Abstract
Enhanced endothelial sodium channel (EnNaC) functioning causes an increase in vessel stiffness. Here, we investigated the regulation of EnNaC in mouse aortic endothelial cells (mAoECs) by the actin cytoskeleton and lipid raft association protein myristoylated alanine-rich C-kinase substrate-like protein 1 (MLP1). We hypothesized that mutation of specific amino acid residues within the effector domain of MLP1 or loss of association between MLP1 and the anionic phospholipid phosphate PIP2 would significantly alter membrane association and EnNaC activity in mAoECs. mAoECs transiently transfected with a mutant MLP1 construct (three serine residues in the effector domain replaced with aspartate residues) showed a significant decrease in EnNaC activity compared with cells transfected with wild-type MLP1. Compared with vehicle treatment, mAoECs treated with the PIP2 synthesis blocker wortmannin showed less colocalization of EnNaC and MLP1. In other experiments, Western blot and densitometric analysis showed a significant decrease in MLP1 and caveolin-1 protein expression in mAoECs treated with wortmannin compared with vehicle. Finally, wortmannin treatment decreased sphingomyelin content and increased membrane fluidity in mAoECs. Taken together, these results suggest that constitutive phosphorylation of MLP1 attenuates the function of EnNaC in aortic endothelial cells by a mechanism involving a decrease in association with MLP1 and EnNaC at the membrane, whereas deletion of PIP2 decreases MLP1 expression and overall membrane fluidity.NEW & NOTEWORTHY In this study, we investigated the functional role of myristoylated alanine-rich C-kinase substrate-like protein 1 (MLP1) phosphorylation in regulating endothelial sodium channel (EnNaC) activity using mouse aortic endothelial cells for the first time. The results from this study will help elucidate the molecular mechanism by which aortic stiffness is regulated by EnNaC.