Calpain‐2 proteolysis of MARCKS and MLP1 regulates ENaC activity (LB273)

Abstract

MARCKS is a ubiquitously expressed protein while MARCKS like protein (MLP1) is more abundantly expressed in mouse kidney. The effector domain of MARCKS and MLP1 is similar and serves as the functional domain for the sequesteration of phosphatidylinositol phosphates which is essential for normal activity of the epithelial sodium channel (ENaC). PIP strip overlay binding assays revealed identical binding of PIP2 and PIP3 by recombinant MARCKS and MLP fusion proteins. The effector domain of MARCKS is known to be regulated by PKC‐mediated phosphorylation of serine residues, binding to calcium‐calmodulin, and calpain‐2 proteolytic cleavage. The purpose of this study was to determine if ENaC activity is affected by calpain‐2‐mediated proteolysis of either ENaC subunits, MARCKS, or MLP. Calpain‐2 inhibition in MPKCCD cells resulted in a decrease in amiloride‐sensitive transepithelial current. However, exogenous application of purified recombinant calpain‐2 to the apical side of MPKCCD cells did not result in any appreciable change in transepithelial current. Algorithms predicting calpain‐2 cleavage sites revealed multiple putative cleavage sites within ENaC, MARCKS, and MLP. In vitro proteolytic assays showed both MARCKS and MLP but not ENaC alpha, beta, or gamma subunits are sensitive to calpain‐2 proteolytic cleavage. Taken together, these results suggest ENaC activity is regulated by calpain‐2 proteolytic cleavage of MARCKS/MLP in mouse kidney.Grant Funding Source: This work was supported by an NIH/NIGMS K12 GM000680 (to DCE) and an NIH/NRSA DK093255‐01 (to AAA).