CLONING AND EXPRESSION OF A RENAL NA-P-I COTRANSPORT SYSTEM FROM FLOUNDER

Starting with the recently published sequence of the rat renal Na-P-i cotransport system, we have cloned a corresponding cDNA from the kidney of winter flounder (Pseudopleuronectes americanus), designated flounder NaPi-II. Expression of the cognate in vitro transcribed RNA in Xenopus laevis oocytes stimulated Na-dependent P-i transport specifically and in a time- and dose-dependent manner. Apparent affinities of Na and P-i, as well as the pH dependency, were very similar to those found for the mammalian systems. The flounder NaPi-II cDNA is 2,424 base pairs long and encodes a protein of 637 amino acids. The hydropathy plot predicts eight transmembrane spanning domains. In these regions the flounder NaPi-II-deduced protein shows high homology (similar to 80% identity, similar to 92% similarity) with the amino acid sequences reported for mammalian NaPi-II proteins. However, in the hydrophilic parts of flounder NaPi-II protein, only minimal similarity could be found between fish and mammalian systems (30% homology, 45% similarity). Northern blot analysis with flounder NaPi-II cDNA as a probe confirmed this finding: even under nonstringent washing conditions, no cross-hybridization with mRNA from rat renal cortex was observed. Interestingly, flounder intestine was found to contain high levels of mRNA. corresponding to NaPi-II. Supplementary bands of 1.9 and 4.2 kb were observed on Northern blots of renal and intestinal tissue. The close functional relationship of the flounder NaPi-II protein with the previously described Na-P-i cotransport systems and the pronounced differences on the level of their primary structures provide the tools for detailed structure-function analysis of Na-P-i cotransport.