Romero M F, Fong P, Berger U V, Hediger M A, Boron W F
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Am J Physiol. 1998 Feb;274(2):F425-32. doi: 10.1152/ajprenal.1998.274.2.F425.
We have recently cloned the renal electrogenic Na(+)-bicarbonate contransporter of the salamander Ambystoma tigrinum (aNBC) (M. F. Romero, M. A. Hediger, E. L. Boulpaep, and W. F. Boron. FASEB J. 10: 89, 1996; and Nature 387: 409-413, 1997). Here we report the cloning of a mammalian homolog of aNBC, named rNBC for rat Na(+)-bicarbonate cotransporter. NBC constitutes the major route for HCO3- reabsorption and assists in Na+ reabsorption across the basolateral membrane of the renal proximal tubule (PT). We used aNBC as a probe to screen a rat kidney cortex cDNA library in lambda gt10 and identified several clones. Each has an initiator Met and a large open-reading frame followed by a 3'-untranslated region of approximately 500 bp. The 7.5-kb mRNA for rNBC is present in kidney, liver, lung, brain, and heart. In situ hybridization with the rNBC probe in the rat kidney revealed staining in the S2 segment of PT. rNBC encodes a protein of 1,035 amino acids, with a predicted molecular mass of 116 kDa. Its deduced amino acid sequence is 86% identical to that of aNBC. Comparison of both the aNBC and rNBC sequences to the GenBank database reveals a low level of amino acid identity (approximately 30%) to the AE family of Cl-/HCO3- exchangers. Injection of rNBC cRNA into Xenopus oocytes leads to expression of an electrogenic Na(+)-HCO3- contransporter that is qualitatively similar to that of aNBC but at a much lower level. Placement of the rNBC cDNA into the context of a Xenopus expression vector produces a substantial increase in rNBC expression. Addition of 1.5% CO2/10 mM HCO3- elicits a hyperpolarization of > 50 mV and a rapid decrease of intracellular pH (pHi), followed by an increase in pHi. Subsequent removal of Na+ in the presence of CO2/HCO3- causes a depolarization of > 50 mV and a concomitant decrease of pHi. Thus rNBC is in the same newly identified family of Na(+)-linked HCO3- transporters as is aNBC.
我们最近克隆了虎纹钝口螈（Ambystoma tigrinum）的肾脏电中性钠-碳酸氢根共转运体（aNBC）（M. F. 罗梅罗、M. A. 赫迪格、E. L. 布尔帕普和W. F. 博龙。《美国实验生物学会联合会杂志》10: 89, 1996；以及《自然》387: 409 - 413, 1997）。在此我们报告了aNBC的一种哺乳动物同源物的克隆，将其命名为rNBC，即大鼠钠-碳酸氢根共转运体。NBC构成了肾脏近端小管（PT）基底外侧膜上HCO₃⁻重吸收的主要途径，并协助Na⁺重吸收。我们用aNBC作为探针筛选λgt10载体中的大鼠肾皮质cDNA文库，鉴定出了几个克隆。每个克隆都有一个起始甲硫氨酸和一个大的开放阅读框，后面跟着一个约500 bp的3'非翻译区。rNBC的7.5 kb mRNA存在于肾脏、肝脏、肺、脑和心脏中。用rNBC探针在大鼠肾脏中进行原位杂交显示在近端小管的S2段有染色。rNBC编码一个1035个氨基酸的蛋白质，预测分子量为116 kDa。其推导的氨基酸序列与aNBC的序列有86%的同一性。将aNBC和rNBC的序列与GenBank数据库进行比较发现，它们与Cl⁻/HCO₃⁻交换体的AE家族的氨基酸同一性水平较低（约30%）。将rNBC的cRNA注射到非洲爪蟾卵母细胞中会导致一种电中性钠-碳酸氢根共转运体的表达，其性质与aNBC相似，但水平要低得多。将rNBC的cDNA插入非洲爪蟾表达载体中会使rNBC的表达大幅增加。添加1.5%的CO₂/10 mM HCO₃⁻会引起超过50 mV的超极化和细胞内pH（pHi）的快速下降，随后pHi升高。随后在有CO₂/HCO₃⁻存在的情况下去除Na⁺会导致超过50 mV的去极化和pHi的相应下降。因此，rNBC与aNBC属于同一个新发现的钠-连接的HCO₃⁻转运体家族。
