the Society for Neuroscience 20041201 50

Protein movements underlying ligand-gated ion channel activation are poorly understood. The binding of agonist initiates a series of conformational movements that ultimately lead to the opening of the ion channel pore. Although little is known about local movements within the GABA-binding site, a recent structural model of the GABA(A) receptor (GABA(A)R) ligand-binding domain predicts that beta2Glu155 is a key residue for direct interactions with the neurotransmitter (Cromer et al., 2002). To el ...[more]

PMID: 15601928

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Project description:BackgroundCardiomyocyte contraction is initiated by influx of extracellular calcium through voltage-gated calcium channels. These oligomeric channels utilize auxiliary beta subunits to chaperone the pore-forming alpha subunit to the plasma membrane, and to modulate channel electrophysiology 1. Several beta subunit family members are detected by RT-PCR in the embryonic heart. Null mutations in mouse beta2, but not in the other three beta family members, are embryonic lethal at E10.5 due to defects in cardiac contractility 2. However, a drawback of the mouse model is that embryonic heart rhythm is difficult to study in live embryos due to their intra-uterine development. Moreover, phenotypes may be obscured by secondary effects of hypoxia. As a first step towards developing a model for contributions of beta subunits to the onset of embryonic heart rhythm, we characterized the structure and expression of beta2 subunits in zebrafish and other teleosts.ResultsCloning of two zebrafish beta2 subunit genes (beta2.1 and beta2.2) indicated they are membrane-associated guanylate kinase (MAGUK)-family genes. Zebrafish beta2 genes show high conservation with mammals within the SH3 and guanylate kinase domains that comprise the "core" of MAGUK proteins, but beta2.2 is much more divergent in sequence than beta2.1. Alternative splicing occurs at the N-terminus and within the internal HOOK domain. In both beta2 genes, alternative short ATG-containing first exons are separated by some of the largest introns in the genome, suggesting that individual transcript variants could be subject to independent cis-regulatory control. In the Tetraodon nigrovidis and Fugu rubripes genomes, we identified single beta2 subunit gene loci. Comparative analysis of the teleost and human beta2 loci indicates that the short 5' exon sequences are highly conserved. A subset of 5' exons appear to be unique to teleost genomes, while others are shared with mammals. Alternative splicing is temporally and spatially regulated in embryo and adult. Moreover, a different subset of spliced beta2 transcript variants is detected in the embryonic heart compared to the adult.ConclusionThese studies refine our understanding of beta2 subunit diversity arising from alternative splicing, and provide the groundwork for functional analysis of beta2 subunit diversity in the embryonic heart.

Dataset Information

Mutation of glutamate 155 of the GABAA receptor beta2 subunit produces a spontaneously open channel: a trigger for channel activation.

Publications

Mutation of glutamate 155 of the GABAA receptor beta2 subunit produces a spontaneously open channel: a trigger for channel activation.

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