Na-Pi cotransport in flounder: same transport system in kidney and intestine

Abstract

The cloning of a renal Na-Pi contransport in system from winter flounder (P eudopleuronectes americanus) has recently been reported. We used this information to answer the questions 1) what is the distribution of the transport protein along the nephron? and 2) how are renal and intestinal transporters related? The distribution of the flounder NaPi-II protein was tested using two antisera raised against partial sequences (amino acids 1-14 and 388-441) of the transporter. Antibody-specific fluorescence was detected at the basolateral membrane of epithelial cells in the proximal tubular segment PII. Two clones corresponding to the renal Na-Pi cotransporter were isolated from a flounder intestinal cDNA library. Their functional properties were determined using Xenopus laevis oocytes. The apparent affinities for Pi [Michaelis constant (K(m)) = 0.063 mM] and Na (K(m) = 45.3 mM), as well as the pH dependency (increasing transport activity with increasing pH), showed the same characteristics in both intestinal and the renal systems. Sequence analysis revealed that the two intestinal clones were 100% homologous to the renal cDNA, Flounder NaPi-II-specific immunofluorescence was observed predominantly at the apical membrane on intestinal cross sections. We report the cloning and expression of the first intestinal Na-Pi cotransport system. This transporter belongs to the small group of proteins that exhibit the same function in the apical and the basolateral membranes of different cells.

Contributions of different NaPi cotransporter isoforms to dietary regulation of P transport in the pyloric caeca and intestine of rainbow trout

“…It is different from that of the mammalian intestinal NaPi cotransporter isoform, which is also Na + -dependent but has higher transport rates at neutral to acidic pH (Berner et al, 1976;Borowitz and Ghishan, 1989;Danisi et al, 1984;Lee et al, 1986;Tenenhouse, 1999;Xu et al, 2002). In chick, pig and sheep intestine, however, Pi transport rate is higher at alkaline pH than acidic pH (Danisi and Murer, 1991), which is in agreement with our data in trout PC and also with data from the intestinal and renal NaPi cotransporter isoforms of zebrafish and flounder (Forster et al, 1997;Graham et al, 2003;Kohl et al, 1996;Nalbant et al, 1999). The amino acid motifs that apparently confer the pH dependency in the NaPi-II intestinal and renal transporter isoforms of mammals (de la Horra et al, 2000) are different from the corresponding sequence in trout PC and intestinal NaPi cotransporter and in zebrafish NaPi cotransporter isoform found in both kidney and intestine.…”

Phosphate Transport Kinetics and Structure-Function Relationships of SLC34 and SLC20 Proteins

“…Its mammalian cousin (SLC34A2 or NaPi-IIb) was then cloned using a mouse 7 embryonic EST clone (Hilfiker, Hattenhauer et al 1998) and localized to the brush border membrane of enterocytes and a number of other organs and tissues, including lung, colon, testes, salivary gland, thyroid gland mammary gland, uterus and liver (Hilfiker, Hattenhauer et al 1998;Frei, Gao et al 2005; Nishimura&Naito 2008). In contrast to the flounder NaPiIIb isoform, which is expressed in both kidney and intestine (Kohl, Herter et al 1996), mammalian NaPi-IIb does not appear to be expressed at the protein level in the kidney. At the primary amino acid sequence level, NaPi-IIb isoforms are distinguished from the renal NaPiIIa by a unique clustering of cysteine residues in their C-terminal region.…”

“…Following the cloning of several NaPi-IIa isoforms, a Na + -P i cotransporter from the kidney and intestine of winter flounder was identified (Werner, Murer et al 1994;Kohl, Herter et al 1996). Its mammalian cousin (SLC34A2 or NaPi-IIb) was then cloned using a mouse 7 embryonic EST clone (Hilfiker, Hattenhauer et al 1998) and localized to the brush border membrane of enterocytes and a number of other organs and tissues, including lung, colon, testes, salivary gland, thyroid gland mammary gland, uterus and liver (Hilfiker, Hattenhauer et al 1998;Frei, Gao et al 2005; Nishimura&Naito 2008).…”

Inhibition of Na+-Dependent Transporters in Cystine-Loaded Human Renal Cells

“…Cultured IHKE-1 cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 1 h. For immunogold scanning electron microscopy, a rabbit polyclonal antibody directed against the NaP i transporter of flounder kidney was employed (28). This antibody is directed against a portion of the protein that shows an 84% identity between the flounder and human NaP i transporter and covers mostly the extracellular loop of the protein.…”