Mass spectrometric detection and characterization of atypical membrane-bound zinc-sensitive phosphatases modulating GABAA receptors.

BACKGROUND: GABAA receptor (GABAAR) function is maintained by an endogenous phosphorylation mechanism for which the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is the kinase. This phosphorylation is specific to the long intracellular loop I2 of the α1 subunit at two identified serine and threonine residues. The phosphorylation state is opposed by an unknown membrane-bound phosphatase, which inhibition favors the phosphorylated state of the receptor and contributes to the maintenance of its function. In cortical nervous tissue from epileptogenic areas in patients with drug-resistant epilepsies, both the endogenous phosphorylation and the functional state of the GABAAR are deficient. METHODOLOGY/PRINCIPAL FINDINGS: The aim of this study is to characterize the membrane-bound phosphatases counteracting the endogenous phosphorylation of GABAAR. We have developed a new analytical tool for in vitro detection of the phosphatase activities in cortical washed membranes by liquid chromatography coupled to mass spectrometry. The substrates are two synthetic phosphopeptides, each including one of the identified endogenous phosphorylation sites of the I2 loop of GABAAR α1 subunit. We have shown the presence of multiple and atypical phosphatases sensitive to zinc ions. Patch-clamp studies of the rundown of the GABAAR currents on acutely isolated rat pyramidal cells using the phosphatase inhibitor okadaic acid revealed a clear heterogeneity of the phosphatases counteracting the function of the GABAAR. CONCLUSION/SIGNIFICANCE: Our results provide new insights on the regulation of GABAAR endogenous phosphorylation and function by several and atypical membrane-bound phosphatases specific to the α1 subunit of the receptor. By identifying specific inhibitors of these enzymes, novel development of antiepileptic drugs in patients with drug-resistant epilepsies may be proposed.

Lability of GABAA receptor function in human partial epilepsy: possible relationship to hypometabolism.

The function of the gamma-aminobutyric acid type A receptor (GABA(A)R) is maintained by endogenous phosphorylation. We have shown that the corresponding kinase is the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), using the locally produced glycolytic ATP. In addition, using cerebral tissue obtained during curative surgery for epilepsy, we showed that both the endogenous phosphorylation and the GABA(A)R function are significantly reduced in the "epileptogenic" cerebral cortex when compared to "control" tissue. This dysfunction likely contributes to seizure generation and/or transition from the interictal to the ictal state. Glucose utilization is decreased in the epileptogenic cortex of patients with partial epilepsy in the interictal state, but the relationship to the disorder remains unclear. We propose that this hypometabolism is related to the deficiency in the endogenous phosphorylation of GABA(A)R and the resulting greater lability of GABAergic inhibition. Several lines of evidences indeed suggest that GABAergic inhibition is costly in terms of metabolic consumption. The deficiency of this glycolysis-dependent mechanism may thus link epileptogenicity to glucose hypometabolism. The antiepileptic effect of ketogenic diets may be mediated by the subsequent rise in the NADH/NAD(+) index, which favors GABA(A)R endogenous phosphorylation and should contribute to restoration of GABAergic inhibition in the epileptogenic zone.

Dysfunction of GABAA receptor glycolysis-dependent modulation in human partial epilepsy.

A reduction in GABAergic neurotransmission has been put forward as a pathophysiological mechanism for human epilepsy. However, in slices of human epileptogenic neocortex, GABAergic inhibition can be clearly demonstrated. In this article we present data showing an increase in the functional lability of GABAergic inhibition in epileptogenic tissue compared with nonepileptogenic human tissue. We have previously shown that the glycolytic enzyme GAPDH is the kinase involved in the glycolysis-dependent endogenous phosphorylation of the alpha1-subunit of GABA(A) receptor, a mechanism necessary for maintaining GABA(A) function. In human epileptogenic cortex obtained during curative surgery of patients with partial seizures, we demonstrate an intrinsic deficiency of GABA(A) receptor endogenous phosphorylation resulting in an increased lability of GABAergic currents in neurons isolated from this tissue when compared with neurons from nonepileptogenic human tissue. This feature was not related to a reduction in the number of GABA(A) receptor alpha1-subunits in the epileptogenic tissue as measured by [(3)H]flunitrazepam photoaffinity labeling. Maintaining the receptor in a phosphorylated state either by favoring the endogenous phosphorylation or by inhibiting a membrane-associated phosphatase is needed to sustain GABA(A) receptor responses in epileptogenic cortex. The increased functional lability induced by the deficiency in phosphorylation can account for transient GABAergic disinhibition favoring seizure initiation and propagation. These findings imply new therapeutic approaches and suggest a functional link to the regional cerebral glucose hypometabolism observed in patients with partial epilepsy, because the dysfunctional GABAergic mechanism depends on the locally produced glycolytic ATP.

Glyceraldehyde-3-phosphate dehydrogenase is a GABAA receptor kinase linking glycolysis to neuronal inhibition.

