Involvement of the TREK-1 channel in human alveolar cell membrane potential and its regulation by inhibitors of the chloride current

K⁺ channels of the alveolar epithelium control the driving force acting on the ionic and solvent flow through the cell membrane contributing to the maintenance of cell volume and the constitution of epithelial lining fluid. In the present work, we analyze the effect of the Cl⁻ channel inhibitors: (4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-inden-5-yl)oxy] butanoic acid (DCPIB) and 9-anthracenecarboxylic acid (9-AC) on the total current in a type II pneumocytes (A549 cell line) model by patch clamp, immunocytochemical, and gene knockdown techniques. We noted that DCPIB and 9-AC promote the activation of K conductance. In fact, they significantly increase the intensity of the current and shift its reversal potential to values more negative than the control. By silencing outward rectifier channel in its anoctamin 6 portion, we excluded a direct involvement of Cl⁻ ions in modulation of I_K and, by means of functional tests with its specific inhibitor spadin, we identified the TREK-1 channel as the presumable target of both drugs. As the activity of TREK-1 has a key role for the correct functioning of the alveolar epithelium, the identification of DCPIB and 9-AC molecules as its activators suggests their possible use to build new pharmacological tools for the modulation of this channel.     

Crosstalk between Na+,K+-ATPase and a volume-regulated anion channel in membrane microdomains of human cancer cells

Low concentrations of cardiac glycosides including ouabain, digoxin, and digitoxin block cancer cell growth without affecting Na⁺,K⁺-ATPase activity, but the mechanism underlying this anti-cancer effect is not fully understood. Volume-regulated anion channel (VRAC) plays an important role in cell death signaling pathway in addition to its fundamental role in the cell volume maintenance. Here, we report cardiac glycosides-induced signaling pathway mediated by the crosstalk between Na⁺,K⁺-ATPase and VRAC in human cancer cells. Submicromolar concentrations of ouabain enhanced VRAC currents concomitantly with a deceleration of cancer cell proliferation. The effects of ouabain were abrogated by a specific inhibitor of VRAC (DCPIB) and knockdown of an essential component of VRAC (LRRC8A), and they were also attenuated by the disruption of membrane microdomains or the inhibition of NADPH oxidase. Digoxin and digitoxin also showed anti-proliferative effects in cancer cells at their therapeutic concentration ranges, and these effects were blocked by DCPIB. In membrane microdomains of cancer cells, LRRC8A was found to be co-immunoprecipitated with Na⁺,K⁺-ATPase α1-isoform. These ouabain-induced effects were not observed in non-cancer cells. Therefore, cardiac glycosides were considered to interact with Na⁺,K⁺-ATPase to stimulate the production of reactive oxygen species, and they also apparently activated VRAC within membrane microdomains, thus producing anti-proliferative effects.