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Renal reabsorption of inorganic phosphate ([P.sub.i]) is mainly mediated by the [Na.sup.+] dependent [P.sub.i]-cotransporter NaPi-IIa that is expressed in the brush-border membrane (BBM) of renal proximal tubules. Regulation and apical expression of NaPi-IIa are known to depend on a network of interacting proteins. Most of the interacting partners identified so far associate with the COOH-terminal PDZ-binding motif (TRL) of NaPi-IIa. In this study [GABA.sub.A] receptor-associated protein (GABARAP) was identified as a novel interacting partner of NaPi-IIa applying a membrane yeast-two-hybrid system (MYTH 2.0) to screen a mouse kidney library with the TRL-truncated cotransporter as bait. GABARAP mRNA and protein are present in renal tubules, and the interaction of NaPi-IIa and GABARAP was confirmed by using glutathione S-transferase pulldowns from BBM and coimmunoprecipitations from transfected HEK293 cells. Amino acids 36-68 of GABARAP were identified as the determinant for the described interaction. The in vivo effects of this interaction were studied in a murine model. [GABARAP.sup.-/-] mice have reduced urinary excretion of [P.sub.i], higher [Na.sup.+] -dependent [sup.32][P.sub.i] uptake in BBM vesicles, and increased expression of NaPi-IIa in renal BBM compared with [GABARAP.sup.+/+] mice. The expression of [Na.sup.+/[H.sup.+] exchanger regulatory factor (NHERF)1, an important scaffold for the apical expression of NaPi-IIa, is also increased in [GABARAP.sup.-/-] mice. The absence of GABARAP does not interfere with the regulation of the cotransporter by either parathyroid hormone or acute changes of dietary [P.sub.i] content. epithelial transport; renal proximal tubules; phosphate homeostasis

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

In the kidney, ~80% of the filtered phosphate ([P.sub.i]) is reabsorbed along the proximal tubule. Changes in renal [P.sub.i] reabsorption are associated with modulation of the sodium-dependent [P.sub.i] cotransporter type IIa (NaPi-IIa) and type IIc (NaPi-IIc) protein abundance in the brush-border membrane (BBM) of proximal tubule cells. NaPi-IIa is mainly regulated by dietary [P.sub.i] intake and parathyroid hormone (PTH). The purpose of the present study was to elucidate the effect of alteration in dietary magnesium ([Mg.sup.2+]) intake on renal [P.sub.i] handling. Urinary [P.sub.i] excretion and renal expression of NaPi-IIa and NaPi-IIc were analyzed in rats fed a normal (0.2%) or high-[Mg.sup.2+] (2.5%) diet. A high-[Mg.sup.2+] diet resulted in decreased renal [P.sub.i] excretion and increased protein expression of NaPi-IIa and NaPi-IIc. Serum FGF-23 (fibroblast growth factor 23) levels were unchanged under a high-[Mg.sup.2+] diet. Serum PTH levels were slightly decreased under a high-[Mg.sup.2+] diet. To examine whether the observed changes in renal [P.sub.i] reabsorption are PTH dependent, expression of NaPi-IIa and NaPi-IIc was also analyzed in parathyroidectomized (PTX) rats fed a normal or high-[Mg.sup.2+] diet. In PTX rats, [Mg.sup.2+] had no significant effect on renal [P.sub.i] excretion or NaPi-IIa protein expression. [Mg.sup.2+] increased NaPi-IIc protein expression in PTX rats. This experiment shows for the first time on the molecular level how [Mg.sup.2+] stimulates renal [P.sub.i] reabsorption. Under a high-[Mg.sup.2+] diet, NaPi-IIa expression is dependent on PTH levels, whereas NaPi-IIc expression seems to be independent of PTH levels. parathyroid hormone

Protein kinases catalyze the phosphorylation of serine/threonine or tyrosine residues, which may directly alter a protein's functional properties. Kinases can also regulate protein functions indirectly, for example, by controlling the [...]

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.sup.+]-dependent [P.sub.i] transporters carry out this task. Remarkably, the two families transport different [P.sub.i] species: whereas type II [Na.sup.+]/ [P.sub.i] cotransporters (SCL34) prefer divalent [HPO.sup.2-.sub.4], type III [Na.sup.+]/[P.sub.i] cotransporters (SLC20) transport monovalent [H.sub.2][PO.sup.-.sub.4]. 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 [P.sub.i] homeostasis, and in salivary and mammary glands, liver, and testes they play a role in modulating the [P.sub.i] content 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 [P.sub.i] homeostasis; 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.sup.+]/[P.sub.i] cotransporter families. phosphate; cotransport; electrophysiology; structure-function