Protein phosphorylation is crucial for regulating synaptic transmission. We describe a novel mechanism for the phosphorylation of the GABA(A) receptor, which mediates fast inhibition in the brain. A protein copurified and coimmunoprecipitated with the phosphorylated receptor alpha1 subunit; this receptor-associated protein was identified by purification and microsequencing as the key glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Molecular constructs demonstrated that GAPDH directly phosphorylates the long intracellular loop of GABA(A) receptor alpha1 subunit at identified serine and threonine residues. GAPDH and the alpha1 subunit were found to be colocalized at the neuronal plasma membrane. In keeping with the GAPDH/GABA(A) receptor molecular association, glycolytic ATP produced locally at plasma membranes was consumed for this alpha1 subunit phosphorylation, possibly within a single macrocomplex. The membrane-attached GAPDH is thus a dual-purpose enzyme, a glycolytic dehydrogenase, and a receptor-associated kinase. In acutely dissociated cortical neurons, the rundown of the GABA(A) responses was essentially attributable to a Mg(2+)-dependent phosphatase activity, which was sensitive to vanadate but insensitive to okadaic acid or fluoride. Rundown was significantly reduced by the addition of GAPDH or its reduced cofactor NADH and nearly abolished by the addition of its substrate glyceraldehyde-3-phosphate (G3P). The prevention of rundown by G3P was abolished by iodoacetamide, an inhibitor of the dehydrogenase activity of GAPDH, indicating that the GABA(A) responses are maintained by a glycolysis-dependent phosphorylation. Our results provide a molecular mechanism for the direct involvement of glycolysis in neurotransmission.

Masking synchronous GABA-mediated potentials controls limbic seizures.

PURPOSE: We determined how CA3-driven interictal discharges block ictal activity generated in the entorhinal cortex during bath application of 4-aminopyridine (4AP, 50 microM). METHODS: Field potential and [K+]o recordings were obtained from mouse combined hippocampus-entorhinal cortex slices maintained in vitro. RESULTS: 4AP induced N-methyl-d-aspartate (NMDA) receptor-dependent ictal discharges that originated in the entorhinal cortex, disappeared over time, but were reestablished by cutting the Schaffer collateral (n = 20) or by depressing CA3 network excitability with local application of glutamatergic receptor antagonists (n = 5). In addition, two types of interictal activity occurred throughout the experiment. The first type was CA3 driven and was abolished by a non-NMDA glutamatergic receptor antagonist. The second type was largely contributed by gamma-aminobutyric acid type A (GABAA) receptor-mediated conductances and persisted during blockade of glutamatergic transmission. The absence of CA3-driven interictal discharges in the entorhinal cortex after Schaffer collateral cut facilitated the GABA-mediated interictal potentials that corresponded to large [K+]o elevations and played a role in ictal discharge initiation. Accordingly, ictal discharges along with GABA-mediated interictal potentials disappeared during GABAA-receptor blockade (n = 7) or activation of mu-opioid receptors that inhibit GABA release (n = 4). CONCLUSIONS: Our findings suggest that CA3-driven interictal events restrain ictal discharge generation in the entorhinal cortex by modulating the size of interictal GABA-mediated potentials that lead to large [K+]o elevations capable of initiating ictal discharges in this structure.

Epileptiform synchronization and GABA(B) receptor antagonism in the juvenile rat hippocampus.

The GABA(B) receptor agonist baclofen enhances the epileptiform activity induced by 4-aminopyridine (4AP) in juvenile rat hippocampal slices. In this study, we used a similar experimental approach (i.e., field potential, intracellular, and [K+]o recordings in the CA3 area of slices obtained from 15-23-day-old rats) to establish whether antagonizing GABA(B) receptors could exert opposite (presumably anticonvulsant) effects. Bath application of 4AP (50 microM) induced spontaneous interictal and ictal discharges along with synchronous GABA receptor-mediated potentials. All types of 4AP-induced synchronous activity occurred more frequently during application of the GABA(B) receptor antagonist p3-amino-propyl,p-diethoxymethylphosphonic acid (CGP 35348) (0.1-1 mM; EC50 = 0.25 mM). Moreover, CGP 35348 augmented the frequency and, to a lesser extent, the duration of the asynchronous synaptic activity recorded intracellularly from CA3 pyramids (n = 19). In medium containing 4AP + ionotropic glutamatergic antagonists (which abolished interictal and ictal activity), CGP 35348 prolonged both GABA-receptor-mediated field potentials and the accompanying intracellular long-lasting depolarizations without modifying their rate (n = 12). The transient elevations in [K+]o associated with GABA receptor-mediated potentials in 4AP-containing medium (n = 7 slices) became larger during CGP 35348 application. Similar findings were obtained when CGP 35348 was applied to medium containing 4AP + ionotropic glutamatergic antagonists (n = 6). Thus, the effect of CGP 35348 on 4AP-induced epileptiform activity is not anticonvulsant and to some extent similar to what was reported in this model during GABA(B) receptor activation. We propose that the facilitation of ictal activity by CGP 35348 is mainly caused by the blockade of presynaptic GABA(B) receptor, leading to an increase in GABA release and subsequent larger [K+]o elevations.