Abstract
BackgroundGABAA 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 FindingsThe 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/SignificanceOur 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.
Highlights
Neuronal inhibition is essentially mediated by GABA type A receptors (GABAAR) forming anionic channels [1]
Synthetic peptides design Four peptides were synthesized by slices by incubation in mg/ml protease-XXIII (Sigma)-Genosys the amino acid sequences of which include the two identified endogenous phosphorylation sites of the I2 loop of GABAA receptor (GABAAR) a1 subunit
The minimal consensus [NXX(T/S)K] of GAPDHrelated phosphorylation site is present in several GABAAR alpha subunits (a1, a2, a3, a5), the I2 loop sequences being different in each subunit
Summary
Neuronal inhibition is essentially mediated by GABA type A receptors (GABAAR) forming anionic channels [1] They are pentameric oligomers assembled with several subunit classes that may have multiple isoforms [1,2,3]. The a1 subunit is highly expressed throughout most brain regions especially in the cortex [5] The function of these receptors can be modulated by reversible post-translational modifications such as phosphorylation-dephosphorylation [6,7,8]. 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 a1 subunit at two identified serine and threonine residues. 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
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