Molecular cloning and functional characterization of swine sodium dependent phosphate cotransporter type II b (NaPi-IIb) gene

摘要

A sodium-dependent phosphate transporter gene, NaPi-IIb, was isolated from swine small intestine using cDNA library screening method. Sequencing analysis revealed that the NaPi-IIb cDNA sequences was 2,016 bp in length and encoded an open-reading frame consisting of 671 amino acids. The cDNA showed 83.1 % sequences identity to the human NaPi-IIb and 78.7 % sequences identity to the chicken NaPi-IIb. Prediction of membrane spanning domains based on the hydrophilic and hydrophobic properties of the amino acids suggested that a putative protein had nine transmembrane domains, with both the NH2 and COOH terminal being intracellular. By northern blot, a ~4.2 kb transcript was found to be abundantly expressed in mall intestine, lung, ovary, mammary glands, liver, kidney, salivary glands, placenta and thymus. Microinjection of swine NaPi-IIb cRNA into Xenopus oocytes demonstrated that the NaPi-IIb showed sodium-dependent Pi cotransport activity, and an approximate 31-fold increase of Pi uptake was seen in cRNA injected oocytes. The swine NaPi-IIb transporter expressed in Xenopus oocytes had a Km for Pi of ~79.35 ± 7.2 μM. Furthermore, the pH dependency characterization of swine NaPi-IIb transporter showed activation at extracellular alkaline-pH.