Search

In dietary potassium deficiency there is a decrease in the transport activity of the type IIa sodium/phosphate cotransporter protein (NaPi) despite an increase in its apical membrane abundance. This novel posttranslational regulation of NaPi activity is mediated by the increased glycosphingolipid content of the potassium-deficient apical membrane. However, the mechanisms by which these lipids modulate NaPi activity have not been determined. We determined if in potassium deficiency NaPi is increasingly partitioned in cholesterol-, sphingomyelin-, and glycosphingolipid-enriched microdomains of the apical membrane and if the increased presence of NaPi in these microdomains modulates its activity. By using a detergent-free density gradient flotation technique, we found that 80% of the apical membrane NaPi partitions into the low density cholesterol-, sphingomyelin-, and GM1-enriched fractions characterized as "lipid raft" fractions. In potassium deficiency, a higher proportion of NaPi was localized in the lipid raft fractions. By combining fluorescence correlation spectroscopy and photon counting histogram methods for control and potassium-deficient apical membranes reconstituted into giant unilamellar vesicles, we showed a 2-fold decrease in lateral diffusion of NaPi protein and a greater than 2-fold increase in size of protein aggregates/clusters in potassium deficiency. Our results indicate that NaPi protein is localized in membrane microdomains, that in potassium deficiency a larger proportion of NaPi protein is present in these microdomains, and that NaPi lateral diffusion is slowed down and NaPi aggregation/clustering is increased in potassium deficiency, both of which could be associated with the decreased Na/Pi cotransport activity in potassium deficiency.

The main nonhormonal mechanism for controlling inorganic phosphate ([P.sub.i]) homeostasis is renal adaptation of the proximal tubular [P.sub.i] transport rate to changes in dietary phosphate content. Opossum kidney (OK) cell line is an in vitro renal model that maintains the ability of renal adaptation to the extracellular [P.sub.i] concentration. We have studied how two competitive inhibitors of [P.sub.i] transport, arsenate [As(V)] and phosphonoformate (PFA), affect adaptation to low and high [P.sub.i] concentrations. OK cells show very high affinity for As(V) (inhibitory constant, [K.sub.i] 0.12 mM) when compared with the rat kidney. As(V) very efficiently reversed the adaptation of OK cells to low [P.sub.i] (0.1 mM), whereas PFA induced adaptation similar to 0.1 mM [P.sub.i]. Adaptation with 2 mM [P.sub.i] or As(V) was characterized by decreases in the maximal velociy ([V.sub.max]) of [P.sub.i] transport and an abundance of the NaPi-IIa [P.sub.i] transporter in the plasma membrane, shown by the protein biotinylation. Conversely, PFA and 0.1 mM [P.sub.i] increased the [V.sub.max] and transporter abundance. Changes in the [V.sub.max] were limited to a 50% variation, which was not paralleled by changes in the concentration of [P.sub.i] or of the inhibitor. OK cells are very sensitive to As(V), but the effects are reversible and noncytotoxic. These effects can be interpreted as As(V) being transported into the cell, thereby mimicking a high [P.sub.i] concentration. PFA blocks the uptake of [P.sub.i] but is not transported, and it therefore simulates a low [P.sub.i] concentration inside the cell. To conclude, a mathematical definition of the adaptation process is reported, thereby explaining the limited changes in [P.sub.i] transport [V.sub.max]. phosphate transport; inorganic phosphate deprivation; NaPi-IIa; renal inorganic phosphate adaptation; inorganic phosphate reabsorption

The principal mediators of renal phosphate ([P.sub.i]) reabsorption are the SLC34 family proteins NaPi-IIa and NaPi-IIc, localized to the proximal tubule (PT) apical membrane. Their abundance is regulated by circulatory factors and dietary [P.sub.i]. Although their physiological importance has been confirmed in knockout animal studies, significant [P.sub.i] reabsorptive capacity remains, which suggests the involvement of other secondary-active [P.sub.i] transporters along the nephron. Here we show that a member of the SLC20 gene family (PIT-2) is localized to the brush-border membrane (BBM) of the PT epithelia and that its abundance, confirmed by Western blot and immunohistochemistry of rat kidney slices, is regulated by dietary [P.sub.i]. In rats treated chronically on a high-[P.sub.i] (1.2%) diet, there was a marked decrease in the apparent abundance of PIT-2 protein in kidney slices compared with those from rats kept on a chronic low-[P.sub.i] (0.1%) diet. In Western blots of BBM from rats that were switched from a chronic low- to high-[P.sub.i] diet, NaPi-IIa showed rapid downregulation after 2 h; PIT-2 was also significantly downregulated at 24 h and NaPi-IIc after 48 h. For the converse dietary regime, NaPi-IIa showed adaptation within 8 h, whereas PIT-2 and NaPi-IIc showed a slower adaptive trend. Our findings suggest that PIT-2, until now considered as a ubiquitously expressed [P.sub.i] housekeeping transporter, is a novel mediator of [P.sub.i] reabsorption in the PT under conditions of acute [P.sub.i] deprivation, but with a different adaptive time course from NaPi-IIa and NaPi-IIc. brush-border membrane; inorganic phosphate; sodium-dependent transport

