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  • 1
    Electronic Resource
    Electronic Resource
    Functional Expression and Purification of Histidine-Tagged Rat Renal Na/Phosphate (NaPi-2) and Na/Sulfate (NaSi-1) Cotransporters (1997)
    Springer
    The journal of membrane biology 160 (1997), S. 111-117 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Renal Na/Pi-cotransport — Renal Na/sulfate-cotransport — Sf9 cells — Purification — Ni2+-chelate chromatography
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Two mammalian sodium-dependent anion-cotransporters (NaPi-2 for phosphate and NaSi-1 for sulfate) have been expressed in Sf9 insect cells using the baculovirus expression system. A histidine tag was introduced at the C-termini in order to facilitate purification by metal-affinity chromatography. Sf9 cells infected with the histidine-tagged Ni/P i -cotransporter exhibited more than 60-fold higher sodium-dependent transport of phosphate compared to noninfected cells. Expressed Na/P i -cotransport exhibited a K m of P i of 0.21 mm and an apparent K m of sodium of 92 mm. Infected cells expressed a 65 kDa polypeptide as detected by Western blotting and immunoprecipitation. Sf9 cells infected with the histidine-tagged NaSi-1 or untagged NaSi-1 protein expressed sodium-dependent sulfate cotransport up to 60-fold higher compared to noninfected cells. Transport of sulfate was highly dependent on sodium exhibiting a K m of SO2− 4 of about 0.3–0.4 mm and a K m of sodium of 55 mm. By Western blotting and immunoprecipitation expressed NaS i -1 proteins were detected at 55–60 kDa. These studies demonstrate that histidine tagged proximal tubular Na-dependent cotransporters for phosphate and sulfate can be expressed functionally in Sf9 cells and that the kinetic characteristics were not altered by the introduction of a histidine tag at the C-termini. Furthermore, it is demonstrated that after solubilization under denaturing conditions histidine-tagged cotransporter proteins can be purified by metal-chelate affinity chromatography.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002329900300
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  • 2
    Electronic Resource
    Electronic Resource
    Effect of Two Tyrosine Mutations on the Activity and Regulation of the Renal Type II Na/P i -Cotransporter Expressed in Oocytes (1999)
    Springer
    The journal of membrane biology 168 (1999), S. 275-282 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Na/Pi-cotransporter — PTH — Endocytosis — Tyrosine-based signals
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The rat renal type II Na/Pi-cotransporter (NaPi2), which is regulated by mechanisms involving endocytosis and lysosomal degradation, contains two sequences that show high homology with two tyrosine (Y)-based consensus motifs previously reported to be involved in such intracellular trafficking: GY402FAM matching the consensus sequence GYXXZ, and Y509RWF matching the motif YXXO. Mutations of any of these two Y nearly abolished the NaPi2 mediated 32P i -uptake after cRNA-injection into oocytes. The mechanisms underlying these defects are however different. Mutation of the Y402 results in a lack of glycosylation and reduced surface expression of the cotransporter, that are specific for the Y402 mutation since substitution of the neighboring F403 did not have any effect. The inhibitory effect of the Y509 mutation is related to a functional inactivation of the protein expressed in the plasma membrane; mutation of the neighboring R510 also led to a decrease in the cotransporter activity. Pharmacological activation of the protein kinase C cascade by DOG induced the retrieval of both wild-type (WT) as well as Y509 cotransporters from the oocyte plasma membrane. These data suggest that the Y402 is important for the surface expression whereas Y509 for the function of the type II Na/P i -cotransporter expressed in oocytes. Y509 seems not to be involved in the membrane retrieval of the cotransporter.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002329900516
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  • 3
    Electronic Resource
    Electronic Resource
    Functional expression of rat renal Na/Pi-cotransport (NaPi-2) in Sf9 cells by the baculovirus system (1995)
    Springer
    The journal of membrane biology 144 (1995), S. 43-48 
    Details
    ISSN: 1432-1424
