Functional characterization of a Na⁺–phosphate cotransporter (NaP_i‐II) from zebrafish and identification of related transcripts

Abstract

Homeostasis of inorganic phosphate (Pé) in vertebrates is achieved by intestinal absorption and tightly controlled renal excretion (Danisi & Murer, 1991;Berndt & Knox, 1992). A protein family of Na¤-dependent phosphate cotransporters, denoted NaPé-II, is crucially involved in both steps Werner et al. 1998). The expression of NaPé-IIrelated transcripts has been demonstrated in kidney, brain and bone of different species (Hisano et al. 1997;Gupta et al. 1997). Only recently, the cloning of a second isoform, denoted NaPé-IIb, has been reported from Xenopus laevis, human and mouse intestine (Ishizuya-Oka et al. 1997;Hilfiker et al. 1998;Feild et al. 1999).A well-established model system for studying different aspects of NaPé-II-mediated Pé transport involves the winter flounder (Werner et al. 1994; Elger et al. 1998). This euryhaline fish expresses identical NaPé-II forms in kidney and intestine (Kohl et al. 1996). Furthermore, the transport protein can be directed to the apical or the basolateral side of renal tubular cells (Elger et al. 1998). This strategy enables the flounder kidney to either reabsorb or secrete Pé depending on physiological requirements. A comparable plasticity in protein sorting has been reported for NaPé-II in osteoclasts, where the transporter was confined to a basal compartment (Gupta et al. 1997). In order to combine the interesting (cell) physiological features of flounder NaPé-II with an established genetic background, we initiated a PCR-based strategy to clone NaPé-II-related cDNA(s) from zebrafish. Considering the predominant expression of flounder NaPé-II in intestine, the approach included the cognate RNA from zebrafish. Unexpectedly, two different NaPé-II-related isoforms and an antisense transcript were found. METHODS AnimalsZebrafish were bred in house. For the extraction of RNA, adult animals of about 4 cm were used. They were rendered hypothermic on ice, decapitated, and the organs of interest were removed. The pH in the gut was estimated by dripping 10-20 ìl of intestinal fluid derived from different segments on pH indicator paper (Merck, Darmstadt, Germany). Female clawed frogs (Xenopus laevis) were

Phosphate Transport Kinetics and Structure-Function Relationships of SLC34 and SLC20 Proteins

“…Moreover, 32 P uptake rates are independent of membrane potential ( Forster, Wagner et al 1997;Forster, Hernando et al 1998;Nalbant, Boehmer et al 1999; 12 Segawa, Kaneko et al 2002;Graham, Nalbant et al 2003;Forster, Virkki et al 2006;Ghezzi, Murer et al 2009)). The apparent affinity constants for total P i (K 0.5 Pi ) are typically <100 µM (pH 7.4 and [Na + ] =100 mM), with the exception of (i) a zebra fish isoform (NaPi-IIb1) (K 0.5 Pi =250 µM) (Graham, Nalbant et al 2003), and (ii) the murine and rat NaPi-IIb that exhibit exceptionally high apparent P i affinities (K 0.5 Pi ≈10 µM) (Forster, Villa-Bellosta&Sorribas 2008).…”

“…However, based on more recent studies and subsequent cloning of other SLC34 isoforms, it is unlikely that this motif represents the only proton interacting site in NaPi-IIa for several reasons. First, although the REK motif is conserved among all NaPi-IIa that display a strong pH dependence (Busch, Waldegger et al 1994;Busch, Wagner et al 1995;Hartmann, Wagner et al 1995;Forster, Hernando et al 1998;Forster, Biber et al 2000), it is present neither in the Zebra fish NaPiIIb1 (Nalbant, Boehmer et al 1999), nor NaPi-IIc, both of which show the phenotypically strong NaPi-IIa pH dependency (Segawa, Kaneko et al 2002). Second, in the flounder NaPiIIb that shows strong pH dependence (Forster, Biber et al 2000) and the zebra fish NaPi-IIb2, that shows weaker pH dependence (Graham, Nalbant et al 2003), the arginine is replaced by a neutral alanine.…”

“…: not determined; PFA: phosphonoformic acid a rat NaPi-IIa, flounder NaPi-IIb (Forster, Loo et al 1999), b mouse NaPi-IIc (Bacconi, Virkki et al 2005) c Xenopus PiT-1 (Ravera, Virkki et al 2007) d flounder NaPi-IIb (Andrini, Meinild et al 2012) e human NaPi-IIc (Segawa, Kaneko et al 2002) f human NaPi-IIa g rat NaPi-IIa (Forster, Hernando et al 1998) h mouse NaPi-IIb (Forster, Virkki et al 2006) i zebra fish NaPi-IIb2 (Graham, Nalbant et al 2003) j flounder NaPi-IIb (Forster, Wagner et al 1997) k zebra fish NaPi-IIb1 (Nalbant, Boehmer et al 1999) l mouse NaPi-IIc (Ghezzi, Murer et al 2009) m human PiT-1, rat PiT-2 (Kavanaugh, Miller et al 1994) n rat NaPi-IIa (Busch, Waldegger et al 1994) o L.V. Virkki, unpublished p rat NaPi-IIa/b/c (Villa-Bellosta&Sorribas 2008) q rat PiT-1,-2 (Villa-Bellosta, r rat NaPi-IIa, flounder NaPi-IIb (Andrini, Ghezzi et al 2008) Figure Legends (A) SLC34 (type II Na + /P i transporters).…”

Renouncing electroneutrality is not free of charge: Switching on electrogenicity in a Na + -coupled phosphate cotransporter

“…Our second strategy involved making a multiple sequence alignment of four currently available mammalian NaPi-IIc sequences and 17 sequences of NaPi-IIa as well as NaPi-IIb isoforms, several of which mediate P i -dependent electrogenic activity when expressed in oocytes (3,(16)(17)(18)(19). The alignment showed that, among the 21 sequences (excluding the N and C termini and ECL-2, where large variations are observed between isoforms), Ϸ75% of residues were 100% identical or in a few cases involved conservative substitutions (Fig.…”

Natural Antisense Transcripts

“…Our group has reported the identification of antisense RNAs related to the epithelial Na/phosphate cotransporter from zebrafish, mice and flounder. 21,59,60 The transporter, denoted NaPi-II, is instrumental in maintaining phosphate homeostasis in vertebrates. 61,62 The antisense transcripts from zebrafish have been cloned and extensively characterized, the mouse homologue(s) has just recently been detected by PCR and sequenced.…”