Boron W F, Fong P, Hediger M A, Boulpaep E L, Romero M F
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut, USA.
Wien Klin Wochenschr. 1997 Jun 27;109(12-13):445-56.
The electrogenic Na/HCO3 cotransporter (symporter) is the major HCO3- transporter of the renal proximal tubule (PiT), located at the basolateral membrane (BLM), and also plays a noteworthy role in Na+ reabsorption. HCO3 transporters are important for regulation of intracellular pH (pHi) in most cells and also thereby regulate blood pH. This electrogenic Na/HCO3 cotransporter was first discovered using perfused Ambystoma tigrinum (salamander) renal, proximal tubules. This novel cotransporter mediates the movement of one Na+ ion with several HCO3- ions, making it electrogenic, is blocked by stilbene compounds, but does not depend on intra- or extracellular Cl-. This and similar cotransporters have been found in a number of tissues and cell types. Recently, we used Xenopus-laevis oocytes to expression clone the salamander renal electrogenic Na Bicarbonate Cotransporter (NBC). Using microelectrodes to monitor membrane potential (Vm) and intracellular pH (pHi), we followed oocyte expression after injecting poly (A)+, fractioned poly (A)+, or cRNA. All experimental solutions contained 100 microM ouabain to block the Na+/K+ pump. Our expression assay was to apply 1.5% CO2/10 mM HCO3- (pH 7.5), allow pHi to stabilize from the CO2-induced acidification, and then remove bath Na+. Removing bath Na+ from native oocytes and water-injected controls, hyperpolarized the oocytes by approximately 5 mV and had no effect on pHi. However, for oocytes injected with poly (A)+ RNA, removing Na+ transiently depolarized the cell by approximately 10 mV and caused pHi to decrease; both effects were blocked by 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) and required HCO3-. Electrophoretic fractionation of the poly (A)+ RNA, enriched the expression signal. From the optimal expression-fraction, we constructed a size-selected cDNA library in pSPORT1. Screening our Ambystoma library yielded a single clone (aNBC). We could detect expression 3 days after injection of NBC cRNA. In aNBC-expressing oocytes, adding CO2/HCO3-elicited a large (> 50mV) and rapid hyperpolarization, followed by a partial relaxation as pHi stabilized. Na+ removal in CO2/HCO3-depolarized the cell by > 40mV and decreased pHi, aNBC encodes a protein of 1035 amino acids with several putative membrane-spanning domains, and has a low level of amino-acid homology (approximately 30% to the AE family of Cl-HCO3 exchangers. aNBC is the first member of a new family of Na(+)-linked HCO3- transporters and, together with the AE family, defines a new superfamily of HCO3- transporters. Using aNBC to screen a rat-kidney cDNA library, we identified a full-length cDNA clone (rNBC), rNBC encodes a protein of 1035 amino acids, is 86% identical to aNBC, and can be functionally expressed in oocytes.
电中性钠/碳酸氢根共转运体（同向转运体）是肾近端小管（PiT）主要的碳酸氢根转运体，位于基底外侧膜（BLM），在钠离子重吸收中也发挥着重要作用。碳酸氢根转运体对大多数细胞内pH（pHi）的调节很重要，进而调节血液pH。这种电中性钠/碳酸氢根共转运体最初是在灌注有虎纹钝口螈（蝾螈）肾近端小管中发现的。这种新型共转运体介导一个钠离子与几个碳酸氢根离子的移动，使其具有电中性，被芪类化合物阻断，但不依赖细胞内或细胞外的氯离子。在许多组织和细胞类型中都发现了这种及类似的共转运体。最近，我们利用非洲爪蟾卵母细胞表达克隆了蝾螈肾电中性钠碳酸氢根共转运体（NBC）。使用微电极监测膜电位（Vm）和细胞内pH（pHi），我们在注射多聚腺苷酸（poly(A)+）、分级分离的poly(A)+或cRNA后追踪卵母细胞的表达情况。所有实验溶液都含有100微摩尔哇巴因以阻断钠钾泵。我们的表达分析是施加1.5%二氧化碳/10毫摩尔碳酸氢根（pH 7.5），使pHi从二氧化碳诱导的酸化中稳定下来，然后去除浴液中的钠离子。从天然卵母细胞和注射水的对照中去除浴液中的钠离子，使卵母细胞超极化约5毫伏，对pHi没有影响。然而，对于注射了poly(A)+RNA的卵母细胞，去除钠离子会使细胞瞬时去极化约10毫伏，并导致pHi降低；这两种效应都被4,4'-二异硫氰酸-2,2'-二苯乙烯二磺酸（DIDS）阻断，且需要碳酸氢根。对poly(A)+RNA进行电泳分级分离，增强了表达信号。从最佳表达分级中，我们在pSPORT1中构建了一个大小选择的cDNA文库。筛选我们的钝口螈文库得到了一个单一克隆（aNBC）。注射NBC cRNA 3天后我们就能检测到表达。在表达aNBC的卵母细胞中，添加二氧化碳/碳酸氢根会引发一个大的（>50毫伏）快速超极化，随后随着pHi稳定而部分松弛。在二氧化碳/碳酸氢根存在的情况下去除钠离子会使细胞去极化>40毫伏并降低pHi，aNBC编码一个含有几个假定跨膜结构域的1035个氨基酸的蛋白质，与氯离子-碳酸氢根交换体的AE家族具有低水平的氨基酸同源性（约30%）。aNBC是钠连接碳酸氢根转运体新家族的第一个成员，与AE家族一起定义了一个新的碳酸氢根转运体超家族。利用aNBC筛选大鼠肾脏cDNA文库，我们鉴定出一个全长cDNA克隆（rNBC），rNBC编码一个1035个氨基酸的蛋白质，与aNBC有86%的同一性，并且可以在卵母细胞中功能性表达。
