Dataset Information

Alphaxalone Binds in Inner Transmembrane ?+-?- Interfaces of ?1?3?2 ?-Aminobutyric Acid Type A Receptors.

ABSTRACT: BACKGROUND:Neurosteroids like alphaxalone are potent anxiolytics, anticonvulsants, amnestics, and sedative-hypnotics, with effects linked to enhancement of ?-aminobutyric acid type A (GABAA) receptor gating in the central nervous system. Data locating neurosteroid binding sites on synaptic ??? GABAA receptors are sparse and inconsistent. Some evidence points to outer transmembrane ?-? interfacial pockets, near sites that bind the anesthetics etomidate and propofol. Other evidence suggests that steroids bind more intracellularly in ?-? interfaces. METHODS:The authors created 12 single-residue ?3 cysteine mutations: ?3T262C and ?3T266C in ?3-M2; and ?3M283C, ?3Y284C, ?3M286C, ?3G287C, ?3F289C, ?3V290C, ?3F293C, ?3L297C, ?3E298C, and ?3F301C in ?3-M3 helices. The authors coexpressed ?1 and ?2L with each mutant ?3 subunit in Xenopus oocytes and electrophysiologically tested each mutant for covalent sulfhydryl modification by the water-soluble reagent para-chloromercuribenzenesulfonate. Then, the authors assessed whether receptor-bound alphaxalone, etomidate, or propofol blocked cysteine modification, implying steric hindrance. RESULTS:Eleven mutant ?3 subunits, when coexpressed with ?1 and ?2L, formed functional channels that displayed varied sensitivities to the three anesthetics. Exposure to para-chloromercuribenzenesulfonate produced irreversible functional changes in ten mutant receptors. Protection by alphaxalone was observed in receptors with ?3V290C, ?3F293C, ?3L297C, or ?3F301C mutations. Both etomidate and propofol protected receptors with ?3M286C or ?3V290C mutations. Etomidate also protected ?3F289C. In ?1?3?2L structural homology models, all these protected residues are located in transmembrane ?-? interfaces. CONCLUSIONS:Alphaxalone binds in transmembrane ?-? pockets of synaptic GABAA receptors that are adjacent and intracellular to sites for the potent anesthetics etomidate and propofol.

SUBMITTER: Ziemba AM

PROVIDER: S-EPMC5771863 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Alphaxalone Binds in Inner Transmembrane β+-α- Interfaces of α1β3γ2 γ-Aminobutyric Acid Type A Receptors.

Ziemba Alexis M AM Szabo Andrea A Pierce David W DW Haburcak Marian M Stern Alex T AT Nourmahnad Anahita A Halpin Elizabeth S ES Forman Stuart A SA

Anesthesiology 20180201 2

<h4>Background</h4>Neurosteroids like alphaxalone are potent anxiolytics, anticonvulsants, amnestics, and sedative-hypnotics, with effects linked to enhancement of γ-aminobutyric acid type A (GABAA) receptor gating in the central nervous system. Data locating neurosteroid binding sites on synaptic αβγ GABAA receptors are sparse and inconsistent. Some evidence points to outer transmembrane β-α interfacial pockets, near sites that bind the anesthetics etomidate and propofol. Other evidence suggest ...[more]

PMID: 29210709

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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.

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.

Dataset Information

Alphaxalone Binds in Inner Transmembrane ?+-?- Interfaces of ?1?3?2 ?-Aminobutyric Acid Type A Receptors.

Publications

Alphaxalone Binds in Inner Transmembrane β+-α- Interfaces of α1β3γ2 γ-Aminobutyric Acid Type A Receptors.

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