ASIC1a/α‐ENaC Hybrid Channels Contribute to Alveolar Fluid Clearance

Abstract

Pneumonia affects 450 million people globally per year with about 4 million deaths. Pneumonia mortality is caused by abnormal alveolar fluid clearance (AFC) preventing normal gas exchange with consequent hypoxemia. Since knockout of α‐ENaC completely prevents new born mice from clearing fluid from their lungs, it is reasonable to conclude that α‐ENaC is responsible for AFC; but if all Na+ reabsorption was via ENaC alone, then amiloride should block all AFC, but it blocks only about half. Single channel measurements from alveolar epithelial cells show why. There is a channel with properties like ENaC expressed in heterologous expression systems, but there is another channel present at almost the same frequency that is non‐selective for Na+ over K+, has a larger conductance, and shorter mean open and closed times. These two types of channels have been called highly selective channels (HSC) and non‐selective cation channels (NSC). HSC channels are made up of three ENaC subunits since knocking down any of the subunits reduces HSC channel number. The composition of NSC channels is unknown, but appears to contain α‐ENaC since knocking down this subunit reduces the number of NSC channels (knocking down β‐ or γ‐ENaC has no effect on NSC number). We now hypothesize that NSC channels consist of at least one α‐ENaC subunit and one or more ASIC1a protein subunits (Acid‐Sensing Ion Channel 1a). ASIC1a is another member of the ENaC/degenerin protein family that is strongly expressed in alveolar epithelial cells. We used molecular biological methods to manipulate the amount of ASIC1a and α‐ENaC in heterologous expression systems and alveolar cells and assessed the effect on HSC and NSC channels using single channel measurements. ASIC1a and α‐ENaC co‐immunoprecipitate from lung cell lysates and are closely associated in mammalian two‐hybrid assays. ASIC1a/α‐ENaC coexpression in FRT cells produces NSC channels that are very similar to NSC channels in mouse lung cells. shRNA knock‐down of ASIC1 in a lung cell line reduces the frequency of NSC channels from 91 % to 10 % of patches with no effect on HSC channels. α‐ENaC silencing reduced HSC frequency from 55% to 8% of patches and NSC channels from 86% to 12% of patches. The frequency of NSC in patches from lung slices is also reduced in mice instilled with ASIC1a shRNA silencing vectors. Therefore, we conclude that NSC channels are ASIC1a/α‐ENaC hybrid channels that contribute to maintaining AFC. The regulation of NSC and HSC channels is so different that it may require a new paradigm to explain normal AFC and AFC in the face of a viral challenge.Support or Funding InformationR37‐DK037963 to DCE; APS/NHLBI STRIDE to PT