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NHERF-1 Modulates Intestinal NaPi transporter NaPi-2b expression in Apical Microvilli.

2011; American Society for Cell Biology; Volume: 22; Linguagem: Inglês

1939-4586

Héctor Giral, Yupanqui Caldas, Luca Lanzanò, Enrico Gratton, Moshe Levi,

Magnesium in Health and Disease

NHERF- 1 Modulates Intestinal NaPi transporter NaPi-2b expression in Apical Microvilli. H. Giral 1 , Y. Caldas 1 , L. Lanzano 2 , E. Gratton 2 , M. Levi 1 ; 1 University of Colorado Denver, Aurora, CO, 2 University of California Irvine, CA The regulation of phosphate (Pi) homeostasis is maintained by the coordinated function of the renal and intestinal phosphate transporters. Several PDZ (PSD-95/discs large/ZO-1 homologous) domain proteins, including NHERF‐1, PDZK1, ShanK2, and PIST play an important role in the regulation of the renal sodium‐phosphate (NaPi) co‐transporters (NaPi‐2a and NaPi‐2c). The main mediator of intestinal sodium dependent transcellular Pi transport, NaPi‐2b, also contains a PDZ‐binding motif consensus in the C‐terminal region. However, interactions of the transporter NaPi‐2b with PDZ proteins have been not described and their potential role in regulation of the intestinal transporter is not known. For this purpose we performed studies with knock‐out (KO) mice models and cell culture to determine a potential role for NHERF‐1 and PDZK1 in the regulation of NaPi‐2b. To study the putative interaction between NaPi‐2b and PDZ proteins we determined the Forster Resonance Energy Transference (FRET) by using Fluorescence Lifetime Imaging Microscopy (FLIM). OK cells, an extensively used proximal tubule model, and CaCo‐2 BBE cells, an enterocyte cell model, were used to perform this technique. First, expression of EGFP‐NaPi‐2b was confirmed in the microvilli of both cell types, and images along a single microvillus were obtained with the novel Modulation Tracking (MT) method. Cells co‐expressing EGFP‐NaPi‐2b and mCherry‐NHERF‐1 were analyzed by FLIM‐FRET technique revealing significant FRET between NaPi‐2b and NHERF‐1. Parallel studies between the pair NaPi‐2b and PDZK1 proteins resulted in non occurrence of FRET. To evaluate the functional significance of these results we study NaPi‐2b expression and activity in NHERF1 KO and PDZK1 KO mice models, where we found that adaptation to a low Pi diet of NaPi2b was markedly impaired in the NHERF‐1 KO mice but not in the PDZK1 KO. Our results therefore suggest an important role of NHERF1 in modulation of NaPi‐2b expression or stability in the microvilli of the mouse intestine. This research was supported by NIH R01 DK066029 to YC, HG, ML, EG and LL; NIH445 P41R03155 to EG and LL; and the R01 DK-080769 to BD.