    Keywords: Renal Na/P i -cotransport ; Sf9 cells ; Infection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract The recently cloned Na/P i -cotransport system NaPi-2 is an apical membrane protein of rat proximal tubular cells and is involved in proximal phosphate reabsorption. To make the protein available for further functional/structural studies, this transport system has been expressed in Sf9 insect cells using a recombinant baculovirus. Sf9 cells infected with NaPi-2 (or 6His tagged NaPi-2) expressed functional Na/P i -cotransport up to 20- to 50-fold over noninfected Sf9 cells. Transport of phosphate in infected cells was highly dependent on sodium, exhibited a K m for P i of 0.114 mm and an apparent K m for Na of 63 mm (Hill coefficient of approximately 3) and was stimulated by high external pH. Infected cells expressed a polypeptide of 65 kDa representing a nonglycosylated form of the 85 kDa mature NaPi-2 transporter as present in proximal tubular brush-border membranes. By confocal microscopy expression of NaPi-2 protein was observed in the plasma membrane, yet submembranous accumulation of NaPi-2 protein could not be excluded. This demonstrates that the rat proximal tubular Na/P i -cotransport system NaPi-2 can be successfully expressed in Sf9 cells with characteristics similar to that in isolated brush-border membranes. The 6His tagging will permit isolation of the NaPi-2 cotransporter in amounts sufficient for structural/functional studies.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00238415
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  • 4
    Electronic Resource
    Electronic Resource
    Molecular Regulation of Renal Phosphate Transport (1996)
    Springer
    The journal of membrane biology 154 (1996), S. 1-9 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Phosphate transport — Dietary phosphate — Parathyroid hormone — Endocytosis — Exocytosis — Microtubules--〉
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002329900127
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  • 5
    Electronic Resource
    Electronic Resource
    Chloride Conductance and P i Transport are Separate Functions Induced by the Expression of NaPi-1 in Xenopus Oocytes (1998)
    Springer
    The journal of membrane biology 164 (1998), S. 71-77 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Cl− Channel — Organic Anions — Phosphate — NPPB
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Expression of the protein NaPi-1 in Xenopus oocytes has previously been shown to induce an outwardly rectifying Cl− conductance (GCl), organic anion transport and Na+-dependent P i -uptake. In the present study we investigated the relation between the NaPi-1 induced GCl and P i -induced currents and transport. NaPi-1 expression induced P i -transport, which was not different at 1–20 ng/oocyte NaPi-1 cRNA injection and was already maximal at 1–2 days after cRNA injection. In contrast, GCl was augmented at increased amounts of cRNA injection (1–20 ng/oocyte) and over a five day expression period. Subsequently all experiments were performed on oocytes injected with 20 ng/oocytes cRNA. P i -induced currents (Ip) could be observed in NaPi-1 expressing oocytes at high concentrations of P i (≥ 1 mm P i ). The amplitudes of Ip correlated well with GCl. Ip was blocked by the Cl− channel blocker NPPB, partially Na+-dependent and completely abolished in Cl− free solution. In contrast, P i -transport in NaPi-1 expressing oocytes was not NPPB sensitive, stronger depending on extracellular Na+ and weakly affected by Cl− substitution. Endogenous P i -uptake in water-injected oocytes amounted in all experiments to 30–50% of the Na+-dependent P i -transport observed in NaPi-1 expressing oocytes. The properties of the endogenous P i -uptake system (K m for P i 〉 1 mm; partial Na+- and Cl−-dependence; lack of NPPB block) were similar to the NaPi-1 induced P i -uptake, but no Ip could be recorded at P i -concentrations ≤3 mm. In summary, the present data suggest that Ip does not reflect charge transfer related to P i -uptake, but a P i -mediated modulation of GCl.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002329900394
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  • 6
    Electronic Resource
    Electronic Resource
    Cleavage of Disulfide Bonds Leads to Inactivation and Degradation of the Type IIa, But not Type IIb Sodium Phosphate Cotransporter Expressed in Xenopus laevis Oocytes (2000)
    Springer
