Renal proximal tubular reabsorption of P(i) is a key element in overall P(i) homeostasis, and it involves a secondary active P(i) transport mechanism. Among the molecularly identified sodium-phosphate (Na/P(i)) cotransport systems a brush-border membrane type IIa Na-P(i) cotransporter is the key player in proximal tubular P(i) reabsorption. Physiological and pathophysiological alterations in renal P(i) reabsorption are related to altered brush-border membrane expression/content of the type IIa Na-P(i) cotransporter. Complex membrane retrieval/insertion mechanisms are involved in modulating transporter content in the brush-border membrane. In a tissue culture model (OK cells) expressing intrinsically the type IIa Na-P(i) cotransporter, the cellular cascades involved in "physiological/pathophysiological" control of P(i) reabsorption have been explored. As this cell model offers a "proximal tubular" environment, it is useful for characterization (in heterologous expression studies) of the cellular/molecular requirements for transport regulation. Finally, the oocyte expression system has permitted a thorough characterization of the transport characteristics and of structure/function relationships. Thus the cloning of the type IIa Na-P(i )cotransporter (in 1993) provided the tools to study renal brush-border membrane Na-P(i) cotransport function/regulation at the cellular/molecular level as well as at the organ level and led to an understanding of cellular mechanisms involved in control of proximal tubular P(i) handling and, thus, of overall P(i) homeostasis.

An isoform of the mammalian renal type II Na͞P i -cotransporter is described. Homology of this isoform to described mammalian and nonmammalian type II cotransporters is between 57 and 75%. Based on major diversities at the C terminus, the new isoform is designed as type IIb Na͞P i -cotransporter. Na͞P i -cotransport mediated by the type IIb cotransporter was studied in oocytes of Xenopus laevis. The results indicate that type IIb Na͞P i -cotransport is electrogenic and in contrast to the renal type II isoform of opposite pH dependence. Expression of type IIb mRNA was detected in various tissues, including small intestine. The type IIb protein was detected as a 108-kDa protein by Western blots using isolated small intestinal brush border membranes and by immunohistochemistry was localized at the luminal membrane of mouse enterocytes. Expression of the type IIb protein in the brush borders of enterocytes and transport characteristics suggest that the described type IIb Na͞P i -cotransporter represents a candidate for small intestinal apical Na͞P icotransport.

We have isolated two cDNA clones, NaPi-2 and NaPi-3, by screening rat kidney cortex and human kidney cortex cDNA libraries, respectively, for expression of sodiumdependent phosphate transport in Xenopus laevis oocytes.

The type IIa Na ؉ -dependent inorganic phosphate (Na/ P i ) cotransporter is localized in the apical membrane of proximal tubular cells and is regulated by an endocytotic pathway. Because molecular processes such as apical sorting, internalization, or subsequent degradation might be assisted by associated proteins, a yeast twohybrid screen against the C-terminal, cytosolic tail of type IIa cotransporter was designed. Most of the potential proteins found belonged to proteins with multiple PDZ modules and were either identical/related to PDZK1 or identical to NHERF-1. Yeast trap truncation assays confined the peptide-protein association to the C-terminal amino acid residues TRL of type IIa cotransporter and to single PDZ domains of each identified protein, respectively. The specificity of these interactions were confirmed in yeast by testing other apical localized transmembraneous proteins. Moreover, the type IIa protein was recovered in vitro by glutathione S-transferase-fused PDZ proteins from isolated renal brush border membranes or from type IIa-expressing oocytes. Further, these PDZ proteins are immunohistochemically detected either in the microvilli or in the subapical compartment of proximal tubular cells. Our results suggest that the type IIa Na/P i cotransporter interacts with various PDZ proteins that might be responsible for the apical sorting, parathyroid hormone controlled endocytosis or the lysosomal sorting of internalized type IIa cotransporter.In kidney, reabsorption of filtered inorganic phosphate (P i ) takes place along the proximal tubules and is controlled by a variety of hormones (e.g. parathyroid hormone, PTH) 1 and other factors (e.g. dietary intake of P i ) (1, 2). Three structurally unrelated sodium-dependent phosphate (Na/P i ) cotransporter families have been identified (1, 3). By immunohistochemistry, it was apparent that members of the type I and the type IIa Na/P i cotransporters are located in the apical membrane of proximal tubular cells (4, 5). Targeted inactivation of the type IIa Na/P i cotransporter gene (npt2) provided strong evidence that ϳ70% of Na-dependent P i transport across the brush border membrane is mediated by the type IIa Na/P i cotransporter (6). Furthermore, the type IIa cotransporter represents the major target for the many factors described to regulate proximal tubular P i reabsorption (2). Additionally, reduced proximal P i -reabsorption, as observed in X-linked hypophosphatemia, is due to a decreased abundance of the type IIa Na/P i cotransporter (7).According to the current mechanistic view, inhibition of proximal tubular Pi-reabsorption, such as by PTH or by a diet of high P i content (acutely given), is achieved by a removal of type IIa cotransporters (2) from the apical membrane. Results obtained from in vivo (rats) and in vitro (OK cells) studies indicated that internalized type IIa Na/P i cotransporters are subjected to degradation in the lysosomes (8, 9). Besides Na/P i cotransport, the activity of the brush border Na/H exchanger, NHE-3, is regulated ...

The voltage dependence of the rat renal type II Na+/Pi cotransporter (NaPi-2) was investigated by expressing NaPi-2 in Xenopus laevis oocytes and applying the two-electrode voltage clamp. In the steady state, superfusion with inorganic phosphate (Pi) induced inward currents (Ip) in the presence of 96 mM Na+ over the potential range −140 ≤ V ≤ +40 mV. With Pi as the variable substrate, the apparent affinity constant (K m Pi) was strongly dependent on Na+, increasing sixfold for a twofold reduction in external Na+. K m Pi increased with depolarizing voltage and was more sensitive to voltage at reduced Na+. The Hill coefficient was close to unity and the predicted maximum Ip (Ipmax) was 40% smaller at 50 mM Na+. With Na+ as the variable substrate, K m Na was weakly dependent on both Pi and voltage, the Hill coefficient was close to 3 and Ipmax was independent of Pi at −50 mV. The competitive inhibitor phosphonoformic acid suppressed the steady state holding current in a Na+-dependent manner, indicating the existence of uncoupled Na+ slippage. Voltage steps induced pre–steady state relaxations typical for Na+-coupled cotransporters. NaPi-2-dependent relaxations were quantitated by a single, voltage-dependent exponential. At 96 mM Na+, a Boltzmann function was fit to the steady state charge distribution (Q-V) to give a midpoint voltage (V0.5) in the range −20 to −50 mV and an apparent valency of ∼0.5 e−. V0.5 became more negative as Na+ was reduced. Pi suppressed relaxations in a dose-dependent manner, but had little effect on their voltage dependence. Reducing external pH shifted V0.5 to depolarizing potentials and suppressed relaxations in the absence of Na+, suggesting that protons interact with the unloaded carrier. These findings were incorporated into an ordered kinetic model whereby Na+ is the first and last substrate to bind, and the observed voltage dependence arises from the unloaded carrier and first Na+ binding step.