Members of the SLC20 family or type III [Na.sup.+]-coupled [P.sub.i] cotransporters (PiT-1, PiT-2) are ubiquitously expressed in mammalian tissue and are thought to perform a housekeeping function for intracellular [P.sub.i] homeostasis. Previous studies have shown that PiT-1 and PiT-2 mediate electrogenic [P.sub.i] co-transport when expressed in Xenopus oocytes, but only limited kinetic characterizations were made. To address this shortcoming, we performed a detailed analysis of SLC20 transport function. Three SLC20 clones (Xenopus PiT-l, human PiT-1, and human PiT-2) were expressed in Xenopus oocytes. Each clone gave robust [Na.sup.+]-dependent [sup.32][P.sub.i] uptake, but only Xenopus PIT-1 showed sufficient activity for complete kinetic characterization by using two-electrode voltage clamp and radionuclide uptake. Transport activity was also documented with [Li.sup.+] substituted for [Na.sup.+]. The dependence of the [P.sub.i]-induced current on [P.sub.i] concentration was Michaelian, and the dependence on [Na.sup.+] concentration indicated weak cooperativity. The dependence on external pH was unique: the apparent [P.sub.i] affinity constant showed a minimum in the pH range 6.2-6.8 of ~0.05 mM and increased to ~0.2 mM at pH 5.0 and pH 8.0. Xenopus PiT-1 stoichiometry was determined by dual [sup.22] [Na-.sup.32][P.sub.i] uptake and suggested a 2:1 [Na.sup.+]:[P.sub.i] stoichiometry. A correlation of 32pi uptake and net charge movement indicated one charge translocation per [P.sub.i]. Changes in oocyte surface pH were consistent with transport of monovalent [P.sub.i]. On the basis of the kinetics of substrate interdependence, we propose an ordered binding scheme of [Na.sup.+]: [H.sub.2]P[O.sub.4]:[Na.sup.+]. Significantly, in contrast to type II [Na.sup.+]-[P.sub.i] cotransporters, the transport inhibitor phosphonoformic acid did not inhibit PiT-1 or PIT-2 activity. [Na.sup.+]-[P.sub.i] cotransport; two-electrode voltage clamp; surface pH electrode; SLC20; retroviral receptor

Phosphate reabsorption in the renal proximal tubule occurs mostly via the type IIa [Na.sup.+]-phosphate cotransporter ([NaP.sub.i]-IIa) in the brush border membrane (BBM). The activity and localization of [NaP.sub.i]-IIa are regulated, among other factors, by parathyroid hormone (PTH). [NaP.sub.i]-IIa interacts in vitro via its last three COOH-terminal amino acids with the PDZ protein [Na.sup.+]/[H.sup.+]-exchanger isoform 3 regulatory factor (NHERF)-1 (NHERF1). Renal phosphate reabsorption in Nherf1-deficient mice is altered, and [NaP.sup.i]-IIa expression in the BBM is reduced. In addition, it has been proposed that NHERF1 and NHERF-2 are important for the coupling of PTH receptors (PTHRs) to phospholipase C (PLC) and the activation of the protein kinase C pathway. We tested the role of NHERF1 in the regulation of [NaP.sub.i]-IIa by PTH in Nherf1-deficient mice. Immunohistochemistry and Western blotting demonstrated that stimulation of apical and basolateral receptors with PTH-(1-34) led to internalization of [NaP.sub.i]-IIa in wild-type and Nherf1-deficient mice. Stimulation of only apical receptors with PTH-(3-34) failed to induce internalization in Nherf1-deficient mice. Expression and localization of apical PTHRs were similar in wild-type and Nherf1-deficient mice. Activation of the protein kinase C- and A-dependent pathways with 1,2-dioctanoyl-sn-glycerol or 8-bromo-cAMP induced normal internalization of [NaP.sub.i]-IIa in wild-type, as well as Nherf1-deficient, mice. Stimulation of PLC activity due to apical PTHRs was impaired in Nherf1-deficient mice. These data suggest that NHERF1 in the proximal tubule is important for PTH-induced internalization of [NaP.sub.i]-IIa and, specifically, couples the apical PTHR to PLC. phosphate cotransporter; PDZ protein; parathyroid hormone; proximal tubule