    The journal of membrane biology 176 (2000), S. 143-149 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Phosphate transport —X. laevis oocytes — TCEP — Disulfide bonds — Degradation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Tris(2-carboxyethyl)phosphine (TCEP) reduces (cleaves) disulfide bonds of the renal proximal tubule type IIa Na/Pi- cotransporter (rat NaPi IIa) and thereby inhibits its function. We tested the effect of TCEP on the murine type IIa Na/P i -cotransporter and the corresponding IIb intestinal isoform both expressed in Xenopus laevis oocytes. After incubation with TCEP the function of NaPi IIa was inhibited and protein amount was decreased. Injection of the lysosomal inhibitor leupeptin prevented degradation of the protein. Exposure of oocytes to TCEP at 0°C led to a reduction in transport function without concomitant loss in Na/Pi IIa protein. In contrast to NaPi type IIa, the type IIb isoform was neither inhibited, nor degraded after incubation with TCEP. These results suggest that cleavage of disulfide bonds led to changes within the confirmation of the type IIa transporter that result in (i) inhibition of the transport activity and (ii) internalization and subsequent lysosomal degradation of transporter protein. Sequence comparisons suggest the involvement/presence of different disulfide bonds in type IIa and type IIb Na/P i -cotransporters.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s00232001083
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  • 7
    Electronic Resource
    Electronic Resource
    Identification of a cDNA/Protein Leading to an Increased P i -uptake in Xenopus laevis Oocytes (1997)
    Springer
    The journal of membrane biology 156 (1997), S. 19 -24 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Na/Pi-cotransport — Expression cloning — Duodenum — Brush border membrane —Xenopus laevis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. In a previous report we documented an increased Na+-dependent transport of inorganic phosphate (P i ) in Xenopus laevis oocytes injected with mRNA isolated from rabbit duodenum (Yagci et al., Pfluegers Arch. 422:211–216, 1992; ref 24). In the present study we have used expression cloning in oocytes to search for the cDNA/mRNA involved in this effect. The identified cDNA (provisionally named PiUS; for P i -uptake stimulator) lead to a 3-4-fold stimulation of Na+-dependent P i -uptake (10ng cRNA injected, 3–5 days of expression). Na+-independent uptake of P i was also affected but transport of sulphate and l-arginine (in the presence or absence of sodium) remained unchanged. The apparent K m -values for the induced Na+-dependent uptake were 0.26 ± 0.04 mm for P i and 14.8 ± 3.0 mm for Na+. The 1796 bp cDNA codes for a protein of 425 amino acids. Hydropathy analysis suggests a lack of transmembrane segments. In vitro translation resulted in a protein of 60 kDa and provided no evidence of glycosylation. In Northern blots a mRNA of ∼2 kb was recognized in various tissues including different intestinal segments, kidney cortex, kidney medulla, liver and heart. Homology searches showed no similarity to proteins involved in membrane transport and its control. In conclusion, we have cloned from a rabbit small intestinal cDNA library a novel cDNA encoding a protein stimulating P i -uptake into Xenopus laevis oocytes, but which is not a P i -transporter itself.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002329900183
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  • 8
    Electronic Resource
    Electronic Resource
    Electrophysiological Characterization of the Flounder Type II Na+/P i Cotransporter (NaPi-5) Expressed in Xenopus laevis Oocytes (1997)
    Springer
    The journal of membrane biology 160 (1997), S. 9-25 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Na+/Pi cotransporter — Proximal tubule — Voltage clamp — Steady-state — Presteady-state —Xenopus laevis oocyte expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract: The two electrode voltage clamp technique was used to investigate the steady-state and presteady-state kinetic properties of the type II Na+/P i cotransporter NaPi-5, cloned from the kidney of winter flounder (Pseudopleuronectes americanus) and expressed in Xenopus laevis oocytes. Steady-state P i -induced currents had a voltage-independent apparent K m for P i of 0.03 mm and a Hill coefficient of 1.0 at neutral pH, when superfusing with 96 mm Na+. The apparent K m for Na+ at 1 mm P i was strongly voltage dependent (increasing from 32 mm at −70 mV to 77 mm at −30 mV) and the Hill coefficient was between 1 and 2, indicating cooperative binding of more than one Na+ ion. The maximum steady-state current was pH dependent, diminishing by 50% or more for a change from pH 7.8 to pH 6.3. Voltage jumps elicited presteady-state relaxations in the presence of 96 mm Na+ which were suppressed at saturating P i (1 mm). Relaxations were absent in non-injected oocytes. Charge was balanced for equal positive and negative steps, saturated at extremes of potential and reversed