Graham C, Nalbant P, Schölermann B, Hentschel H, Kinne R K H, Werner A
School of Cell and Molecular Biosciences, University of Newcastle, Newcastle upon Tyne, NE2 4HH, United Kingdom.
Am J Physiol Renal Physiol. 2003 Apr;284(4):F727-36. doi: 10.1152/ajprenal.00356.2002. Epub 2002 Dec 17.
Zebrafish (Danio rerio) express two isoforms of the type IIb Na-dependent P(i) cotransporter (NaPi). 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 its functional characterization. RT-PCR with renal RNA and degenerate NaPi-IIb-specific primers resulted in a specific fragment. 3'-Rapid amplification of cDNA ends yielded a product that contained typical NaPi-IIb characteristics such as a cysteine-rich COOH terminus and a PDZ (PSD95- Dlg-zona occludens-1) 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 ). The combined clone displayed a high degree of homology with published type IIb cotransporter sequences. Specific antibodies were raised against a COOH-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 was 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.
斑马鱼（Danio rerio）表达两种IIb型钠依赖性磷酸转运体（NaPi）的同工型。之前已经克隆并鉴定了NaPi-IIb1型。在此，我们报告了从斑马鱼肾脏中克隆NaPi-IIb2转录本、其定位及其功能特性。用肾脏RNA和简并性NaPi-IIb特异性引物进行逆转录聚合酶链反应（RT-PCR）产生了一个特异性片段。3'-cDNA末端快速扩增产生了一个产物，该产物包含典型的NaPi-IIb特征，如富含半胱氨酸的COOH末端和一个PDZ（PSD95-Dlg-紧密连接蛋白-1）结合基序。几种方法在克隆该转录本的5'末端时均未成功；产物缺乏读码框起始密码子。缺失的信息从一个EST（基因银行登录号）中获得。合并后的克隆与已发表的IIb型共转运体序列显示出高度同源性。针对NaPi-IIb1和NaPi-IIb2同工型的COOH末端表位制备了特异性抗体。免疫组织化学定位显示这两种同工型在斑马鱼肾脏、肠道上皮以及胆管的顶端表达。新克隆在卵母细胞中表达，并通过双电极电压钳技术测定其功能。尽管结构上有很强的相似性，但发现新的NaPi-IIb2克隆的功能与NaPi-IIb1有显著差异。发现NaPi-IIb2对电压非常敏感，对钠和磷酸盐的亲和力均高于NaPi-IIb1。此外，NaPi-IIb2对外界pH的敏感性明显低于NaPi-IIb1。强大的结构相似性但功能不同使得这些斑马鱼转运体成为II型NaPi共转运体家族中功能重要区域分子定位的理想模型。
