斑马鱼钠-磷酸盐共转运体（NaPi-II）的功能特性及相关转录本的鉴定

Functional characterization of a Na+-phosphate cotransporter (NaPi-II) from zebrafish and identification of related transcripts.

Nalbant P, Boehmer C, Dehmelt L, Wehner F, Werner A

Max-Planck-Institute for Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.

J Physiol. 1999 Oct 1;520 Pt 1(Pt 1):79-89. doi: 10.1111/j.1469-7793.1999.00079.x.

Abstract

We report the molecular identification of a Na+-Pi (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 Km for Pi was 250 microM (96 mM Na+, -60 mV), and voltage-dependent binding of Na+ exhibited a Km of 67.1 mM (1 mM Pi, -60 mV). 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 Pi 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.

摘要

我们报道了斑马鱼肠道中NaPi-II蛋白家族的一种Na⁺-Pi（无机磷酸盐）共转运系统的分子鉴定。采用与PCR相关的策略，从肠道来源的RNA中分离出一个DNA片段。通过cDNA末端快速扩增（3'-和5'-RACE）得到了完整序列（2607 bp），其中包含一个1893 bp的开放阅读框。
NaPi-II相关蛋白在非洲爪蟾卵母细胞中表达，并通过电生理方法分析产生的转运活性。Pi的表观Km为250 μM（96 mM Na⁺，-60 mV），Na⁺的电压依赖性结合表现出Km为67.1 mM（1 mM Pi，-60 mV）。
有趣的是，总体转运活性几乎不受保持电位变化的影响。在超极化条件下，对Na⁺的表观亲和力降低，而Pi结合没有电压依赖性。转运活性在低pH下受到抑制，这是肾脏NaPi-II同工型的特征。
通过逆转录PCR研究了NaPi-II相关同工型的表达。在肠道、肝脏、眼睛和肾脏中可以检测到mRNA。出乎意料的是，鉴定出了第二种NaPi-II相关同工型，发现它在肾脏、肠道、肝脏、大脑、眼睛中表达，在睾丸中表达尤为突出。此外，一个较短的扩增子被证明是与NaPi-II肠道同工型相关的反义转录本。