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Neurosteroids allosterically modulate binding of the anesthetic etomidate to gamma-aminobutyric acid type A receptors.

ABSTRACT: Photoaffinity labeling of gamma-aminobutyric acid type A (GABA(A))-receptors (GABA(A)R) with an etomidate analog and mutational analyses of direct activation of GABA(A)R by neurosteroids have each led to the proposal that these structurally distinct general anesthetics bind to sites in GABA(A)Rs in the transmembrane domain at the interface between the beta and alpha subunits. We tested whether the two ligand binding sites might overlap by examining whether neuroactive steroids inhibited etomidate analog photolabeling. We previously identified (Li, G. D., Chiara, D. C., Sawyer, G. W., Husain, S. S., Olsen, R. W., and Cohen, J. B. (2006) J. Neurosci. 26, 11599-11605) azietomidate photolabeling of GABA(A)R alpha1Met-236 and betaMet-286 (in alphaM1 and betaM3). Positioning these two photolabeled amino acids in a single type of binding site at the interface of beta and alpha subunits (two copies per pentamer) is consistent with a GABA(A)R homology model based upon the structure of the nicotinic acetylcholine receptor and with recent alphaM1 to betaM3 cross-linking data. Biologically active neurosteroids enhance rather than inhibit azietomidate photolabeling, as assayed at the level of GABA(A)R subunits on analytical SDS-PAGE, and protein microsequencing establishes that the GABA(A)R-modulating neurosteroids do not inhibit photolabeling of GABA(A)R alpha1Met-236 or betaMet-286 but enhance labeling of alpha1Met-236. Thus modulatory steroids do not bind at the same site as etomidate, and neither of the amino acids identified as neurosteroid activation determinants (Hosie, A. M., Wilkins, M. E., da Silva, H. M., and Smart, T. G. (2006) Nature 444, 486-489) are located at the subunit interface defined by our etomidate site model.

SUBMITTER: Li GD

PROVIDER: S-EPMC2673245 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Neurosteroids allosterically modulate binding of the anesthetic etomidate to gamma-aminobutyric acid type A receptors.

Li Guo-Dong GD Chiara David C DC Cohen Jonathan B JB Olsen Richard W RW

The Journal of biological chemistry 20090312 18

Photoaffinity labeling of gamma-aminobutyric acid type A (GABA(A))-receptors (GABA(A)R) with an etomidate analog and mutational analyses of direct activation of GABA(A)R by neurosteroids have each led to the proposal that these structurally distinct general anesthetics bind to sites in GABA(A)Rs in the transmembrane domain at the interface between the beta and alpha subunits. We tested whether the two ligand binding sites might overlap by examining whether neuroactive steroids inhibited etomidat ...[more]

PMID: 19282280

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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.

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Neurosteroids allosterically modulate binding of the anesthetic etomidate to gamma-aminobutyric acid type A receptors.

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Neurosteroids allosterically modulate binding of the anesthetic etomidate to gamma-aminobutyric acid type A receptors.

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