Glutathionylation of the β1 Subunit Prevents the E1Na3 to E2P Forward Reaction in the Na+, K+ ATPase

Abstract

Exposure of voltage clamped cardiomyocytes to receptor-coupled oxidant signalling has been associated with a decrease in electrogenic Na+, K+ pump current activity vattributed to glutathionylation of the pump's β1 subunit. The mechanism by which glutathionylation, a reversible oxidative modification, affects turnover is not understood. We investigated the effect of β1 subunit glutathionylation on the E1Na3 → E2P forward reaction using the fluorescent probe anthryl-ouabain (AO) that binds to E2P species.We induced glutathionylation in Na+, K+ ATPase enriched pig kidney membrane fragments by exposure to the chemical oxidant peroxynitrite. The Na+, K+ ATPase was stabilised in the E1Na3 poise by incubation in 100 mM NaCl and 20 mM Histidine. Glutathionylation of the β1 subunit was confirmed by immunoblotting techniques. MgATP was added to initiate the E1Na3 → E2P conversion and the E2P species was detected by the binding of AO. Addition of MgATP induced an increase in AO fluorescence indicative of a shift to E2P. The increase in fluorescence was blocked by the exposure to peroxynitrite. When glutathionylation was reversed by exposure to glutaredoxin 1, confirmed by the immublotting technique, the shift to E2P was restored as indicated by AO fluorescence.In an independent series of experiments we utilised baseline glutathionylation of a fraction of the Na+, K+ ATPase that is detectable without exposure to oxidants. We followed the E1Na3 → E2P conversion with RH421 fluorescence. As expected, addition of MgATP markedly increased fluorescence. We added 2.5 mM dithiothreitol (DTT) to rapidly reverse glutathionylation. This further increased RH421 fluorescence indictating that DTT made additional Na+, K+ ATPase available to undergo the E1Na3 → E2P conversion.We conclude that glutathionylation of the β1 subunit blocks the forward E1Na3 → E2P reaction.