The function of the NaPiIIa renal sodium-phosphate transporter is regulated through a complex network of interacting proteins. Several PDZ domain-containing proteins interact with its COOH terminus while the small membrane protein MAP17 interacts with its N[H.sub.2] end. To elucidate the function of MAP17, we identified its interacting proteins using both bacterial and mammalian two-hybrid systems. Several PDZ domain-containing proteins, including the four NHERF proteins, as well as NaPiIIa and NHE3, were found to bind to MAP17. The interactions of MAP17 with the NHERF proteins and with NaPiIIa were further analyzed in opossum kidney (OK) cells. Expression of MAP 17 alone had no effect on the NaPiIIa apical membrane distribution, but coexpression of MAP 17 and NHERF3 or NHERF4 induced internalization of NaPiIIa, MAP17, and the PDZ protein to the trans-Golgi network (TGN). This effect was not observed when MAP17 was cotransfected with NHERF1/2 proteins. Inhibition of protein kinase C (PKC) prevented expression of the three proteins in the TGN. Activation of PKC in OK cells transfected only with MAP17 induced complete degradation of MAP17 and NaPiIIa. When lysosomal degradation was prevented, both proteins accumulated in the TGN. When the dopamine D1-like receptor was activated with fenoldopam, both NaPiIIa and MAP17 also accumulated in the TGN. Finally, cotransfection of MAPI7 and NHERF3 prevented the adaptive upregulation of phosphate transport activity in OK cells in response to low extracellular phosphate. Therefore, the interaction between MAP17, NHERF3/4, and NaPiIIa in the TGN could be an important intermediate or alternate path in the internalization of NaPiIIa. phosphate transport; PDZ; PDZK1; protein interaction; opossum kidney cells

We previously showed an inverse correlation between membrane cholesterol content and Na-[P.sub.i] cotransport activity during the aging process and adaptation to alterations in dietary [P.sub.i] in the rat (Levi M, Jameson DM, and van der Meer BW. Am J Physiol Renal Fluid Electrolyte Physiol 256: F85-F94, 1989). The purpose of the present study was to determine whether alterations in cholesterol content per se modulate Na-[P.sub.i] cotransport activity and apical membrane Na-[P.sub.i] protein expression in opossum kidney (OK) cells. Acute cholesterol depletion achieved with [beta]-methyl cyclodextrin ([beta]-MCD) resulted in a significant increase in Na-[P.sub.i] cotransport activity accompanied by a moderate increase in apical membrane Na-[P.sub.i] protein abundance and no alteration of total cellular Na-[P.sub.i] protein abundance. Conversely, acute cholesterol enrichment achieved with [beta]-MCD/cholesterol resulted in a significant decrease in Na-[P.sub.i] cotransport activity with a moderate decrease in apical membrane Na-[P.sub.i] protein abundance and no change of the total cellular Na-[P.sub.i] protein abundance. In contrast, chronic cholesterol depletion, achieved by growing cells in lipoprotein-deficient serum (LPDS), resulted in parallel and significant increases in Na-[P.sub.i] cotransport activity and apical membrane and total cellular Na-[P.sub.i] protein abundance. Cholesterol depletion also resulted in a significant increase in membrane lipid fluidity and alterations in lipid microdomains as determined by laurdan fluorescence spectroscopy and imaging. Chronic cholesterol enrichment, achieved by growing cells in LPDS followed by loading with low-density lipoprotein, resulted in parallel and significant decreases in Na-[P.sub.i] cotransport activity and apical membrane and total cellular Na-[P.sub.i] protein abundance. Our results indicate that in OK cells acute and chronic alterations in cholesterol content per se modulate Na-[P.sub.i] cotransport activity by diverse mechanisms that also include significant interactions of Na-[P.sub.i] protein with lipid microdomains. two-photon fluorescence microscopy; filipin; lipid microdomains; laurdan; opossum kidney cells

