The Na[+]/H[+] exchanger 1 activity is regulated by Reactive Oxygen Species

The NHE1 Na[+]/H[+] exchanger plays a key role in regulating pH and intracellular volume. Its role is paradoxical during ischemia-reperfusion where its activation causes an overload in sodium and indirectly in calcium which can cause disorders during reperfusion, particularly in excitable tissues such as the heart. This exchanger is regulated by intracellular pH, cell volume and by a multitude of signaling pathways. Several studies and preliminary results have indicated a possible regulation by reactive oxygen species that are produced during ischemia-reperfusion. The objective of this study is to analyze by which mechanisms these reactive oxygen species and in particular the superoxide anion O2.- regulate the activity of NHE1 and to understand which regions and which amino acids of NHE1 are important for this regulation.The activity of NHE1 wild type was measured in the presence of O2.- donors (Menadione) or in inhibition of O2.- production (Diphenyliodonium (DPI) NaDPH oxidase inhibitor). NHE1 activity was quantified by lithium flux as a function of intracellular pH. Menadione thus caused an activation of NHE1 while DPI decreased the activity of NHE1, demonstrating that NHE1 is therefore regulated by O2.-. This activation by Menadione and inhibition by DPI are lost in the Cysless mutant of NHE1 deprived of all its cysteines. Cysteines therefore play a key role in the regulation of NHE1 by O2.-. We then tested the inhibition by DPI on mutants of NHE1 on each of its cysteines, which made it possible to identify more precisely 3 key cysteines in this mechanism.In order to deepen the structural groups of cysteines potentially involved in this regulation, an analysis of possible covalent modifications was carried out including the search for nitrosylation by biotin switch, the search for disulfide bridges by exposure to iodoacetamide, and glutathionylation. This showed the presence of nitrosylation of the cysteines, but no disulphide bridges.At the same time, we observed a very different adhesion and migration profile of cells depending on the type of NHE1 cysteine mutant expressed. We therefore analyzed the expression of adhesion proteins and the binding of NHE1 to the cytoskeleton. By coimmunoprecipitation of NHE1 with the ERM complex (Ezrin/Radixin/Moesin) we showed that the cysteines of this transporter are crucial for its binding to the ERM complex and therefore to cortical actin.This thesis has therefore made it possible to show that the activity of NHE1 is regulated by the superoxide anion O2.- and that this regulation involves certain cysteines of NHE1 that we have identified. We also showed that some of the cysteines of NHE1 are crucial for the interaction with the actin cytoskeleton and play a key role in cell adhesion and migration. All of this work identifies a key connection between the production of reactive oxygen species, the regulation of intracellular pH and motility and adhesion.