Renal type IIa [Na.sup.+]-coupled inorganic phosphate ([P.sub.i]) cotransporters (NaPi-IIa) mediate divalent [P.sub.i] transport in an electrogenic manner, whereas the renal type IIc isoform (NaPi-IIc) is electroneutral, yet it shows high sequence identity with NaPi-IIa. Dual uptake ([sup.32][P.sub.i]/[sup.22]Na) assays confirmed that NaPi-IIc displayed [Na.sup.+]-coupled [P.sub.i] cotransport with a 2:1 ([Na.sup.+]:Pi) stoichiometry compared with 3:1 established for NaPi-IIa. This finding suggested that the electrogenicity of NaPi-IIa arises from the interaction of an additional [Na.sup.+] ion compared with NaPi-IIc. To identify the molecular elements responsible for the functional difference between isoforms, we used chimera and amino acid replacement approaches. Transport activity of chimeras constructed with NaPi-IIa and NaPi-IIc indicated that residues within the first six transmembrane domains were essential for the electrogenicity of NaPi-IIa. Sequence comparison between electrogenic and electroneutral isoforms revealed differences in the charge and polarity of residues clustered in three areas, one of which included part of the predicted third transmembrane domain. Here, substitution of three residues with their NaPi-IIa equivalents in NaPi-IIc ($189A, $191A, and G195D) resulted in a transporter that displayed a 1:1 charge/[P.sub.i] coupling, a 3:1 [Na.sup.+]:[P.sub.i] stoichiometry, and transient currents that resembled pre-steady-state relaxations. The mutant's weaker voltage dependency and 10-fold lower apparent [P.sub.i] affinity compared with NaPi-IIa indicated that other residues important for the NaPi-IIa kinetic fingerprint exist. Our findings demonstrate that, through a minimal number of side chain substitutions, we can effect a switch from electroneutral to electrogenic cotransporter function, concomitant with the appearance of a cosubstrate interaction site.

The type IIa [Na.sup.+]-Pi cotransporter (Na[P.sub.i]-IIa) and the [Na.sup.+]/[H.sup.+] exchanger regulatory factor-1 (NHERF1) colocalize in the apical membrane of proximal tubular cells. Both proteins interact in vitro. Herein the interaction between Na[P.sub.i]-IIa and NHERF1 is further documented on the basis of coimmunoprecipitation and co-pull-down assays. Na[P.sub.i]-IIa is endocytosed and degraded in lysosomes upon parathyroid hormone (PTH) treatment. To investigate the effect of PTH on the Na[P.sub.i]-IIa-NHERF1 association, we first compared the localization of both proteins after PTH treatment. In mouse proximal tubules and OK cells, Na[P.sub.i]-IIa was removed from the apical membrane after hormonal treatment; however, NHERF1 remained at the membrane. Moreover, PTH treatment led to degradation of Na[P.sub.i]-IIa without changes in the amount of NHERF1. The effect of PTH on the Na[P.sub.i]-IIa-NHERF1 interaction was further studied using coimmunoprecipitation. PTH treatment reduced the amount of Na[P.sub.i]-IIa coimmunoprecipitated with NHERF antibodies. PTH-induced internalization of Na[P.sub.i]-IIa requires PKA and PKC; therefore, we next analyzed whether PTH induces changes in the phosphorylation state of either partner. NHERF1 was constitutively phosphorylated. Moreover, in mouse kidney slices, PTH induced an increase in NHERF1 pbosphorylation; independent activation of PKA or PKC also resulted in increased phosphorylation of NHERF1 in kidney slices. However, Na[P.sub.i]-IIa was not phosphorylated either basally or after exposure to PTH. Our study supports an interaction between NHERF1 and Na[P.sub.i]-IIa on the basis of their brash-border membrane colocalization and in vitro coimmunoprecipitation/co-pull-down assays. Furthermore, PTH weakens this interaction as evidenced by different in situ and in vivo behavior. The PTH effect takes place in the presence of increased phosphorylation of NHERF1. proximal tubule; opossum kidney cells; phosphorylation; endocytosis

We have characterized the kinetics of substrate transport in the renal type IIa human sodium-phosphate cotransporter (NaPi-IIa). The transporter was expressed in Xenopus laevis oocytes, and steady-state and pre-steady-state currents and substrate uptakes were characterized by voltage-clamp and isotope flux. First, by measuring simultaneous uptake of a substrate ([sup.32][P.sub.i], [sup.22]Na) and charge in voltage-clamped oocytes, we established that the human NaPi-IIa isoform operates with a Na:Pi:charge stoichiometry of 3:1:1 and that the preferred transported [P.sub.i] species is HP[O.sup.2-.sub.4]. We then probed the complex interrelationship of substrates, pH, and voltage in the NaPi-IIa transport cycle by analyzing both steady-state and pre-steady-state currents. Steady-state current measurements show that the apparent HP[O.sup.2-.sub.4] affinity is voltage dependent and that this voltage dependency is abrogated by lowering the pH or the [Na.sup.+] concentration. In contrast, the voltage dependency of the apparent [Na.sup.+] affinity increased when pH was lowered. De-steady-state current analysis shows that [Na.sup.+] ions bind first and influence the preferred orientation of the transporter in the absence of [P.sub.i]. Pre-steady-state charge movement was partially suppressed by complete removal of [Na.sup.+] from the bath, by reducing extracellular pH (both in the presence and absence of [Na.sup.+]), or by adding [P.sub.i] (in the presence of 100 mM Na). None of these conditions suppressed charge movement completely. The results allowed us to modify previous models for the transport cycle of NaPi-II transporters by including voltage dependency of HP[O.sup.2-.sub.4] binding and proton modulation of the first [Na.sup.+] binding step. Xenopus laevis oocyte; electrophysiology; two-electrode voltage clamp; stoichiometry; structure-function

