Mal protein stabilizes luminal membrane PLC-β3 and negatively regulates ENaC in mouse cortical collecting duct cells.

Abnormally high epithelial Na channel (ENaC) activity in the aldosterone-sensitive distal nephron and collecting duct leads to hypertension. Myelin and lymphocyte (Mal) is a lipid raft-associated protein that has been previously shown to regulate Na-K-2Cl cotransporter and aquaporin-2 in the kidney, but it is not known whether it regulates renal ENaC. ENaC activity is positively regulated by the anionic phospholipid phosphate phosphatidylinositol 4,5-bisphosphate (PIP2). Members of the myristoylated alanine-rich C-kinase substrate (MARCKS) family increase PIP2 concentrations at the plasma membrane, whereas hydrolysis of PIP2 by phospholipase C (PLC) reduces PIP2 abundance. Our hypothesis was that Mal protein negatively regulates renal ENaC activity by stabilizing PLC protein expression at the luminal plasma membrane. We investigated the association between Mal, MARCKS-like protein, and ENaC. We showed Mal colocalizes with PLC-β3 in lipid rafts and positively regulates its protein expression, thereby reducing PIP2 availability at the plasma membrane. Kidneys of 129Sv mice injected with MAL shRNA lentivirus resulted in increased ENaC open probability in split-open renal tubules. Overexpression of Mal protein in mouse cortical collecting duct (mpkCCD) cells resulted in an increase in PLC-β3 protein expression at the plasma membrane. siRNA-mediated knockdown of MAL in mpkCCD cells resulted in a decrease in PLC-β3 protein expression and an increase in PIP2 abundance. Moreover, kidneys from salt-loaded mice showed less Mal membrane protein expression compared with non-salt-loaded mice. Taken together, Mal protein may play an essential role in the negative feedback of ENaC gating in principal cells of the collecting duct.