Cloning and expression of a renal Na-Pi cotransport system from flounder

Starting with the recently published sequence of the rat renal Na-Pi 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 Pi transport specifically and in a time- and dose-dependent manner. Apparent affinities of Na and Pi, 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 (approximately 80%, identity, approximately 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-Pi cotransport systems and the pronounced differences on the level of their primary structures provide the tools for detailed structure-function analysis of Na-Pi cotransport.