at the holding potential. Fitting the charge transfer to a Boltzmann relationship typically gave a midpoint voltage (V 0.5) close to zero and an apparent valency of approximately 0.6. The maximum steady-state transport rate correlated linearly with the maximum P i -suppressed charge movement, indicating that the relaxations were NaPi-5-specific. The apparent transporter turnover was estimated as 35 sec−1. The voltage dependence of the relaxations was P i -independent, whereas changes in Na+ shifted V 0.5 to −60 mV at 25 mm Na+. Protons suppressed relaxations but contributed to no detectable charge movement in zero external Na+. The voltage dependent presteady-state behavior of NaPi-5 could be described by a 3 state model in which the partial reactions involving reorientation of the unloaded carrier and binding of Na+ contribute to transmembrane charge movement.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002329900291
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  • 9
    Electronic Resource
    Electronic Resource
    Cysteine Residues and the Structure of the Rat Renal Proximal Tubular Type II Sodium Phosphate Cotransporter (Rat NaPi IIa) (2000)
    Springer
    The journal of membrane biology 176 (2000), S. 133-141 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Phosphate transport — NaPi — Cysteine residues — TCEP — MTS-reagents — Disulfide bonds
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The rat renal Na/P i cotransporter type IIa (rat NaPi IIa) is a 637 amino acid protein containing 12 cysteine residues. We examined the effect of different cysteine modifying methanethiosulfonate (MTS)-reagents and the disulfide bond reducing agent tris(2-carboxyethyl)phosphine (TCEP) on the transport activity of wild-type and 12 single cysteine substitution mutants of rat NaPi IIa expressed in Xenopus laevis oocytes. The transport activity of the wild-type protein was resistant to three membrane impermeant MTS-reagents (MTSEA, MTSET and MTSES). In contrast, membrane permeant methyl methanethiosulfonate (MMTS) and TCEP inhibited the transport activity of both the wild-type, as well as all the single mutant proteins. This indicated the existence of more than one functionally important cysteine residue, not accessible extracellularly, and at least 2 disulfide bridges. To identify the disulfide bridges, three double mutants lacking 2 of the 3 cysteine residues predicted to be extracellular in different combinations were examined. This led to the identification of one disulfide bridge between C306 and C334; reconsideration of the topological model predictions suggested a second disulfide bridge between C225 and C520. Evaluation of a fourth double mutant indicated that at least one of two disulfide bridges (C306 and C334; C225 and C520) has to be formed to allow the surface expression of a functional cotransporter. A revised secondary structure is proposed which includes two partially repeated motifs that are connected by disulfide bridges formed between cysteine pairs C306-C334 and C225-C520.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s00232001082
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  • 10
    Electronic Resource
    Electronic Resource
    Transport ofl-cysteine by rat renal brush border membrane vesicles (1983)
    Springer
    The journal of membrane biology 73 (1983), S. 25-37 
    Details
    ISSN: 1432-1424
    Keywords: l-cysteine transport ; renal proximal tubule ; brush border membrane vesicles ; cotransport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Brush border membranes were isolated from rat renal cortex by a divalent cation precipitation method.l-35S-cysteine uptake into the vesicles was measured by a rapid filtration method. Only minimal binding of the amino acid to the vesicles was observed. Sodium stimulatesl-cysteine uptake specifically. Anion replacement experiments, experiments in the presence of potassium/valinomycin-induced diffusion potential as well as experiments with a potential-sensitive fluorescent dye document an electrogenic sodium-dependent uptake mechanism forl-cysteine. Tracer replacement experiments as well as the fluorescence experiments indicate a preferential transport ofl-cysteine. Transport ofl-cysteine is inhibited byl-alanine andl-phenylalanine but not byl-glutamic acid and thel-basic amino acids. Initial, linear influx kinetics provide evidence for the existence of two transport sites. The results suggest (a) sodium-dependent mechanism(s) forl-cysteine shared by other neutral amino acids.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01870338
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