Renal reabsorption is the main mechanism that controls mannose homeostasis. This takes place through a specific Na-coupled uphill transport system, the molecular identity of which is unknown. We prepared and screened a size-selected rat kidney cortex cDNA library through the expression of mannose transport in Xenopus laevis oocytes. We have identified a membrane protein that induces high-affinity and specific Na-dependent transport of D-mannose and D-glucose in X. laevis oocytes, most likely through stimulation of the capacity of an endogenous transport system of the oocyte. Sequencing has revealed that the cDNA encodes the counterpart of the human membrane-associated protein MAP17, previously known by its overexpression in renal, colon, lung, and breast carcinomas. We show that MAP17 is a 12.2-kDa nonglycosylated membrane protein that locates to the brush-border plasma membrane and the Golgi apparatus of transfected cells and that it is expressed in the proximal tubules of the kidney cortex and in the spermatids of the seminiferous tubules. It spans twice the cell membrane, with both termini inside the cell, and seems to form homodimers through intracellular [Cys.sup.55], a residue also involved in transport expression. MAP17 is responsible for mannose transport expression in oocytes by rat kidney cortex mRNA. The induced transport has the functional characteristics of a Na-glucose cotransporter (SGLT), because D-glucose and [alpha]-methyl-D-glucopyranoside are also accepted substrates that are inhibited by phloridzin. The corresponding transporter from the proximal tubule remains to be identified, but it is different from the known mammalian SGLT-1, -2, and -3. expression cloning; kidney cortex; mannose reabsorption

An essential role in phosphate homeostasis is played by Na/Pi cotransporter IIa that is localized in the brush borders of renal proximal tubular cells. Recent studies identified several PDZ proteins interacting with the COOH-terminal tail of NaPi-IIa, such as PDZK1 and NHERF-1. Here, by using yeast two-hybrid screen of mouse kidney cDNA library, we attempted to find proteins interacting with the N[H.sub.2]-terminal part of NaPi-IIa. We identified MAP17, a 17-kDa membrane protein that has been described to be associated with various human carcinomas, but it is also expressed in normal kidneys. Results obtained by various in vitro analyses suggested that MAP17 interacts with the fourth domain of PDZK1 but not with other PDZ proteins localized in proximal tubular brush borders. As revealed by immunofluorescence, MAP17 was abundant in S1 but almost absent in S3 segments. No alterations of the apical abundance of MAP17 were observed after maneuvers undertaken to change the content of NaPi-IIa (parathyroid hormone treatment, different phosphate diets). In agreement, no change in the amount of MAP17 mRNA was observed. Results obtained from transfection studies using opossum kidney cells indicated that the apical localization of MAP17 is independent of PDZK1 but that MAP17 is required for apical localization of PDZK1. In summary, we conclude that MAP17 1) interacts with PDZK1 only, 2) associates with the N[H.sub.2] terminus of NaPi-IIa within the PDZK1/NaPi-IIa/ MAP17 complex, and 3) acts as an apical anchoring site for PDZK1. interacting proteins; Na/Pi cotransport; PDZ proteins; NHERF-1; opossum kidney cells

Low dietary phosphate, P(sub i), in rabbits results in a specific increase in apical renal proximal tubular Na-P(sub i) cotransport system. NaP(sub i)-1 and NaP(sub i)-6 are two different NaP(sub i) cotransport systems, but only the latter is regulated by dietary P(sub i). NaPi-6 cDNA encodes proteins to function as a brush border membrane Na-Pi cotransport.

Analysis of the cellular pathways that mediate chronic and acute adaptations of Pi transporter to dietary Pi modifications in rat renal cells shows that rats that undergo chronic administration of low Pi-diet have a higher brush border membrane (BBM) Na-Pi cotransport rate than the rats on high Pi-diet. The high cotransport corresponds to the enhancement of BBM NaPi-2 protein and mRNA concentration in the rats with chronic feedings of low Pi-diet. Rats with acute high-Pi diet exhibit a low Na-Pi cotransport rate and a reduction in NaPi2-protein but not NaPi-2 mRNA levels compared to chronically low Pi-diet fed rats.

Rat kidney cortex and human kidney cortex cDNA libraries were screened to isoalte two cDNA clones, NaPi-2 and NaPi-3 for the expression of sodium-dependent phosphate transport in Xenopus laevis oocytes. In NaPi-2/3-linked proteins, there is a minimum of eight membrane-spanning areas. In vitro translation yields proteins of the anticipated size and indicates glycosylation. Two intimately connected proteins which are concerned with human and rat renal brush border Na/Pi cotransport have been structurally determined.