Electrogenic Na/HCO3 cotransporter (NBCe1) variants expressed in Xenopus oocytes: functional comparison and roles of the amino and carboxy termini.
Electrogenic Na/HCO3 cotransporter (NBCe1) variants expressed in Xenopus oocytes: functional comparison and roles of the amino and carboxy termini.
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DOI：
10.1085/jgp.200609520
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发表时间：
2006-06
期刊：
影响因子：
--
通讯作者：
Bevensee MO
中科院分区：
文献类型：
--
作者：
McAlear SD;Liu X;Williams JB;McNicholas-Bevensee CM;Bevensee MO
使用pH和电压敏感的微电极，以及双电极电压钳和macropatch技术，我们比较了三个NBCe 1变体（NBCe 1-A，-B，和-C）的功能特性与不同的氨基和/或羧基末端在非洲爪蟾卵母细胞中表达。表达大鼠脑NBCe 1-B并暴露于CO2/HCO 3 −溶液的卵母细胞显示出产电Na+/HCO 3 −共转运蛋白的所有特征：（a）初始CO2诱导酸化后的DIDS敏感性pHi恢复，（B）瞬时超极化，（c）电压钳条件下的瞬时Na+依赖性外向电流（−60 mV）。所有三种变体都具有相似的外部HCO 3 −依赖性（表观KM为4-6 mM）和外部Na+依赖性（表观KM为21-36 mM），以及相似的电压依赖性。然而，表达NBCe 1-A的电压钳位卵母细胞（-60 mV）显示出HCO 3-刺激的NBC电流峰值，比表达最不相似的C变体的卵母细胞中观察到的电流大4.3倍。在电流-电压关系中也观察到较大的NBCe 1-A电流。质膜表达水平评估单卵母细胞化学发光与血凝素标记的NBCs是相似的三个变种。在全细胞实验（Vm = −60 mV）中，去除NBCe 1-A的独特氨基末端使HCO 3 −诱导的平均NBC电流降低55%，而去除NBCe 1-C的不同氨基末端使平均NBC电流增加2.7倍。在macropatch实验中观察到类似的模式。因此，NBCe 1-A的独特氨基末端刺激转运蛋白活性，而B和C变体的不同氨基末端抑制活性。一个或多个胞质因子也可能有助于NBCe 1活性的基础上的差异macropatch和全细胞电流。氨基末端影响转运蛋白功能，羧基末端影响质膜表达。去除NBCe 1-C的整个胞质羧基末端或A/B变体的不同羧基末端，由于质膜上的低表达而导致NBC活性的丧失。
Using pH- and voltage-sensitive microelectrodes, as well as the two-electrode voltage-clamp and macropatch techniques, we compared the functional properties of the three NBCe1 variants (NBCe1-A, -B, and -C) with different amino and/or carboxy termini expressed in Xenopus laevis oocytes. Oocytes expressing rat brain NBCe1-B and exposed to a CO2/HCO3 − solution displayed all the hallmarks of an electrogenic Na+/HCO3 − cotransporter: (a) a DIDS-sensitive pHi recovery following the initial CO2-induced acidification, (b) an instantaneous hyperpolarization, and (c) an instantaneous Na+-dependent outward current under voltage-clamp conditions (−60 mV). All three variants had similar external HCO3 − dependencies (apparent KM of 4–6 mM) and external Na+ dependencies (apparent KM of 21–36 mM), as well as similar voltage dependencies. However, voltage-clamped oocytes (−60 mV) expressing NBCe1-A exhibited peak HCO3 −-stimulated NBC currents that were 4.3-fold larger than the currents seen in oocytes expressing the most dissimilar C variant. Larger NBCe1-A currents were also observed in current–voltage relationships. Plasma membrane expression levels as assessed by single oocyte chemiluminescence with hemagglutinin-tagged NBCs were similar for the three variants. In whole-cell experiments (Vm = −60 mV), removing the unique amino terminus of NBCe1-A reduced the mean HCO3 −-induced NBC current 55%, whereas removing the different amino terminus of NBCe1-C increased the mean NBC current 2.7-fold. A similar pattern was observed in macropatch experiments. Thus, the unique amino terminus of NBCe1-A stimulates transporter activity, whereas the different amino terminus of the B and C variants inhibits activity. One or more cytosolic factors may also contribute to NBCe1 activity based on discrepancies between macropatch and whole-cell currents. While the amino termini influence transporter function, the carboxy termini influence plasma membrane expression. Removing the entire cytosolic carboxy terminus of NBCe1-C, or the different carboxy terminus of the A/B variants, causes a loss of NBC activity due to low expression at the plasma membrane.
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