Cross-Linking of Catalytically Essential Vicinal Thiols at Active Sites of the Cerebral Sodium Transporter Inactivates its Electrogenic Function

The inactivation of the electrogenic function of the transmembrane sodium transporter in oxidative stress conditions has been intrinsically linked with the oxidation of its catalytically essential thiols. However, the spatial proximity of these catalytically relevant thiols is yet to be fully elucidated and thus still open. Herein, the influence of a thiol cross-linking [diamide, DA (0.1-2mM)] and a thiol alkylating [iodoacetamide, IA (0.1-5mM)] agent on the activity of the synaptosomal Na ⁺ /K ⁺ -ATPase were determined. In addition, the ability of dithiothreitol to either prevent or reverse the inhibition imposed by the thiol modifiers on the enzyme activity was also evaluated. The results showed that the thiol cross-linker inactivates the electrogenic function of the synaptosomal Na ⁺ /K ⁺ -ATPase when exposed to the thiols located at either the nucleotide or cation-binding sites. Conversely, irrespective of the exposed active sites, the thiol alkylating agents have no overt effect on the activity of the pump. Furthermore, dithiothreitol markedly prevented but did not reverse the inactivation of the electrogenic pump caused by cross-linking of its critical thiols. Interestingly, both the thiol cross-linker and alkylating agents markedly oxidize dithiothreitol in a time and concentration-dependent fashion. Consequently, within the limit of the present data, it appears that the catalytically relevant thiols of the transmembrane electrogenic pump located at the cationic and nucleotide binding sites, are in close proximity sufficient enough to allow for their cross-linking.