ENaC activity is regulated by calpain-2 proteolysis of MARCKS proteins


  • Authors: Montgomery, D. S., Yu, L., Ghazi, Z. M., Thai, T. L., Al-Khalili, O., Ma, H. P., Eaton, D. C., Alli, A. A.
  • Year: 2017
  • Journal: Am J Physiol Cell Physiol 313 C42-C53
  • Applications: in vitro / DNA / jetPRIME
  • Cell type: mpkCCDcl4
    Description: Mouse kidney cortical collecting duct cell line.


We previously demonstrated a role for the myristoylated alanine-rich C kinase substrate (MARCKS) to serve as an adaptor protein in the anionic phospholipid phosphate-dependent regulation of the epithelial sodium channel (ENaC). Both MARCKS and ENaC are regulated by proteolysis. Calpains are a family of ubiquitously expressed intracellular Ca(2+)-dependent cysteine proteases involved in signal transduction. Here we examine the role of calpain-2 in regulating MARCKS and ENaC in cultured renal epithelial cells and in the mouse kidney. Using recombinant fusion proteins, we show that MARCKS, but not the ENaC subunits, are a substrate of calpain-2 in the presence of Ca(2+) Pharmacological inhibition of calpain-2 alters MARCKS protein expression in light-density sucrose gradient fractions from cell lysates of mouse cortical collecting duct cells. Calpain-dependent cleaved products of MARCKS are detectable in cultured renal cells. Ca(2+) mobilization and calpain-2 inhibition decrease the association between ENaC and MARCKS. The inhibition of calpain-2 reduces ENaC activity as demonstrated by single-channel patch-clamp recordings and transepithelial current measurements. These results suggest that calpain-2 proteolysis of MARCKS promotes its interaction with lipids and ENaC at the plasma membrane to allow for the phosphatidylinositol 4,5-bisphosphate (PIP2)-dependent regulation of ENaC activity in the kidney.