Functional characterization of a Na+-phosphate cotransporter (NaPi-II) from zebrafish and identification of related...

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.