Graham, C., Nalbant, P., Scholermann, B., Hentschel, H., Kinne, R.K., and Werner, A. (2003) Characterization of a type IIb sodium-phosphate cotransporter from zebrafish (Danio rerio) kidney. American journal of physiology. Renal physiology. 284(4):F727-736.
Zebrafish (Danio rerio) express two isoforms of the type IIb NaPi cotransporter. Type NaPi-IIb1 has previously been cloned and characterized. Here, we report the cloning of the NaPi-IIb2 transcript from zebrafish kidney, its localization and functional characterization. RT-PCR with renal RNA and degenerate NaPi-IIb specific primers resulted in a specific fragment. 3' RACE yielded a product which contained typical NaPi-IIb characteristics such as a cysteine rich C-terminus and a PDZ binding motif. Several approaches were unsuccessful at cloning the 5' end of the transcript; products lacked an in frame start codon. The missing information was obtained from an EST (GenBank accession number AW423104). The combined clone displayed a high degree of homology with published type IIb cotransporter sequences. Specific antibodies were raised against a C-terminal epitope of both NaPi-IIb1 and NaPi-IIb2 isoforms. Immunohistochemical mapping revealed apical expression of both isoforms in zebrafish renal and intestinal epithelia, as well as in bile ducts. The novel clone was expressed in oocytes and function assayed by the two electrode voltage clamp technique. The function of the new NaPi-IIb2 clone was found to be significantly different from NaPi-IIb1 despite strong structural similarities. NaPi-IIb2 was found to be strongly voltage-sensitive with higher affinities for both sodium and phosphate than NaPi-IIb1. Also, NaPi-IIb2 was significantly less-sensitive to external pH than NaPi-IIb1. The strong structural similarity but divergent function makes these zebrafish transporters ideal models for the molecular mapping of functionally important regions in the type II NaPi-cotransporter family.