Identification of a mammalian silicon transporter.

Silicon (Si) has long been known to play a major physiological and structural role in certain organisms, including diatoms, sponges, and many higher plants, leading to the recent identification of multiple proteins responsible for Si transport in a range of algal and plant species. In mammals, despite several convincing studies suggesting that silicon is an important factor in bone development and connective tissue health, there is a critical lack of understanding about the biochemical pathways that enable Si homeostasis. Here we report the identification of a mammalian efflux Si transporter, namely Slc34a2 (also termed NaPiIIb), a known sodium-phosphate cotransporter, which was upregulated in rat kidney following chronic dietary Si deprivation. Normal rat renal epithelium demonstrated punctate expression of Slc34a2, and when the protein was heterologously expressed in Xenopus laevis oocytes, Si efflux activity (i.e., movement of Si out of cells) was induced and was quantitatively similar to that induced by the known plant Si transporter OsLsi2 in the same expression system. Interestingly, Si efflux appeared saturable over time, but it did not vary as a function of extracellular [Formula: see text] or Na+ concentration, suggesting that Slc34a2 harbors a functionally independent transport site for Si operating in the reverse direction to the site for phosphate. Indeed, in rats with dietary Si depletion-induced upregulation of transporter expression, there was increased urinary phosphate excretion. This is the first evidence of an active Si transport protein in mammals and points towards an important role for Si in vertebrates and explains interactions between dietary phosphate and silicon.

Cation Interactions and Membrane Potential Induce Conformational Changes in NaPi-IIb.

Voltage-dependence of Na(+)-coupled phosphate cotransporters of the SLC34 family arises from displacement of charges intrinsic to the protein and the binding/release of one Na(+) ion in response to changes in the transmembrane electric field. Candidate coordination residues for the cation at the Na1 site were previously predicted by structural modeling using the x-ray structure of dicarboxylate transporter VcINDY as template and confirmed by functional studies. Mutations at Na1 resulted in altered steady-state and presteady-state characteristics that should be mirrored in the conformational changes induced by membrane potential changes. To test this hypothesis by functional analysis, double mutants of the flounder SLC34A2 protein were constructed that contain one of the Na1-site perturbing mutations together with a substituted cysteine for fluorophore labeling, as expressed in Xenopus oocytes. The locations of the mutations were mapped onto a homology model of the flounder protein. The effects of the mutagenesis were characterized by steady-state, presteady-state, and fluorometric assays. Changes in fluorescence intensity (ΔF) in response to membrane potential steps were resolved at three previously identified positions. These fluorescence data corroborated the altered presteady-state kinetics upon perturbation of Na1, and furthermore indicated concomitant changes in the microenvironment of the respective fluorophores, as evidenced by changes in the voltage dependence and time course of ΔF. Moreover, iodide quenching experiments indicated that the aqueous nature of the fluorophore microenvironment depended on the membrane potential. These findings provide compelling evidence that membrane potential and cation interactions induce significant large-scale structural rearrangements of the protein.

Molecular cloning and functional characterization of swine sodium dependent phosphate cotransporter type II b (NaPi-IIb) gene.

A sodium-dependent phosphate transporter gene, NaPi-IIb, was isolated from swine small intestine using cDNA library screening method. Sequencing analysis revealed that the NaPi-IIb cDNA sequences was 2,016 bp in length and encoded an open-reading frame consisting of 671 amino acids. The cDNA showed 83.1 % sequences identity to the human NaPi-IIb and 78.7 % sequences identity to the chicken NaPi-IIb. Prediction of membrane spanning domains based on the hydrophilic and hydrophobic properties of the amino acids suggested that a putative protein had nine transmembrane domains, with both the NH(2) and COOH terminal being intracellular. By northern blot, a ~4.2 kb transcript was found to be abundantly expressed in mall intestine, lung, ovary, mammary glands, liver, kidney, salivary glands, placenta and thymus. Microinjection of swine NaPi-IIb cRNA into Xenopus oocytes demonstrated that the NaPi-IIb showed sodium-dependent Pi cotransport activity, and an approximate 31-fold increase of Pi uptake was seen in cRNA injected oocytes. The swine NaPi-IIb transporter expressed in Xenopus oocytes had a Km for Pi of ~79.35 ± 7.2 µM. Furthermore, the pH dependency characterization of swine NaPi-IIb transporter showed activation at extracellular alkaline-pH.

In silico analysis and immunohistochemical characterization of NaPi2b protein expression in ovarian carcinoma with monoclonal antibody Mx35.

