Parathyroid hormone regulation of Na⁺,K⁺-ATPase requires the PDZ 1 domain of sodium hydrogen exchanger regulatory factor-1 in opossum kidney cells

It was demonstrated that expression of murine sodium hydrogen exchanger regulatory factor (NHERF-1) lacking the ezrin-binding domain blocks parathyroid hormone (PTH) regulation of Na⁺,K⁺-ATPase in opossum kidney (OK) cells. The hypothesis that the NHERF-1 PDZ domains contribute to PTH regulation of Na⁺,K⁺-ATPase was tested by comparison of PTH regulation of Na⁺,K⁺-ATPase in wild-type OK (OK-WT) cells, NHERF-deficient OKH cells, OK-WT transfected with siRNA for NHERF (NHERF siRNA OK-WT), and OKH cells that were stably transfected with full-length NHERF-1 or constructs with mutated PDZ domains. OKH cells and NHERF siRNA OK-WT showed decreased expression of NHERF-1 but equivalent expression of ezrin and Na⁺,K⁺-ATPase α₁ subunit when compared with OK-WT cells. PTH decreased Na⁺,K⁺-ATPase activity and stimulated phosphorylation of the Na₊,K⁺-ATPase α₁ in OK-WT cells but not in NHERF-deficient cells. Rubidium (⁸⁶Rb) uptake was equivalent in OK-WT, OKH, and OKH cells that were transfected with all but the double PDZ domain mutants. PTH decreased ⁸⁶Rb uptake significantly in OK-WT but not in OKH cells. PTH also significantly inhibited ⁸⁶Rb uptake in OKH cells that were transfected with full-length NHERF-1 or NHERF-1 with mutated PDZ 2 but not in OKH cells that were transfected with mutated PDZ 1. Transfection with NHERF expressing both mutated PDZ domains resulted in diminished basal ⁸⁶Rb uptake that was not inhibited further by PTH. PTH stimulated protein kinase Cα activity and α₁ subunit phosphorylation in OK-WT but not in NHERF-deficient cells. Transfection of OKH cells with NHERF constructs that contained an intact PDZ1 domain restored PTH-stimulated protein kinase Cα activity and α₁ subunit phosphorylation. These results demonstrate that NHERF-1 is necessary for PTH-mediated inhibition of Na ⁺,K⁺-ATPase activity and that the inhibition is mediated through the PDZ1, not PDZ2, domain.