Redox Regulation of Epithelial Sodium Channels in the Alveolar Epithilium and Microvascular Endothelium

Dr. Charles A. Downs’ laboratory uses electrophysiology (single channel patch clamp recordings), and a variety of biochemical and molecular techniques, to investigate the effect of redox state on direct and indirect regulation of the epithelial sodium channel (ENaC) in all cells types that comprise the alveolus.

Appropriate lung fluid balance is requisite for life, and alveolar flooding seriously impairs gas exchange leading to respiratory failure and, if untreated, death. Under physiological conditions, lung fluid balance is maintained through activity of the amiloride-sensitive ENaC. ENaC is formed from three subunit proteins: α, β, and γENaC; although overexpression models clearly show that α-ENaC alone can transport Na+. In the lung, ENaC functions to absorb Na+ from the apical surface thereby increasing intracellular [Na+]. The basolaterally located Na+/K+-ATPase pump then extrudes Na+ from the cytosol to the interstitial space. This creates an osmotic gradient which removes water from the alveolar lumen that is subsequently absorbed by the lymphatics and vasculature. The importance of ENaC to lung fluid balance is highlighted in seminal work illustrating that α-ENaC knockout mice die within 24 hours of birth due to an inability to clear lung fluid. Because ENaC plays the critical role in maintaining lung fluid balance, it is important to gain a better understanding of the signal transduction pathways that regulate ENaC. In addition to leading to a better understanding of mechanisms underlying lung fluid balance, this work aims to provide pharmaceutical targets for persons suffering from acute respiratory distress syndrome (ARDS), a fatal form of respiratory failure with a paucity of treatments and no cure.

Oxidative stress occurs in ARDS; however, the effect of oxidative stress on the regulation of ENaC is poorly understood. Indeed, Dr. Down’s laboratory has demonstrated that physiologic oxidant signaling, particularly from the family of NADPH oxidases (NOX), and extracellular redox state (GSH/GSSG) affect ENaC activity and expression. However, under pathological conditions the effect is unclear. Dr. Down’s lab plans to use proteomics, coupled with single channel patch clamp recordings and in vivo measurements of lung fluid clearance, to identify differentially expressed proteins, subjected to various redox states, and their effect on ENaC activity/expression from all cells that comprise the alveolus. Dr. Down’s lab also plans to use investigational compounds, under various redox states, to assess for an effect on ENaC structure, function, and expression. A future goal is to use planar lipid bilayer electrical recording to assess purified ENaC structure and function to investigate pathobiological mechanisms in pulmonary vascular diseases in collaboration with several laboratories in the Division of Translational and Regenerative Medicine, including Drs. Yuan, Arce, Garcia, and Black. 

Figure: Extracellular shifts in glutathione (GSH)/glutathione disulfide (GSSG) glutathione redox (Eh) potential affect ENaC open probability (Po). A) Representative portions of a trace taken from a single channel recording of T2 cell (-10mV (-Vp)) is shown. Closed states (c) are indicated by dashed line and # indicates a break from continual recording. B) Point amplitude histograms for data presented in A; peaks in the distribution represent 0 or 1 open channel. C) Current voltage relationship of representative trace shown in A, with a calculated conductance (γ=26.6 pS). D) Dot plot of 12 separate T2 cell shown effect of control (-210 mV), GSSG (-75mV) and then GSH (-213 mV) sequentially added on ENaC Po. *=P<0.05. E) GSSG (-75 mV) similarly decreases ENaC Po in T1 cells. F) Percent control values for individual cells (circles), before and after adding a mixture of GSH and GSSG (redox potentials ranging from -300 to -100 mV), calculated by dividing Po before and after addition of GSH/GSSG and multiplied by 100. Open squares with error bars represent mean ± S.E. for each redox potential. Linear regression was performed to determine the best fit relationship between redox potential and ENaC activity.