Role of Na⁺/H⁺exchanger regulatory factor 1 in forward trafficking of the type IIa Na⁺-P_icotransporter

Abstract

Ketchem CJ, Khundmiri SJ, Gaweda AE, Murray R, Clark BJ, Weinman EJ, Lederer ED. Role of Na ϩ /H ϩ exchanger regulatory factor 1 in forward trafficking of the type IIa Na ϩ -Pi cotransporter. Am J Physiol Renal Physiol 309: F109 -F119, 2015. First published May 20, 2015 doi:10.1152 doi:10. /ajprenal.00133.2015 ϩ /H ϩ exchanger regulatory factor (NHERF1) plays a critical role in the renal transport of phosphate by binding to Na ϩ -Pi cotransporter (NpT2a) in the proximal tubule. While the association between NpT2a and NHERF1 in the apical membrane is known, the role of NHERF1 to regulate the trafficking of NpT2a has not been studied. To address this question, we performed cell fractionation by sucrose gradient centrifugation in opossum kidney (OK) cells placed in low-P i medium to stimulate forward trafficking of NpT2a. Immunoblot analysis demonstrated expression of NpT2a and NHERF1 in the endoplasmic reticulum (ER)/Golgi. Coimmunoprecipitation demonstrated a NpT2a-NHERF1 interaction in the ER/Golgi. Low-P i medium for 4 and 8 h triggered a decrease in NHERF1 in the plasma membrane with a corresponding increase in the ER/Golgi. Time-lapse total internal reflection fluorescence imaging of OK cells placed in low-P i medium, paired with particle tracking and mean square displacement analysis, indicated active directed movement of NHERF1 at early and late time points, whereas NpT2a showed active movement only at later times. Silence of NHERF1 in OK cells expressing green fluorescent protein (GFP)-NpT2a resulted in an intracellular accumulation of GFPNpT2a. Transfection with GFP-labeled COOH-terminal (TRL) PDZbinding motif deleted or wild-type NpT2a in OK cells followed by cell fractionation and immunoprecipitation confirmed that the interaction between NpT2a and NHERF1 was dependent on the TRL motif of NpT2a. We conclude that appropriate trafficking of NpT2a to the plasma membrane is dependent on the initial association between NpT2a and NHERF1 through the COOH-terminal TRL motif of NpT2a in the ER/Golgi and requires redistribution of NHERF1 to the ER/Golgi. endoplasmic reticulum/Golgi; Na ϩ /H ϩ exchanger regulatory factor 1; sodium phosphate cotransporter 2a; plasma membrane; trafficking DESPITE THE IMPORTANCE of Na ϩ -P i cotransporter (NpT2a) in the regulation of total body phosphate homeostasis, our understanding of the factors regulating the apical membrane expression of this transporter remains incomplete. Major physiological regulators of NpT2a expression include parathyroid hormone (PTH), dopamine, fibroblast growth factor-23, and high-phosphate diet, which decrease apical membrane expression by stimulation of endocytosis and degradation of NpT2a as well as decreased mRNA expression (40). A low-phosphate diet has the opposite effect, stimulating insertion of NpT2a into the apical membrane and inhibiting endocytosis (22, 31). Thus, the regulation of the protein is dynamic and responsive to multiple physiological regulatory mechanisms. NpT2a is an intrinsic membrane protein for which hydropathy analysis predi...

Low dose ouabain stimulates Na K ATPase α1 subunit association with angiotensin II type 1 receptor in renal proximal tubule cells

“…HKC11 cells were transfected with the indicated plasmids by electroporation using Neon Transfection System (Invitrogen) in OPTI-MEM following manufacturer’s instruction for rat proximal tubular cells and as described previously [24]. …”

“…FRET imaging experiments were performed in living cells exactly as described previously [3,24]. Briefly, HKC11 cells were transfected with either mCherry-NKA or mCherry-AT1R (acceptor) for 24 h followed by 15 min treatment with Bodipy-ouabain (donor).…”

“…Cells were viewed and analyzed with an Olympus FRET/TIRF microscope (Center Valley, PA). FRET image acquisition and analysis was performed using SlideBook software version 4.2 as described previously [3,24]. The software is based on the three-filter “micro-FRET” image subtraction method described by Jiang and Sorkin [25].…”

“…Membrane TIRF was performed as described previously [3,24]. Briefly, HKC11 cells were grown to 60% confluence in a dish with a collagen coated coverslip bottom (no.…”

Protein-DNA Interactions at the Opossum Npt2a Promoter are Dependent upon NHERF-1

“…Npt2a is localized in the proximal tubule brush border membrane (BBM) and Npt2a protein level in the BBM is regulated by dietary phosphate and parathyroid hormone (PTH). Our group and others have established a critical role for Na+/H+ exchanger regulatory factor 1 (NHERF-1) in the expression and trafficking of Npt2a protein to the brush border membrane [1][2][3][4]. We have also shown the loss of NHERF-1, either by siRNA silencing in OK cells or using the NHERF-1 deficient OK-H clonal line, resulted in loss (95% reduction) of Npt2a mRNA expression and Npt2a protein expression with significant decrease in functional phosphate uptake.…”