Citation

  • Authors: Sluysmans S. et al.
  • Year: 2022
  • Journal: J Biol Chem 14 102138
  • Applications: in vitro / DNA / jetOPTIMUS
  • Cell type: mCCD

Method

For transfection of mCCD cells, cells at 60-80% confluence were transfected one day after seeding, using jetOPTIMUS (Polyplus) following the manufacturer’s guidelines, and processed for IF 72 hr later.

Abstract

The Plasma Membrane Calcium ATPase (PMCA) extrudes calcium from the cytosol to the extracellular space to terminate calcium-dependent signaling. Although the distribution of PMCA is crucial for its function in calcium homeostasis and signaling, the molecular mechanisms that regulate the localization of PMCA isoforms are not well understood. PLEKHA7 is a junctional protein implicated by genetic studies in hypertension and the regulation of calcium handling. PLEKHA7 recruits the small adaptor protein PDZD11 to adherens junctions, and together they control the trafficking and localization of plasma membrane-associated proteins, including the Menkes copper ATPase. Since PDZD11 binds to the C-terminal domain of b-isoforms of PMCA, PDZD11 and its interactor PLEKHA7 could control the localization and activity of PMCA. Here we test this hypothesis using cultured cell model systems. We showed using immunofluorescence microscopy and a surface biotinylation assay show that KO of either PLEKHA7 or PDZD11 in mouse kidney collecting duct (mCCD) epithelial cells resulted in increased accumulation of endogenous PMCA at lateral cell-cell contacts and PDZ-dependent ectopic apical localization of exogenous PMCA4x/b isoform. In HeLa cells, co-expression of PDZD11 reduced membrane accumulation of overexpressed PMCAx/b and analysis of cytosolic calcium transients showed an effect of PDZD11 on the amplitude of the calcium peaks induced by the expression of PMCA4x/b, but not PMCA4x/a, which lacks the PDZ-binding motif. Moreover, KO of PDZD11 in either endothelial (bEnd.3) or epithelial (mCCD) cells increased the rate of calcium extrusion. Collectively, these results suggest that the PLEKHA7-PDZD11 complex modulates calcium homeostasis by regulating the localization of PMCA.

Pubmed