OP40 - Oxygen sensing by the Na,K-ATPase: the cellular mechanism unraveled

Abstract

Reduction in ATP generation under conditions of limited oxygen supply triggers acute decrease in ATP consumption by turning off the Na,K-pump. This adaptive response is effective when followed by “channel arrest”, but may be lethal when not associated with reduction in passive Na and K permeability. Oxygen sensing by the Na,K-ATPase is mediated by way of reversible thiol modifications. We have identified the four regulatory cysteine residues within the ATP binding site of the catalytic α subunit that, when S-glutathionylated, prevent binding of ATP to the enzyme. S-glutathionylation may only occur under conditions of mild ATP depletion associated with GSSG accumulation in the enzyme proximity. Identification of the residues was performed by mass spectrometry on purified Na,K-ATPase which was completely inactivated by treatment with GSSG. In silico modelling and titration experiments using isothermal scanning calorimetry confirmed competition of GSSG and ATP for the ATP binding domain. Finally, a set of HEK293 cell lines transfected with murine α1 subunit protein with point mutations of the candidate cysteines for alanines was produced and the oxygen-insensitivity of mutant murine Na,K-ATPase was proven. The lack of oxygen-sensitive made the transfected host cells less hypoxia-tolerant. We furthermore advanced in identification of the source of hypoxia-induced free radical production triggering GSSG production which is required for making the Na,K-ATPase oxygen-sensitive. A burst of superoxide anion production originating from the mitochondria was observed in several cell types including cerebellar granule cells during the first 10-15 minutes of hypoxia. The latter could be inhibited by silencing or pharmacological inhibition of the mitochondrial Na/Ca exchanger by CGP-37157. Administration of CGP-37157 preserved the Na,K-ATPase function in hypoxic cerebellar granule cells, decreased the intracellular GSSG levels and reduced S-glutathionylation of the α subunit.