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Phosphatase inhibitors remove the run-down of gamma-aminobutyric acid type A receptors in the human epileptic brain.

ABSTRACT: The properties of gamma-aminobutyric acid (GABA) type A receptors (GABA(A) receptors) microtransplanted from the human epileptic brain to the plasma membrane of Xenopus oocytes were compared with those recorded directly from neurons, or glial cells, in human brains slices. Cell membranes isolated from brain specimens, surgically obtained from six patients afflicted with drug-resistant temporal lobe epilepsy (TLE) were injected into frog oocytes. Within a few hours, these oocytes acquired GABA(A) receptors that generated GABA currents with an unusual run-down, which was inhibited by orthovanadate and okadaic acid. In contrast, receptors derived from membranes of a nonepileptic hippocampal uncus, membranes from mouse brain, or recombinant rat alpha 1 beta 2 gamma 2-GABA receptors exhibited a much less pronounced GABA-current run-down. Moreover, the GABA(A) receptors of pyramidal neurons in temporal neocortex slices from the same six epileptic patients exhibited a stronger run-down than the receptors of rat pyramidal neurons. Interestingly, the GABA(A) receptors of neighboring glial cells remained substantially stable after repetitive activation. Therefore, the excessive GABA-current run-down observed in the membrane-injected oocytes recapitulates essentially what occurs in neurons, rather than in glial cells. Quantitative RT-PCR analyses from the same TLE neocortex specimens revealed that GABA(A)-receptor beta 1, beta 2, beta 3, and gamma 2 subunit mRNAs were significantly overexpressed (8- to 33-fold) compared with control autopsy tissues. Our results suggest that an abnormal GABA-receptor subunit transcription in the TLE brain leads to the expression of run-down-enhanced GABA(A) receptors. Blockage of phosphatases stabilizes the TLE GABA(A) receptors and strengthens GABAergic inhibition. It may be that this process can be targeted to develop new treatments for intractable epilepsy.

SUBMITTER: Palma E

PROVIDER: S-EPMC454185 | biostudies-literature | 2004 Jul

REPOSITORIES: biostudies-literature

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Phosphatase inhibitors remove the run-down of gamma-aminobutyric acid type A receptors in the human epileptic brain.

Palma E E Ragozzino D A DA Di Angelantonio S S Spinelli G G Trettel F F Martinez-Torres A A Torchia G G Arcella A A Di Gennaro G G Quarato P P PP Esposito V V Esposito V V Cantore G G Miledi R R Eusebi F F

Proceedings of the National Academy of Sciences of the United States of America 20040624 27

The properties of gamma-aminobutyric acid (GABA) type A receptors (GABA(A) receptors) microtransplanted from the human epileptic brain to the plasma membrane of Xenopus oocytes were compared with those recorded directly from neurons, or glial cells, in human brains slices. Cell membranes isolated from brain specimens, surgically obtained from six patients afflicted with drug-resistant temporal lobe epilepsy (TLE) were injected into frog oocytes. Within a few hours, these oocytes acquired GABA(A) ...[more]

PMID: 15218107

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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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Phosphatase inhibitors remove the run-down of gamma-aminobutyric acid type A receptors in the human epileptic brain.

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Phosphatase inhibitors remove the run-down of gamma-aminobutyric acid type A receptors in the human epileptic brain.

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