INTRODUCTION: Ovarian adenocarcinoma is frequently detected at the late stage, when therapy efficacy is limited and death occurs in up to 50% of the cases. A potential novel treatment for this disease is a monoclonal antibody that recognizes phosphate transporter sodium-dependent phosphate transporter protein 2b (NaPi2b). MATERIALS AND METHODS: To better understand the expression of this protein in different histologic types of ovarian carcinomas, we immunostained 50 tumor samples with anti-NaPi2b monoclonal antibody MX35 and, in parallel, we assessed the expression of the gene encoding NaPi2b (SCL34A2) by in silico analysis of microarray data. RESULTS: Both approaches detected higher expression of NaPi2b (SCL34A2) in ovarian carcinoma than in normal tissue. Moreover, a comprehensive analysis indicates that SCL34A2 is the only gene of the several phosphate transporters genes whose expression differentiates normal from carcinoma samples, suggesting it might exert a major role in ovarian carcinomas. Immunohistochemical and mRNA expression data have also shown that 2 histologic subtypes of ovarian carcinoma express particularly high levels of NaPi2b: serous and clear cell adenocarcinomas. Serous adenocarcinomas are the most frequent, contrasting with clear cell carcinomas, rare, and with worse prognosis. CONCLUSION: This identification of subgroups of patients expressing NaPi2b may be important in selecting cohorts who most likely should be included in future clinical trials, as a recently generated humanized version of MX35 has been developed.

Characterization of the chicken small intestine type IIb sodium phosphate cotransporter.

Intestinal absorption and renal resorption play a critical role in overall phosphorus homeostasis in chickens. Using RNase-ligase-mediated rapid amplification of cDNA ends PCR, we obtained a cDNA from the broiler small intestine that encodes a type IIb Na-dependent phosphate transporter. The cDNA has an open reading frame of 2,022 bp and predicts a 674-amino acid protein with a molecular mass of approximately 74 kDa. Prediction of membrane spanning domains based on the hydrophilic and hydrophobic properties of the amino acids suggests 8 transmembrane domains, with both the NH(2) and COOH termini being intracellular. The Na-inorganic phosphate (Pi) IIb cotransporter has relative high homology with other type II Na-Pi cotransporters but low homology with the type I or type III Na-Pi cotransporters. Northern blot analysis demonstrated the presence of a single mRNA transcript present predominantly in the small intestine, with the highest expression in the duodenum, followed by the jejunum and ileum. In situ hybridization indicated that the Na-Pi cotransporter mRNA is expressed throughout the vertical cryptvillus axis of the small intestine. Reduction of P in the diet of chicks from hatch to 4 d of age resulted in a significant induction of Na-Pi cotransporter mRNA expression in the small intestine. Further study is needed to elucidate its physiological role in intestinal phosphate absorption in chickens.

Proximal tubular handling of phosphate: A molecular perspective.

Members of the SLC34 gene family of solute carriers encode for three Na+-dependent phosphate (P i) cotransporter proteins, two of which (NaPi-IIa/SLC34A1 and NaPi-IIc/SLC34A3) control renal reabsorption of P i in the proximal tubule of mammals, whereas NaPi-IIb/SCLC34A2 mediates P i transport in organs other than the kidney. The P i transport mechanism has been extensively studied in heterologous expression systems and structure-function studies have begun to reveal the intricacies of the transport cycle at the molecular level using techniques such as cysteine scanning mutagenesis, and voltage clamp fluorometry. Moreover, sequence differences between the three types of cotransporters have been exploited to obtain information about the molecular determinants of hormonal sensitivity and electrogenicity. Renal handling of P i is regulated by hormonal and non-hormonal factors. Changes in urinary excretion of P i are almost invariably mirrored by changes in the apical expression of NaPi-IIa and NaPi-IIc in proximal tubules. Therefore, understanding the mechanisms that control the apical expression of NaPi-IIa and NaPi-IIc as well as their functional properties is critical to understanding how an organism achieves P i homeostasis.

Mapping conformational changes of a type IIb Na+/Pi cotransporter by voltage clamp fluorometry.

The fluorescence of a fluorophore depends on its environment, and if attached to a protein it may report on conformational changes. We have combined two-electrode voltage clamp with simultaneous fluorescence measurements to detect conformational changes in a type IIb Na(+)/P(i) cotransporter expressed in Xenopus oocytes. Four novel Cys, labeled with a fluorescent probe, yielded voltage- and substrate-dependent changes in fluorescence (F). Neither Cys substitution nor labeling significantly altered the mutant electrogenic properties. Different F responses to voltage and substrate were recorded at the four sites. S155C, located in an intracellular re-entrant loop in the first half of the protein, and E451C, located in an extracellular re-entrant loop in the second half of the protein, both showed Na(+), Li(+), and P(i)-dependent F signals. S226C and Q319C, located at opposite ends of a large extracellular loop in the middle of the protein, mainly responded to changes in Na(+) and Li(+). Hyperpolarization increased F for S155C and S226C but decreased F for Q319C and E451C. The labeling and F response of S155C, confirmed that the intracellular loop containing Ser-155 is re-entrant as it is accessible from the extracellular milieu. The behavior of S155C and E451C indicates a strong involvement of the two re-entrant loops in conformational changes during the transport cycle. Moreover, the data for S226C and Q319C suggest that also the large extracellular loop is associated with transport function. Finally, the reciprocal voltage dependences of the S155C-E451C and S226C-Q319C pairs suggest reciprocal conformational changes during the transport cycle for their respective local environments.