Ouabain Modulates the Functional Interaction Between Na,K-ATPase and NMDA Receptor.

The N-methyl-D-aspartate (NMDA) receptor plays an essential role in glutamatergic transmission and synaptic plasticity and researchers are seeking for different modulators of NMDA receptor function. One possible mechanism for its regulation could be through adjacent membrane proteins. NMDA receptors coprecipitate with Na,K-ATPase, indicating a potential interaction of these two proteins. Ouabain, a mammalian cardiotonic steroid that specifically binds to Na,K-ATPase and affects its conformation, can protect from some toxic effects of NMDA receptor activation. Here we have examined whether NMDA receptor activity and downstream effects can be modulated by physiological ouabain concentrations. The spatial colocalization between NMDA receptors and the Na,K-ATPase catalytic subunits on dendrites of cultured rat hippocampal neurons was analyzed with super-resolution dSTORM microscopy. The functional interaction was analyzed with calcium imaging of single hippocampal neurons exposed to 10 µM NMDA in presence and absence of ouabain and by determination of the ouabain effect on NMDA receptor-dependent long-term potentiation. We show that NMDA receptors and the Na,K-ATPase catalytic subunits alpha1 and alpha3 exist in same protein complex and that ouabain in nanomolar concentration consistently reduces the calcium response to NMDA. Downregulation of the NMDA response is not associated with internalization of the receptor or with alterations in its state of Src phosphorylation. Ouabain in nanomolar concentration elicits a long-term potentiation response. Our findings suggest that ouabain binding to a fraction of Na,K-ATPase molecules that cluster with the NMDA receptors will, via a conformational effect on the NMDA receptors, cause moderate but consistent reduction of NMDA receptor response at synaptic activation.

Influence of Oxidative Stress on Catalytic and Non-glycolytic Functions of Glyceraldehyde-3-phosphate Dehydrogenase.

BACKGROUND: Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) is a unique enzyme that, besides its main function in glycolysis (catalysis of glyceraldehyde-3-phosphate oxidation), possesses a number of non-glycolytic activities. The present review summarizes information on the role of oxidative stress in the regulation of the enzymatic activity as well as non-glycolytic functions of GAPDH. METHODS: Based on the analysis of literature data and the results obtained in our research group, mechanisms of the regulation of GAPDH functions through the oxidation of the sulfhydryl groups in the active site of the enzyme have been suggested. RESULTS: Mechanism of GAPDH oxidation includes consecutive oxidation of the catalytic Cysteine (Cys150) into sulfenic, sulfinic, and sulfonic acid derivatives, resulting in the complete inactivation of the enzyme. The cysteine sulfenic acid reacts with reduced glutathione (GSH) to form a mixed disulfide (S-glutathionylated GAPDH) that further reacts with Cys154 yielding the disulfide bond in the active site of the enzyme. In contrast to the sulfinic and sulfonic acids, the mixed disulfide and the intramolecular disulfide bond are reversible oxidation products that can be reduced in the presence of GSH or thioredoxin. CONCLUSION: Oxidation of sulfhydryl groups in the active site of GAPDH is unavoidable due to the enhanced reactivity of Cys150. The irreversible oxidation of Cys150 is prevented by Sglutathionylation and disulfide bonding with Cys154. The oxidation/reduction of the sulfhydryl groups in the active site of GAPDH can be used for regulation of glycolysis and numerous side activities of this enzyme including the induction of apoptosis.