Dataset Information

Neurosteroid analog photolabeling of a site in the third transmembrane domain of the ?3 subunit of the GABA(A) receptor.

ABSTRACT: Accumulated evidence suggests that neurosteroids modulate GABA(A) receptors through binding interactions with transmembrane domains. To identify these neurosteroid binding sites directly, a neurosteroid-analog photolabeling reagent, (3?,5?)-6-azi-pregnanolone (6-AziP), was used to photolabel membranes from Sf9 cells expressing high-density, recombinant, His(8)-?3 homomeric GABA(A) receptors. 6-AziP inhibited (35)S-labeled t-butylbicyclophosphorothionate binding to the His(8)-?3 homomeric GABA(A) receptors in a concentration-dependent manner (IC(50) = 9 ± 1 ?M), with a pattern consistent with a single class of neurosteroid binding sites. [(3)H]6-AziP photolabeled proteins of 30, 55, 110, and 150 kDa, in a concentration-dependent manner. The 55-, 110-, and 150-kDa proteins were identified as His(8)-?3 subunits through immunoblotting and through enrichment on a nickel affinity column. Photolabeling of the ?3 subunits was stereoselective, with [(3)H]6-AziP producing substantially greater labeling than an equal concentration of its diastereomer [(3)H](3?,5?)-6-AziP. High-resolution mass spectrometric analysis of affinity-purified, 6-AziP-labeled His(8)-?3 subunits identified a single photolabeled peptide, ALLEYAF-6-AziP, in the third transmembrane domain. The identity of this peptide and the site of incorporation on Phe301 were confirmed through high-resolution tandem mass spectrometry. No other sites of photoincorporation were observed despite 90% sequence coverage of the whole ?3 subunit protein, including 84% of the transmembrane domains. This study identifies a novel neurosteroid binding site and demonstrates the feasibility of identifying neurosteroid photolabeling sites by using mass spectrometry.

SUBMITTER: Chen ZW

PROVIDER: S-EPMC3422701 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Neurosteroid analog photolabeling of a site in the third transmembrane domain of the β3 subunit of the GABA(A) receptor.

Chen Zi-Wei ZW Manion Brad B Townsend R Reid RR Reichert David E DE Covey Douglas F DF Steinbach Joe Henry JH Sieghart Werner W Fuchs Karoline K Evers Alex S AS

Molecular pharmacology 20120530 3

Accumulated evidence suggests that neurosteroids modulate GABA(A) receptors through binding interactions with transmembrane domains. To identify these neurosteroid binding sites directly, a neurosteroid-analog photolabeling reagent, (3α,5β)-6-azi-pregnanolone (6-AziP), was used to photolabel membranes from Sf9 cells expressing high-density, recombinant, His(8)-β3 homomeric GABA(A) receptors. 6-AziP inhibited (35)S-labeled t-butylbicyclophosphorothionate binding to the His(8)-β3 homomeric GABA(A) ...[more]

PMID: 22648971

Similar Datasets

Project description:BackgroundDisruption of normal brain development is implicated in numerous psychiatric disorders with neurodevelopmental origins, including autism spectrum disorder (ASD). Widespread abnormalities in brain structure and functions caused by dysregulations of neurodevelopmental processes has been recently shown to exert adverse effects across generations. An imbalance between excitatory/inhibitory (E/I) transmission is the putative hypothesis of ASD pathogenesis, supporting by the specific implications of inhibitory γ-aminobutyric acid (GABA)ergic system in autistic individuals and animal models of ASD. However, the contribution of GABAergic system in the neuropathophysiology across generations of ASD is still unknown. Here, we uncover profound alterations in the expression and function of GABAA receptors (GABAARs) in the amygdala across generations of the VPA-induced animal model of ASD.MethodsThe F2 generation was produced by mating an F1 VPA-induced male offspring with naïve females after a single injection of VPA on embryonic day (E12.5) in F0. Autism-like behaviors were assessed by animal behavior tests. Expression and functional properties of GABAARs and related proteins were examined by using western blotting and electrophysiological techniques.ResultsSocial deficit, repetitive behavior, and emotional comorbidities were demonstrated across two generations of the VPA-induced offspring. Decreased synaptic GABAAR and gephyrin levels, and inhibitory transmission were found in the amygdala from two generations of the VPA-induced offspring with greater reductions in the F2 generation. Weaker association of gephyrin with GABAAR was shown in the F2 generation than the F1 generation. Moreover, dysregulated NMDA-induced enhancements of gephyrin and GABAAR at the synapse in the VPA-induced offspring was worsened in the F2 generation than the F1 generation. Elevated glutamatergic modifications were additionally shown across generations of the VPA-induced offspring without generation difference.ConclusionsTaken together, these findings revealed the E/I synaptic abnormalities in the amygdala from two generations of the VPA-induced offspring with GABAergic deteriorations in the F2 generation, suggesting a potential therapeutic role of the GABAergic system to generational pathophysiology of ASD.

Project description:The Zn-dependent membrane-located protease YvjB has previously been shown to serve as a target receptor for LsbB, a class II leaderless lactococcal bacteriocin. Although yvjB is highly conserved in the genus Lactococcus, the bacteriocin appears to be active only against the subspecies L. lactis subsp. lactis Comparative analysis of the YvjB proteins of a sensitive strain (YvjBMN) and a resistant strain (YvjBMG) showed that they differ from each other in 31 positions. In this study, we applied site-directed mutagenesis and performed directed binding studies to provide biochemical evidence that LsbB interacts with the third transmembrane helix of YvjB in susceptible cells. The site-directed mutagenesis of LsbB and YvjB proteins showed that certain amino acids and the length of LsbB are responsible for the bacteriocin activity, most probably through adequate interaction of these two proteins; the essential amino acids in LsbB responsible for the activity are tryptophan (Trp(25)) and terminal alanine (Ala(30)). It was also shown that the distance between Trp(25) and terminal alanine is crucial for LsbB activity. The crucial region in YvjB for the interaction with LsbB is the beginning of the third transmembrane helix, particularly amino acids tyrosine (Tyr(356)) and alanine (Ala(353)). In vitro experiments showed that LsbB could interact with both YvjBMN and YvjBMG, but the strength of interaction is significantly less with YvjBMG In vivo experiments with immunofluorescently labeled antibody demonstrated that LsbB specifically interacts only with cells carrying YvjBMN IMPORTANCE: The antimicrobial activity of LsbB bacteriocin depends on the correct interaction with the corresponding receptor in the bacterial membrane of sensitive cells. Membrane-located bacteriocin receptors have essential primary functions, such as cell wall synthesis or sugar transport, and it seems that interaction with bacteriocins is suicidal for cells. This study showed that the C-terminal part of LsbB is crucial for the bacteriocin activity, most probably through adequate interaction with the third transmembrane domain of the YvjB receptor. The conserved Tyr(356) and Ala(353) residues of YvjB are essential for the function of this Zn-dependent membrane-located protease as a bacteriocin receptor.

Dataset Information

Neurosteroid analog photolabeling of a site in the third transmembrane domain of the ?3 subunit of the GABA(A) receptor.

Publications

Neurosteroid analog photolabeling of a site in the third transmembrane domain of the β3 subunit of the GABA(A) receptor.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets