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Analyzing the mechanisms that facilitate the subtype-specific assembly of γ-aminobutyric acid type A receptors.

ABSTRACT: Impaired inhibitory signaling underlies the pathophysiology of many neuropsychiatric and neurodevelopmental disorders including autism spectrum disorders and epilepsy. Neuronal inhibition is regulated by synaptic and extrasynaptic γ-aminobutyric acid type A receptors (GABA _A Rs), which mediate phasic and tonic inhibition, respectively. These two GABA _A R subtypes differ in their function, ligand sensitivity, and physiological properties. Importantly, they contain different α subunit isoforms: synaptic GABA _A Rs contain the α1-3 subunits whereas extrasynaptic GABA _A Rs contain the α4-6 subunits. While the subunit composition is critical for the distinct roles of synaptic and extrasynaptic GABA _A R subtypes in inhibition, the molecular mechanism of the subtype-specific assembly has not been elucidated. To address this issue, we purified endogenous α1- and α4-containing GABA _A Rs from adult murine forebrains and examined their subunit composition and interacting proteins using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and quantitative analysis. We found that the α1 and α4 subunits form separate populations of GABA _A Rs and interact with distinct sets of binding proteins. We also discovered that the β3 subunit, which co-purifies with both the α1 and α4 subunits, has different levels of phosphorylation on serines 408 and 409 (S408/9) between the two receptor subtypes. To understand the role S408/9 plays in the assembly of α1- and α4-containing GABA _A Rs, we examined the effects of S408/9A (alanine) knock-in mutation on the subunit composition of the two receptor subtypes using LC-MS/MS and quantitative analysis. We discovered that the S408/9A mutation results in the formation of novel α1α4-containing GABA _A Rs. Moreover, in S408/9A mutants, the plasma membrane expression of the α4 subunit is increased whereas its retention in the endoplasmic reticulum is reduced. These findings suggest that S408/9 play a critical role in determining the subtype-specific assembly of GABA _A Rs, and thus the efficacy of neuronal inhibition.

SUBMITTER: Choi C

PROVIDER: S-EPMC9574402 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Analyzing the mechanisms that facilitate the subtype-specific assembly of γ-aminobutyric acid type A receptors.

Choi Catherine C Smalley Joshua L JL Lemons Abigail H S AHS Ren Qiu Q Bope Christopher E CE Dengler Jake S JS Davies Paul A PA Moss Stephen J SJ

Frontiers in molecular neuroscience 20221003

Impaired inhibitory signaling underlies the pathophysiology of many neuropsychiatric and neurodevelopmental disorders including autism spectrum disorders and epilepsy. Neuronal inhibition is regulated by synaptic and extrasynaptic γ-aminobutyric acid type A receptors (GABA A Rs), which mediate phasic and tonic inhibition, respectively. These two GABA A R subtypes differ in their function, ligand sensitivity, and physiological properties. Importantly, th ...[more]

PMID: 36263376

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Project description:Impaired inhibitory signaling underlies the pathophysiology of many neuropsychiatric and neurodevelopmental disorders including autism spectrum disorders and epilepsy. Neuronal inhibition is regulated by synaptic and extrasynaptic γ-aminobutyric acid type A receptors (GABAARs), which mediate phasic and tonic inhibition, respectively. These two GABAAR subtypes differ in their function, ligand sensitivity, and physiological properties. Importantly, they contain different α subunit isoforms: synaptic GABAARs contain the α1-3 subunits whereas extrasynaptic GABAARs contain the α4-6 subunits. While the subunit composition is critical for the distinct roles of synaptic and extrasynaptic GABAAR subtypes in inhibition, the molecular mechanism of the subtype-specific assembly has not been elucidated. To address this issue, we purified endogenous α1- and α4-containing GABAARs from adult murine forebrains and examined their subunit composition and interacting proteins using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and quantitative analysis. We found that the α1 and α4 subunits form separate populations of GABAARs and interact with distinct sets of binding proteins. We also discovered that the β3 subunit, which co-purifies with both the α1 and α4 subunits, has different levels of phosphorylation on serines 408 and 409 (S408/9) between the two receptor subtypes. To understand the role S408/9 plays in the assembly of α1- and α4-containing GABAARs, we examined the effects of S408/9A (alanine) knock-in mutation on the subunit composition of the two receptor subtypes using LC-MS/MS and quantitative analysis. We discovered that the S408/9A mutation results in the formation of novel α1α4-containing GABAARs. Moreover, in S408/9A mutants, the plasma membrane expression of the α4 subunit is increased whereas its retention in the endoplasmic reticulum is reduced. These findings suggest that S408/9 play a critical role in determining the subtype-specific assembly of GABAARs, and thus the efficacy of neuronal inhibition.

Project description:ObjectiveHuman hypothalamic hamartomas (HHs) are highly associated with treatment-resistant gelastic seizures. HHs are intrinsically epileptogenic, although the basic cellular mechanisms responsible for seizure activity are unknown. Altered gamma-aminobutyric acid (GABA) function can contribute to epileptogenesis in humans and animal models. Recently, functional GABA(A) receptor (GABA(A) R) rundown has been described in surgically resected human temporal lobe epilepsy tissue. We asked whether functional GABA(A) R rundown also occurs in human HH neurons.MethodsGABA(A) R-mediated currents were measured using perforated patch-clamp recordings in single neurons acutely dissociated from surgically resected HH tissue. In addition, functional GABA(A) Rs were expressed in Xenopus oocytes after microinjection with membrane fractions from either HH or control hypothalamus, and were studied with 2-electrode voltage-clamp recordings.ResultsPerforated patch-clamp recordings in dissociated HH neurons showed that repetitive exposure to GABA (5 consecutive exposures to 0.1 mM GABA with 1-second duration and at 20-second intervals) induced a time-dependent rundown of whole-cell currents in small HH neurons, whereas large HH neurons showed much less rundown using the same protocol. Functional rundown was not observed in HH neurons with repetitive exposure to glycine or glutamate. Two-electrode voltage-clamp recordings (6 consecutive exposures to 1 mM GABA with 10-second duration and at 40-second intervals) induced GABA current rundown in Xenopus oocytes microinjected with HH membrane proteins, but not in the oocytes expressing hypothalamic membrane proteins derived from human autopsy controls. Functional rundown of GABA currents was significantly attenuated by intracellular application of adenosine triphosphate or the nonspecific phosphatase inhibitor, okadaic acid.InterpretationNeurons from surgically resected human HH demonstrate functional rundown of GABA(A) R-mediated transmembrane currents in response to GABA agonist exposure. Rundown may be a marker for impaired GABAergic function and a contributing mechanism for seizure genesis within HH tissue.

Project description:γ-aminobutyric acid type A receptors (GABAARs), the major inhibitory neurotransmitter receptors in the mammalian central nervous system, are arguably the most challenging member of the pentameric Cys-loop receptors to study due to their heteromeric structure. When two or more subunits are expressed together in heterologous systems, receptors of variable subunit type, ratio, and orientation can form, precluding accurate interpretation of data from functional studies. Subunit concatenation is a technique that involves the linking of individual subunits and in theory allows the precise control of the uniformity of expressed receptors. In reality, the resulting concatemers from widely used constructs are flexible in their orientation and may therefore assemble with themselves or free GABAAR subunits in unexpected ways. In this study, we examine functional responses of receptors from existing concatenated constructs and describe refinements necessary to allow expression of uniform receptor populations. We find that dimers from two commonly used concatenated constructs, β-23-α and α-10-β, assemble readily in both the clockwise and the counterclockwise orientations when coexpressed with free subunits. Furthermore, we show that concatemers formed from new tetrameric α-10-β-α-β and α-10-β-α-γ constructs also assemble in both orientations with free subunits to give canonical αβγ receptors. To restrict linker flexibility, we systematically shorten linker lengths of dimeric and pentameric constructs and find optimized constructs that direct the assembly of GABAARs only in one orientation, thus eliminating the ambiguity associated with previously described concatemers. Based on our data, we revisit some noncanonical GABAAR configurations proposed in recent years and explain how the use of some concatenated constructs may have led to wrong conclusions. Our results help clarify current contradictions in the literature regarding GABAAR subunit stoichiometry and arrangement. The lessons learned from this study may guide future efforts in understanding other related heteromeric receptors.

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Analyzing the mechanisms that facilitate the subtype-specific assembly of γ-aminobutyric acid type A receptors.

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Analyzing the mechanisms that facilitate the subtype-specific assembly of <i>γ</i>-aminobutyric acid type A receptors.

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