Functional characterization of a Na+-phosphate cotransporter (NaPi-II) from zebrafish and identification of related transcripts.
1. We report the molecular identification of a Na+-Pi (inorganic phosphate) cotransport system of the NaPi-II protein family from zebrafish intestine. Following a PCR-related strategy, a DNA fragment from intestine-derived RNA was isolated. Rapid amplification of cDNA ends (3'- and 5'-RACE) resulted in the complete sequence (2607 bp) containing an open reading frame of 1893 bp. 2. The NaPi-II-related protein was expressed in Xenopus laevis oocytes and the resulting transport activity was analysed by electrophysiological means. The apparent Km for Pi was 250 microM (96 mM Na+, -60 mV), and voltage-dependent binding of Na+ exhibited a Km of 67.1 mM (1 mM Pi, -60 mV). 3. Interestingly, the overall transport activity was almost insensitive to changes in the holding potential. The apparent affinity for Na+ decreased under hyperpolarizing conditions, whereas Pi binding showed no voltage dependence. Transport activity was inhibited at low pH, which is characteristic for renal NaPi-II isoforms. 4. The expression of the NaPi-II-related isoform was addressed by reverse-transcription PCR. The mRNA could be detected in intestine, liver, eye and kidney. Unexpectedly, a second NaPi-II-related isoform was identified and found to be expressed in kidney, intestine, liver, brain, eye and prominently in testis. In addition, a shorter amplicon was demonstrated to be an antisense transcript related to the NaPi-II intestinal isoform.
Nalbant
P
P
Max-Planck-Institute for Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Boehmer
C
C
Dehmelt
L
L
Wehner
F
F
Werner
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004594:Electrophysiology; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D007700:Kinetics; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D016372:RNA, Antisense / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D050608:Sodium-Phosphate Cotransporter Proteins; D050614:Sodium-Phosphate Cotransporter Proteins, Type IIb; D027981:Symporters; D014018:Tissue Distribution; D014158:Transcription, Genetic / Q000235:genetics; D014982:Xenopus laevis; D015027:Zebrafish / Q000502:physiology*; D029961:Zebrafish Proteins
D002352:Carrier Proteins; D016372:RNA, Antisense; D012333:RNA, Messenger; D050608:Sodium-Phosphate Cotransporter Proteins; D050614:Sodium-Phosphate Cotransporter Proteins, Type IIb; D027981:Symporters; D029961:Zebrafish Proteins; C493560:slc34a2a protein, zebrafish; C493561:slc34a2b protein, zebrafish