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bioRxiv · 10.1101/2024.06.25.600595

Activity and function of the endothelial sodium channel is regulated by the effector domain of MARCKS like protein 1 in mouse aortic endothelial cells

Yu, L.·Bala, N.·Nguyen, V.-A. L.·Kessler, L.·LaDisa, J. F.·Alli, A. A.
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Abstract

The endothelial sodium channel (EnNaC) plays an important role in regulating vessel stiffness. Here, we investigated the regulation of EnNaC in mouse aortic endothelial cells (mAoEC) 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 mAoEC. mAoEC 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 to cells transfected with wildtype MLP1. Compared to vehicle treatment, mAoEC 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 caveloin-1 protein expression in mAoEC treated with wortmannin compared to vehicle. Finally, wortmannin treatment decreased sphingomyelin content and increased membrane fluidity in mAoEC. Taken together, our results suggest 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, while deletion of PIP2 decreases MARCKS expression and overall membrane fluidity.

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Yu, L., Bala, N., Nguyen, V.-A. L., Kessler, L., LaDisa, J. F., Alli, A. A.. 2024-06-26. Activity and function of the endothelial sodium channel is regulated by the effector domain of MARCKS like protein 1 in mouse aortic endothelial cells. https://doi.org/10.1101/2024.06.25.600595

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