Inhibition of αENaC expression and ENaC activity following blockade of the circadian clock-regulatory kinases CK1δ/ε

Richards J, Greenlee MM, Jeffers LA, Cheng KY, Guo L, Eaton DC, Gumz ML. Inhibition of alpha ENaC expression and ENaC activity following blockade of the circadian clock-regulatory kinases CK1 delta/epsilon. Am J Physiol Renal Physiol 303: F918-F927, 2012. First published July 25, 2012; doi:10.1152/ajprenal.00678.2011.-Increasing evidence suggests that the circadian clock plays an important role in the control of renal function and blood pressure. We previously showed that the circadian clock protein Period (Per)1, positively regulates the expression of the rate limiting subunit of the renal epithelial sodium channel (alpha ENaC), which contributes to blood pressure regulation. Casein kinases 1 delta and 1 epsilon (CK1 delta/epsilon) are critical regulators of clock proteins. CK1 delta/epsilon must phosphorylate the circadian clock protein Per1 in order for the latter to enter the nucleus. We used a commercially available CK1 delta/epsilon inhibitor, PF670462, to test the effect of CK1 delta/epsilon blockade and inhibited Per1 nuclear entry on alpha ENaC in a model of the renal cortical collecting duct (mpkCCD(c14) cells). CK1 delta/epsilon blockade prevented Per1 and Clock from interacting with an E-box from the alpha ENaC promoter. CK1 delta/epsilon inhibition reduced alpha ENaC mRNA levels by <60%. A similar decrease in alpha ENaC mRNA was observed following siRNA-mediated CK1 delta/epsilon knock-down. Inhibition of CK1 delta/epsilon effectively prevented the transcriptional response of alpha ENaC to aldosterone, suggesting an interaction between the circadian clock and aldosterone-mediated regulation of alpha ENaC. CK1 delta/epsilon inhibition significantly reduced alpha ENaC but increased Caveolin-1 membrane protein levels; transepithelial current, a measure of ENaC activity, was decreased. Importantly, single channel analysis in amphibian renal cells demonstrated a dramatic decrease in the number of patches with observable ENaC current following CK1 delta/epsilon inhibition. The present study shows for the first time that CK1 delta/epsilon inhibition and impaired Per1 nuclear entry results in decreased alpha ENaC expression and ENaC activity, providing further support for direct control of ENaC by the circadian clock.