The [Na.sup.+]/phosphate cotransporter NaPi-IIa (SLC34A1) is the major transporter mediating the reabsorption of [P.sub.i] in the proximal tubule. Expression and activity of NaPi-IIa is regulated by several factors, including parathyroid hormone, dopamine, metabolic acidosis, and dietary [P.sub.i] intake. Dopamine induces natriuresis and phosphaturia in vivo, and its actions on several [Na.sup.+]-transporting systems such as NHE3 and [Na.sup.+]-[K.sup.+]-ATPase have been investigated in detail. Using freshly isolated mouse kidney slices, perfused proximal tubules, and cultured renal epithelial cells, we examined the acute effects of dopamine on NaPi-IIa expression and localization. Incubation of isolated kidney slices with the selective [D.sub.1]-like receptor agonists fenoldopam (10 [micro]M) and SKF-38393 (10 [micro]M) for 1 h induced NaPi-IIa internalization and reduced expression of NaPi-IIa in the brush border membrane (BBM). The [D.sub.2]-like selective agonist quinpirole (1 [micro]M) had no effect. The [D.sub.1] and [D.sub.2] agonists did not affect the renal [Na.sup.+]/sulfate cotransporter NaSi in the BBM of the proximal tubule. Studies with isolated perfused proximal tubules demonstrated that activation of luminal, but not basolateral, [D.sub.1]-like receptors caused NaPi-IIa internalization. In kidney slices, inhibition of PKC (1 [micro]M chelerythrine) or ERK1/2 (20 [micro]M PD-098089) pathways did not prevent the fenoldopam-induced intemalization. Inhibition with the PKA blocker H-89 (10 [micro]M) abolished the effect of fenoldopam. Immunoblot demonstrated a reduction of NaPi-IIa protein in BBMs from kidney slices treated with fenoldopam. Incubation of opossum kidney cells transfected with NaPi-IIa-green fluorescent protein chimera shifted fluorescence from the apical membrane to an intracellular pool. In summary, dopamine induces internalization of NaPi-IIa by activation of luminal [D.sub.1]-like receptors, an effect that is mediated by PKA. protein kinase A: proximal tubule; brush border membrane

Hepatocytes and cholangiocytes release ATP into bile, where it is rapidly degraded into adenosine and [P.sub.i]. In rat, biliary [P.sub.i] concentration (0.01 mM) is ~100-fold and 200-fold lower than in hepatocytes and plasma, respectively, indicating active reabsorption of biliary [P.sub.i]. We aimed to functionally characterize canalicular [P.sub.i] reabsorption in rat liver and to identify the involved [P.sub.i] transport system(s). [P.sub.i] transport was determined in isolated rat canalicular liver plasma membrane (LPM) vesicles using a rapid membrane filtration technique. Identification of putative [P.sub.i] transporters was performed with RT-PCR from liver mRNA. Phosphate transporter protein expression was confirmed by Western blotting in basolateral and canalicular LPM and by immunofluorescence in intact liver. Transport studies in canalicular LPM vesicles demonstrated sodium-dependent [P.sub.i] uptake. Initial [P.sub.i] uptake rates were saturable with increasing [P.sub.i] concentrations, exhibiting an apparent [K.sub.m] value of ~11 [micro]M. [P.sub.i] transport was stimulated by an acidic extravesicular pH and by an intravesicular negative membrane potential. These data are compatible with transport characteristics of sodium-phosphate cotransporters NaPi-IIb, PiT-1, and PiT-2, of which the mRNAs were detected in rat liver. On the protein level, NaPi-IIb was detected at the canalicular membrane of hepatocytes and at the brush-border membrane of cholangiocytes. In contrast, PiT-1 and PiT-2 were detected at the basolateral membrane of hepatocytes. We conclude that NaPi-IIb is most probably involved in the reabsorption of [P.sub.i] from primary hepatic bile and thus might play an important role in the regulation of biliary [P.sub.i] concentration. NaPi; PiT; bile formation; transport