title: Mass spectrometric detection and characterization of atypical membrane-bound zinc-sensitive phosphatases modulating GABAA receptors.
abstract: 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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors: Inserm, Infantile Epilepsies and Brain Plasticity U1129, Paris, France; University Paris Descartes, Paris, France; CEA, Gif sur Yvette, France.
authors: Mounia
authors: M
authors: SidAhmed-Mezi
authors: University Paris Descartes, Paris, France; Inserm, Centre de Psychiatrie et de Neurosciences U894, Paris, France.
authors: Irène
authors: I
authors: Kurcewicz
authors: University Paris Descartes, Paris, France; Inserm, Centre de Psychiatrie et de Neurosciences U894, Paris, France.
authors: Christiane
authors: C
authors: Rose
authors: University Paris Descartes, Paris, France; Inserm, Centre de Psychiatrie et de Neurosciences U894, Paris, France.
authors: Jacques
authors: J
authors: Louvel
authors: Institut de Recherche Pierre Fabre, Neurologie & Psychiatrie, Castres, France.
authors: Pierre
authors: P
authors: Sokoloff
authors: Inserm, Infantile Epilepsies and Brain Plasticity U1129, Paris, France; University Paris Descartes, Paris, France; CEA, Gif sur Yvette, France.
authors: René
authors: R
authors: Pumain
authors: Inserm, Infantile Epilepsies and Brain Plasticity U1129, Paris, France; University Paris Descartes, Paris, France; CEA, Gif sur Yvette, France.
authors: Jacques J
authors: JJ
authors: Laschet
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology*; D002540:Cerebral Cortex / Q000166:cytology / Q000502:physiology; D055724:Electrophysiological Phenomena; D057075:Enzyme Assays / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D019319:Okadaic Acid / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism*
chemical_list: D010748:Phosphopeptides; D011963:Receptors, GABA-A; D019319:Okadaic Acid; D010744:Phosphoric Monoester Hydrolases; D015032:Zinc