Phosphate is an essential component of life and must be actively transported into cells against its electrochemical gradient. In vertebrates, two unrelated families of Na+-dependent Pitransporters carry out this task. Remarkably, the two families transport different Pispecies: whereas type II Na+/Picotransporters (SCL34) prefer divalent HPO42−, type III Na+/Picotransporters (SLC20) transport monovalent H2PO4−. The SCL34 family comprises both electrogenic and electroneutral members that are expressed in various epithelia and other polarized cells. Through regulated activity in apical membranes of the gut and kidney, they maintain body Pihomeostasis, and in salivary and mammary glands, liver, and testes they play a role in modulating the Picontent of luminal fluids. The two SLC20 family members PiT-1 and PiT-2 are electrogenic and ubiquitously expressed and may serve a housekeeping role for cell Pihomeostasis; however, also more specific roles are emerging for these transporters in, for example, bone mineralization. In this review, we focus on recent advances in the characterization of the transport kinetics, structure-function relationships, and physiological implications of having two distinct Na+/Picotransporter families.

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Over the past 25 years, successive cloning of SLC34A1, SLC34A2 and SLC34A3, which encode the sodium-dependent inorganic phosphate (P i ) cotransport proteins 2a-2c, has facilitated the identification of molecular mechanisms that underlie the regulation of renal and intestinal P i transport. P i and various hormones, including parathyroid hormone and phosphatonins, such as fibroblast growth factor 23, regulate the activity of these P i transporters through transcriptional, translational and post-translational mechanisms involving interactions with PDZ domain-containing proteins, lipid microdomains and acute trafficking of the transporters via endocytosis and exocytosis. In humans and rodents, mutations in any of the three transporters lead to dysregulation of epithelial P i transport with effects on serum P i levels and can cause cardiovascular and musculoskeletal damage, illustrating the importance of these transporters in the maintenance of local and systemic P i homeostasis. Functional and structural studies have provided insights into the mechanism by which these proteins transport P i , whereas in vivo and ex vivo cell culture studies have identified several small molecules that can modify their transport function. These small molecules represent potential new drugs to help maintain P i homeostasis in patients with chronic kidney disease -a condition that is associated with hyperphosphataemia and severe cardiovascular and skeletal consequences.

Polarized epithelial cells are responsible for the vectorial transport of solutes and have a key role in maintaining body fluid and electrolyte homeostasis. Such cells contain structurally and functionally distinct plasma membrane domains. Brush border and basolateral membranes of renal and intestinal epithelial cells can be separated using a number of different separation techniques, which allow their different transport functions and receptor expressions to be studied. In this communication, we report a proteomic analysis of these two membrane segments, apical and basolateral, obtained from the rat renal cortex isolated by two different methods: differential centrifugation and free-flow electrophoresis. The study was aimed at assessing the nature of the major proteins isolated by these two separation techniques. Two analytical strategies were used: separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at the protein level or by cation-exchange high-performance liquid chromatography (HPLC) after proteolysis (i.e., at the peptide level). Proteolytic peptides derived from the proteins present in gel pieces or from HPLC fractions after proteolysis were sequenced by on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several hundred proteins were identified in each membrane section. In addition to proteins known to be located at the apical and basolateral membranes, several novel proteins were also identified. In particular, a number of proteins with putative roles in signal transduction were identified in both membranes. To our knowledge, this is the first reported study to try and characterize the membrane proteome of polarized epithelial cells and to provide a data set of the most abundant proteins present in renal proximal tubule cell membranes.

In a previous report we documented an increased Na(+)-dependent transport of inorganic phosphate (P(i)) in Xenopus laevis oocytes injected with mRNA isolated from rabbit duodenum (Yagci et al., Pfluegers Arch. 422:211-216, 1992; ref 24). In the present study we have used expression cloning in oocytes to search for the cDNA/mRNA involved in this effect. The identified cDNA (provisionally named PiUS; for P(i)-uptake stimulator) lead to a 3-4-fold stimulation of Na(+)-dependent P(i)-uptake (10ng cRNA injected, 3-5 days of expression). Na(+)-independent uptake of P(i) was also affected but transport of sulphate and L-arginine (in the presence or absence of sodium) remained unchanged. The apparent K(m)-values for the induced Na(+)-dependent uptake were 0.26 +/- 0.04 mM for P(i) and 14.8 +/- 3.0 mM for Na+. The 1796 bp cDNA codes for a protein of 425 amino acids. Hydropathy analysis suggests a lack of transmembrane segments. In vitro translation resulted in a protein of 60 kDa and provided no evidence of glycosylation. In Northern blots a mRNA of approximately 2 kb was recognized in various tissues including different intestinal segments, kidney cortex, kidney medulla, liver and heart. Homology searches showed no similarity to proteins involved in membrane transport and its control. In conclusion, we have cloned from a rabbit small intestinal cDNA library a novel cDNA encoding a protein stimulating P(i)-uptake into Xenopus laevis oocytes, but which is not a P(i)-transporter itself.