Abstract
In renal proximal tubule (PT) cells, sodium-phosphate cotransporter IIa (NaPiIIa) is normally concentrated within the apical membrane where it reabsorbs ∼70% of luminal phosphate (Pi). NaPiIIa activity is acutely regulated by moderating its abundance within the apical membrane. Under low-Pi conditions, NaPiIIa is retained within the apical membrane. Under high-Pi conditions, NaPiIIa is retrieved from the apical membrane and trafficked to the lysosomes for degradation. The present study investigates the role of Shank2 in regulating the distribution of NaPiIIa. In opossum kidney cells, a PT cell model, knockdown of Shank2 in cells maintained in low-Pi media resulted in a marked decrease in NaPiIIa abundance. After being transferred into high-Pi media, live-cell imaging showed that mRFP-Shank2E and GFP-NaPiIIa underwent endocytosis and trafficked together through the subapical domain. Fluorescence cross-correlation spectroscopy demonstrated that GFP-NaPiIIa and mRFP-Shank2 have indistinguishable diffusion coefficients and migrated through the subapical domain in temporal synchrony. Raster image cross-correlation spectroscopy demonstrated these two proteins course through the subapical domain in temporal-spatial synchrony. In the microvilli of cells under low-Pi conditions and in the subapical domain of cells under high-Pi conditions, fluorescence lifetime imaging microscopy-Forster resonance energy transfer analysis of Cer-NaPiIIa and EYFP-Shank2E found these fluors reside within 10 nm of each other. Demonstrating a complexity of functions, in cells maintained under low-Pi conditions, Shank2 plays an essential role in the apical retention of NaPiIIa while under high-Pi conditions Shank2 remains associated with NaPiIIa and escorts NaPiIIa through the cell interior.
Highlights
SiRNA; live-cell imaging; fluorescence lifetime imaging microscopyForster resonance energy transfer; endocytosis; fluorescence correlation spectroscopy; raster image correlation spectroscopy
In intact rat renal proximal tubule (PT) cells and cultured opossum kidney (OK) cells, low-Pi conditions in the serum or culture medium induce NaPiIIa and Shank2 to concentrate within the apical microvilli with little of either protein found in the cell interior [12, 28]
To determine if Shank2 contributed to the microvillar retention of NaPiIIa, the expression levels of Shank2 were either knocked down by transfecting Shank2 siRNAs or were elevated by transfecting Flag-Shank2 cDNA into OK cells. qPCR analyses showed that compared with cells that were untransfected or transfected with scrambled siRNAs, OK cells receiving Shank2 siRNAs had significantly lower Shank2 mRNA levels (Fig. 1A; n ϭ 3)
Summary
SiRNA; live-cell imaging; fluorescence lifetime imaging microscopyForster resonance energy transfer; endocytosis; fluorescence correlation spectroscopy; raster image correlation spectroscopy. Under low serum Pi/PTH conditions, NaPiIIa activity is increased primarily by increasing the abundance of NaPiIIa within the apical membrane [12, 14]. The COOH-terminal tail of NaPiIIa interacts with at least four specific PDZ domain-containing proteins [18, 20, 28], including EBP50 (a.k.a. NHERF-1), E3KARP (a.k.a. NHERF2), PDZK1 (a.k.a. NHERF-3), and Shank (a.k.a. ProSAP1; CortBP1). These PDZ domain proteins are positioned to moderate the distribution of NaPiIIa within PT cells and impact serum Pi homeostasis. Foreshadowing its importance within epithelial cells, the PDZ domain of Shank binds to key transporter and ion channel proteins, including the cystic fibrosis transmembrane conductance regulator (CFTR) ClϪ channel, the sodium-proton exchanger 3 (NHE3), and NaPiIIa [19, 23, 28]. Rat renal PT cells maintained under low serum Pi conditions have both Shank http://www.ajpcell.org
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
