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

Measuring Local Policy to Advance Fair Housing and Climate Goals Through a Comprehensive Assessment of Land Use Entitlements

California’s legislature has passed several laws that intervene in local land-use regulation in order to increase desperately needed housing production—particularly affordable housing production. Some of these new laws expand local reporting requirements concerning zoning and planning laws, and the application of those laws apply to proposed housing development. This emphasis on measurement requires the state to develop a housing data strategy to support both enforcement of existing law and effective policymaking in the future. Our Comprehensive Assessment of Land Use Entitlements Study (CALES) predates, but aligns with and supports, this state-led effort to improve local reporting. For the cities that it covers, CALES provides verified and cleaned data indicating how cities apply local and state law. In this symposium contribution, we use the CALES data to illustrate the importance of collecting a range of precise objective data on how cities apply law, and then offer a simple but sufficiently comprehensive measurement of regulation that can help identify when a local land use regime may be operating to exclude affordable housing

Searching for Ground Truth: a stepping stone in automating genre classification

This paper examines genre classification of documents and its role in enabling the effective automated management of digital documents by digital libraries and other repositories. We have previously presented genre classification as a valuable step toward achieving automated extraction of descriptive metadata for digital material. Here, we present results from experiments using human labellers, conducted to assist in genre characterisation and the prediction of obstacles which need to be overcome by an automated system, and to contribute to the process of creating a solid testbed corpus for extending automated genre classification and testing metadata extraction tools across genres. We also describe the performance of two classifiers based on image and stylistic modeling features in labelling the data resulting from the agreement of three human labellers across fifteen genre classes.

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