Dataset Information

Pentavalent arsenate transport by zebrafish phosphate transporter NaPi-IIb1.

ABSTRACT: Arsenate is a pentavalent form of arsenic that shares similar chemical properties to phosphate. It has been shown to be taken up by phosphate transporters in both eukaryotic and prokaryotic microbes such as yeast and Escherichia coli. Recently, the arsenate uptake in vertebrate cells was reported to be facilitated by mammalian type II sodium/phosphate transporter with different affinities. As arsenate is the most common form of arsenic exposure in aquatic system, identifying the uptake pathway of arsenate into aquatic animals is a crucial step in the elucidation of the entire metabolic pathway of arsenic. In this study, the ability of a zebrafish phosphate transporter, NaPi-IIb1 (SLC34a2a), to transport arsenate was examined. Our results demonstrate that a type II phosphate transporter in zebrafish, NaPi-IIb1, can transport arsenate in vitro when expressed in Xenopus laevis oocytes. NaPi-IIb1 mediates a high-affinity arsenate transport, with a K(m) of 0.22?mM. The natural substrate of NaPi-IIb1, dibasic phosphate, inhibits arsenate transport. Arsenate transport via NaPi-IIb1 is coupled with Na(+) and exhibits sigmoidal kinetics with a Hill coefficient of 3.24?±?0.19. Consistent with these in vitro studies, significant arsenate accumulation is observed in all examined zebrafish tissues where NaPi-IIb1 is expressed, particularly intestine, kidney, and eye, indicating that zebrafish NaPi-IIb1 is likely the transport protein that is responsible for arsenic accumulation in vivo.

SUBMITTER: Beene LC

PROVIDER: S-EPMC3174731 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Pentavalent arsenate transport by zebrafish phosphate transporter NaPi-IIb1.

Beene Lauren C LC Halluer Janell J Yoshinaga Masafumi M Hamdi Mohammad M Liu Zijuan Z

Zebrafish 20110819 3

Arsenate is a pentavalent form of arsenic that shares similar chemical properties to phosphate. It has been shown to be taken up by phosphate transporters in both eukaryotic and prokaryotic microbes such as yeast and Escherichia coli. Recently, the arsenate uptake in vertebrate cells was reported to be facilitated by mammalian type II sodium/phosphate transporter with different affinities. As arsenate is the most common form of arsenic exposure in aquatic system, identifying the uptake pathway o ...[more]

PMID: 21854209

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Project description:The rat renal brush border membrane sodium/phosphate co-transporter NaPi-2 was analysed in Western blots with polyclonal antibodies raised against its N-terminal and C-terminal segments. Under reducing conditions, proteins of 45-49 and 70-90 kDa (p45 and p70) were detected with N-terminal antibodies, and proteins of 40 and 70-90 kDa (p40 and p70) were detected with C-terminal antibodies. p40 and p45 apparently result from a post-translational cleavage of NaPi-2 but remain linked through one or more disulphide bonds. Glycosidase digestion showed that both polypeptides are glycosylated; the cleavage site could thus be located between Asn-298 and Asn-328, which have been shown to constitute the only two N-glycosylated residues in NaPi-2. In the absence of reducing agents, both N-terminal and C-terminal antibodies detected p70 and a protein of 180 kDa (p180), suggesting the presence of p70 dimers. Much higher concentrations of beta-mercaptoethanol were required to produce a given effect in intact membrane vesicles than in solubilized proteins, indicating that the affected disulphide bonds are not exposed at the surface of the co-transporter. Phosphate transport activity decreased with increasing concentrations of reducing agents [beta-mercaptoethanol, dithiothreitol and tris-(2-carboxyethyl)phosphine] and was linearly correlated with the amount of p180 detected. The target sizes estimated from the radiation-induced loss of intensity of p40, p70 and p180 were all approx. 190 kDa, suggesting that NaPi-2 exists as an oligomeric protein in which the subunits are sufficiently close to one another to allow substantial energy transfer between the monomers. When protein samples were pretreated with beta-mercaptoethanol [2.5% and 5% (v/v) to optimize the detection of p40 and p70] before irradiation, target sizes estimated from the radiation-induced loss of intensity of p40 and p70 were 74 and 92 kDa respectively, showing the presence of disulphide bridges in the molecular structure of NaPi-2.

Project description:Phosphate plays essential biological roles and its plasma level in humans requires tight control to avoid bone loss (insufficiency) or vascular calcification (excess). Intestinal absorption and renal reabsorption of phosphate are mediated by members of the SLC34 family of sodium-coupled transporters (NaPi-IIa,b,c) whose membrane expression is regulated by various hormones, circulating proteins, and phosphate itself. Consequently, NaPi-II proteins are also potentially important pharmaceutical targets for controlling phosphate levels. Their crucial role in Pi homeostasis is underscored by pathologies resulting from naturally occurring SLC34 mutations and SLC34 knockout animals. SLC34 isoforms have been extensively studied with respect to transport mechanism and structure-function relationships; however, the three-dimensional structure is unknown. All SLC34 transporters share a duplicated motif comprising a glutamine followed by a stretch of threonine or serine residues, suggesting the presence of structural repeats as found in other transporter families. Nevertheless, standard bioinformatic approaches fail to clearly identify a suitable template for molecular modeling. Here, we used hydrophobicity profiles and hidden Markov models to define a structural repeat common to all SLC34 isoforms. Similar approaches identify a relationship with the core regions in a crystal structure of Vibrio cholerae Na(+)-dicarboxylate transporter VcINDY, from which we generated a homology model of human NaPi-IIa. The aforementioned SLC34 motifs in each repeat localize to the center of the model, and were predicted to form Na(+) and Pi coordination sites. Functional relevance of key amino acids was confirmed by biochemical and electrophysiological analysis of expressed, mutated transporters. Moreover, the validity of the predicted architecture is corroborated by extensive published structure-function studies. The model provides key information for elucidating the transport mechanism and predicts candidate substrate binding sites.

Dataset Information

Pentavalent arsenate transport by zebrafish phosphate transporter NaPi-IIb1.

Publications

Pentavalent arsenate transport by zebrafish phosphate transporter NaPi-IIb1.

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