Functional characterization of a Na+-phosphate cotransporter (NaP1-II) from zebrafish and identification of related transcripts
Functional characterization of a Na+-phosphate cotransporter (NaP1-II) from zebrafish and identification of related transcripts
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DOI：
10.1111/j.1469-7793.1999.00079.x
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发表时间：
1999-10-01
影响因子：
5.5
通讯作者：
Werner, A
中科院分区：
文献类型：
--
作者：
Nalbant, P;Boehmer, C;Werner, A
1.本文报道了斑马鱼肠道Na ~+-P-i（无机磷酸盐）共转运蛋白家族的分子鉴定。在PCR相关策略之后，分离了来自马槟榔衍生的RNA的DNA片段。cDNA末端的快速扩增（3 '-和5'-RACE）产生包含1893 bp的开放阅读框的完整序列（2607 bp）。NaPi-II相关蛋白在非洲爪蟾卵母细胞中表达，并通过电生理手段分析所产生的运输活性。P-i的表观K-m为250 μ m（96 mM Na+ -60 mV），Na+的电压依赖性结合显示K-m为67.1 mM（1 mM P-i，-60 mM）。有趣的是，整体运输活动几乎是不敏感的保持电位的变化。在超极化条件下，对Na+的表观亲和力降低，而P-1结合没有显示电压依赖性。在低pH下转运活性被抑制，这是肾脏NaPi-II亚型的特征。通过逆转录PCR解决NaPi-II相关亚型的表达。在肠、肝、眼和肾中均可检测到该mRNA。出乎意料的是，第二个NaPi-II相关的同种型被鉴定并发现在肾、肠、肝、脑、眼中表达，并且在睾丸中显著表达。此外，一个较短的扩增子被证明是一个反义转录相关的NaPi-II肠亚型。
1. We report the molecular identification or a Na+-P-i (inorganic phosphate) cotransport system of the NaPi-II protein family from zebrafish intestine. Following a PCR-related strategy, a DNA fragment from intestine-derived RNA was isolated. Rapid amplification of cDNA ends (3'- and 5'-RACE) resulted in the complete sequence (2607 bp) containing an open reading frame of 1893 bp.2. The NaPi-II-related protein was expressed in Xenopus laevis oocytes and the resulting transport activity was analysed by electrophysiological means. The apparent K-m for P-i was 250 mu m (96 mM Na+ -60 mV), and voltage-dependent binding of Na+ exhibited a K-m of 67.1 mM (1 mM P-i, -60 mM).3. Interestingly, the overall transport activity was almost insensitive to changes in the holding potential. The apparent affinity for Na+ decreased under hyperpolarizing conditions, whereas P-i binding showed no voltage dependence. Transport activity was inhibited at low pH, which is characteristic for renal NaPi-II isoforms.4. The expression of the NaPi-II-related isoform was addressed by reverse-transcription PCR. The mRNA could be detected in intestine, liver, eye and kidney. Unexpectedly, a second NaPi-II-related isoform was identified and found to be expressed in kidney, intestine, liver, brain, eye and prominently in testis. In addition, a shorter amplicon was demonstrated to be an antisense transcript related to the NaPi-II intestinal isoform.