Expression of the Na/Pi-cotransporter Type IIb in Sf9 Cells: Functional Characterization and Purification

In mammals the type IIb Na/Pi-cotransporter is expressed in various tissues such as intestine, brain, lung and testis. The type IIb cotransporter shows 51% homology with the renal type IIa Na/Pi-cotransporter, for which a detailed model of the secondary structure has emerged based on recent structure/function studies. To make the type IIb Na/Pi-cotransporter available for future structural studies, we have expressed this cotransporter in Sf9 cells. Sf9 cells were infected with recombinant baculovirus containing 6His NaPi-IIb. Infected cells expressed a polypeptide of ~90 kDa, corresponding to a partially glycosylated form of the type IIb cotransporter. Transport studies demonstrated that the type IIb protein expressed in Sf9 cells mediates transport of phosphate in a Na-dependent manner with similar kinetic characteristics (apparent K ms for sodium and phosphate and pH dependence) as previously described. Solubilization experiments demonstrated that, in contrast to the type IIa cotransporter, the type IIb can be solubilized by nonionic detergents and that solubilized type IIb Na/Pi-cotransporter can be purified by Ni-NTA chromatograph

What goes in must come out - the small intestine modulates renal phosphate excretion

In a recent article in PNAS, Berndt et al. describe a novel and rapid regulation of renal phosphate excretion by phosphate instilled into the small intestine [1]. In a series of elegant experiments, renal phosphate clearance was measured before and during the infusion of a small amount of phosphate into the distal duodenum of rats. Twenty minutes after the infusion, massive phosphaturia was observed. This effect was specific for phosphate and was not seen when phosphate was instilled into the stomach or when NaCl was applied. Phosphaturia occurred without a measurable increase in serum phosphate an

Regulation of phosphate transport in proximal tubules

Homeostasis of inorganic phosphate (Pi) is primarily an affair of the kidneys. Reabsorption of the bulk of filtered Pi occurs along the renal proximal tubule and is initiated by apically localized Na+-dependent Pi cotransporters. Tubular Pi reabsorption and therefore renal excretion of Pi is controlled by a number of hormones, including phosphatonins, and metabolic factors. In most cases, regulation of Pi reabsorption is achieved by changing the apical abundance of Na+/Pi cotransporters. The regulatory mechanisms involve various signaling pathways and a number of proteins that interact with Na+/Pi cotransporter

3D modeling of indoor environments by a mobile robot with a laser scanner and panoramic camera

We present a method to acquire a realistic, visually convincing 3D model of indoor office environments based on a mobile robot that is equipped with a laser range scanner and a panoramic camera. The data of the 2D laser scans are used to solve the SLAM problem and to extract walls. Textures for walls and floor are built from the images of a calibrated panoramic camera. Multi-resolution blending is used to hide seams in the generated textures

Functionally Important Residues in the Predicted 3rd Transmembrane Domain of the Type IIa Sodium-phosphate Cotransporter (NaPi-IIa)

The type IIa Na+/Pi, cotransporter (NaPi-IIa) mediates electrogenic transport of three Na+ and one divalent Pi ion (and one net positive charge) across the cell membrane. Sequence comparison of electrogenic NaPi-IIa and IIb isoforms with the electroneutral NaPi-IIc isoform pointed to the third transmembrane domain (TMD-3) as a possibly significant determinant of substrate binding. To elucidate the role of TMD-3 in the topology and mechanism underlying NaPi-IIa function we subjected it to cysteine scanning mutagenesis. The constructs were expressed in Xenopus oocytes and Pi transport kinetics were assayed by electrophysiology and radiotracer uptake. Cys substitution resulted in only marginally altered kinetics of Pi transport in those mutants providing sufficient current for analysis. Only one site, at the extracellular end of TMD-3, appeared to be accessible to methanethiosulfonate reagents. However, additional mutations carried out at D224 (replaced by E, G or N) and N227 (replaced by D or Q) resulted in markedly altered voltage and substrate dependencies of the Pi-dependent currents. Replacing Asp-224 (highly conserved in electrogenic a and b isoforms) with Gly (the residue found in the electroneutral c isoform) resulted in a mutant that mediated electroneutral Na+-dependent Pi transport. Since electrogenic NaPi-II transports 3 Na+/transport cycle, whereas electroneutral NaPi-IIc only transports 2, we speculate that this loss of electrogenicity might result from the loss of one of the three Na+ binding sites in NaPi-II

Segment-specific expression of sodium-phosphate cotransporters NaPi-IIa and -IIc and interacting proteins in mouse renal proximal tubules

Sodium-dependent phosphate cotransport in renal proximal tubules (PTs) is heterogeneous with respect to proximal tubular segmentation (S1 vs. S3) and nephron generation (superficial vs. juxtamedullary). In the present study, S1 and S3 segments of superficial and juxtamedullary nephrons were laser-microdissected and mRNA and protein expression of the Na/Pi-cotransporters NaPi-IIa and NaPi-IIc and the PDZ proteins NHERF-1 and PDZK1 determined. Expression of NaPi-IIa mRNA decreased axially in juxtamedullary nephrons. There was no effect of dietary Pi content on NaPi-lla mRNA expression in any proximal tubular segment. The abundance of the NaPi-IIa cotransporter in the brush-border membrane showed inter- and intranephron heterogeneity and increased in response to a low-Pi diet (5days), suggesting that up-regulation of NaPi-lla occurs via post-transcriptional mechanisms. In contrast, NaPi-IIc mRNA and protein was up-regulated by the low-Pi diet in all nephron generations analysed. NHERF-1 and PDZK1, at both mRNA and protein levels, were distributed evenly along the PTs and did not change after a low-Pi die

Expression of renal and intestinal Na/Pi cotransporters in the absence of GABARAP

We have recently shown that the abundance of the renal sodium (Na)/inorganic phosphate (Pi) cotransporter NaPi-IIa is increased in the absence of the GABA(A) receptor-associated protein (GABARAP). Accordingly, GABARAP-deficient mice have a reduced urinary excretion of Pi. However, their circulating levels of Pi do not differ from wild-type animals, suggesting the presence of a compensatory mechanism responsible for keeping serum Pi values constant. Here, we aimed first to identify the molecular basis of this compensation by analyzing the expression of Na/Pi cotransporters known to be expressed in the kidney and intestine. We found that, in the kidney, the upregulation of NaPi-IIa is not accompanied by changes on the expression of either NaPi-IIc or PiT2, the other cotransporters known to participate in renal Pi reabsorption. In contrast, the intestinal expression of NaPi-IIb is downregulated in mutant animals, suggesting that a reduced intestinal absorption of Pi could contribute to maintain a normophosphatemic status despite the increased renal retention. The second goal of this work was to study whether the alterations on the expression of NaPi-IIa induced by chronic dietary Pi are impaired in the absence of GABARAP. Our data indicate that, in response to high Pi diets, GABARAP-deficient mice downregulate the expression of NaPi-IIa to levels comparable to those seen in wild-type animals. However, in response to low Pi diets, the upregulation of NaPi-IIa is greater in the mutant mice. Thus, both the basal expression and the dietary-induced upregulation of NaPi-IIa are increased in the absence of GABARAP