Abstract:

1. We report the molecular identification of a Na+-P(i) (inorganic phosphate) cotransport system of the NaP(i)-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. The NaP(i)-II-related protein was expressed in Xenopus laevis oocytes and the resulting transport activity was analysed by electrophysiological means. The apparent K(m) for P(i) was 250 μM (96 mM Na+, -60 mV), and voltage-dependent binding of Na+ exhibited a K(m) of 67.1 mM (1 mM P(i), -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 P(i) binding showed no voltage dependence. Transport activity was inhibited at low pH, which is characteristic for renal NaP(i)-II isoforms. 4. The expression of the NaP(i)-II-related isoform was addressed by reverse-transcription PCR. The mRNA could be detected in intestine, liver, eye and kidney. Unexpectedly, a second NaP(i)-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 NaP(i)-II intestinal isoform.