Functional characterization of human NBC4 as an electrogenic Na+-HCO3- cotransporter (NBCe2)
Functional characterization of human NBC4 as an electrogenic Na+-HCO3- cotransporter (NBCe2)
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DOI：
10.1152/ajpcell.00589.2001
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发表时间：
2002-06-01
影响因子：
5.5
通讯作者：
Boron, WF
中科院分区：
文献类型：
--
作者：
Virkki, LV;Wilson, DA;Boron, WF
我们对Na+驱动的碳酸氢盐转运蛋白（NBC） 4进行了功能表征，该转运蛋白最初是由Pushkin等人从人类心脏中克隆出来的（Pushkin A, Abuladze N, Newman D, Lee I, Xu G, and Kurtz I.生物化学生物物理学报，1493:215-218,2000）。在GenBank中目前存在的四个NBC4变体中，我们自己的克隆努力只产生了变体c。AF293337)在非洲爪蟾卵母细胞中的表达，并利用微电极检测膜电位（V-m）和pH调节功能。将表达nbc4c的卵母细胞暴露在含有5% CO2和33 mM HCO3-的溶液中，会引起大的超极化，表明该转运体是电致的。最初由二氧化碳引起的细胞内pH值（pH(i)）下降，随后缓慢恢复，并通过去除外部Na+而逆转。表达nbc4c的卵母细胞的双电极电压钳显示出较大的HCO3-和Na+依赖电流。当我们箝位V-m远离NBC4c的估计反转电位（E-rev）时，pH(i)回收率大幅增加。200 mma 4,4'-二异硫氰基二苯乙烯-2,2'-二磺酸（DIDS）阻断了电流和pHi回收率。我们通过测量不同细胞外Na+浓度（[Na+](o)）值下的E-rev来估计转运体的HCO3-:Na+化学计量学。Erev与log[Na+](o)呈线性关系，斜率为54.8 mV/ log[Na+](o)。这一观察结果，以及绝对E-rev值，符合2:1的化学计量。综上所述，NBC4c（我们建议将其称为NBCe2-c）的行为与第一个电致NBC NBCe1相似。
We have functionally characterized Na+-driven bicarbonate transporter (NBC) 4, originally cloned from human heart by Pushkin et al. (Pushkin A, Abuladze N, Newman D, Lee I, Xu G, and Kurtz I. Biochem Biophys Acta 1493: 215-218, 2000). Of the four NBC4 variants currently present in GenBank, our own cloning efforts yielded only variant c. We expressed NBC4c (GenBank accession no. AF293337) in Xenopus laevis oocytes and assayed membrane potential (V-m) and pH regulatory function with microelectrodes. Exposing an NBC4c-expressing oocyte to a solution containing 5% CO2 and 33 mM HCO3- elicited a large hyperpolarization, indicating that the transporter is electrogenic. The initial CO2-induced decrease in intracellular pH (pH(i)) was followed by a slow recovery that was reversed by removing external Na+. Two-electrode voltage clamp of NBC4c-expressing oocytes revealed large HCO3- and Na+-dependent currents. When we voltage clamped V-m far from NBC4c's estimated reversal potential (E-rev), the pH(i) recovery rate increased substantially. Both the currents and pHi recovery were blocked by 200 muM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). We estimated the transporter's HCO3- :Na+ stoichiometry by measuring E-rev at different extracellular Na+ concentration ([Na+](o)) values. A plot of Erev against log[Na+](o) was linear, with a slope of 54.8 mV/ log[Na+](o). This observation, as well as the absolute E-rev values, are consistent with a 2: 1 stoichiometry. In conclusion, the behavior of NBC4c, which we propose to call NBCe2-c, is similar to that of NBCe1, the first electrogenic NBC.