Demaurex N, Orlowski J, Brisseau G, Woodside M, Grinstein S
Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.
J Gen Physiol. 1995 Jul;106(1):85-111. doi: 10.1085/jgp.106.1.85.
Na+/H+ exchange in vertebrates is thought to be electroneutral and insensitive to the membrane voltage. This basic concept has been challenged by recent reports of antiport-associated currents in the turtle colon epithelium (Post and Dawson, 1992, 1994). To determine the electrogenicity of mammalian antiporters, we used the whole-cell patch clamp technique combined with microfluorimetric measurements of intracellular pH (pHi). In murine macrophages, which were found by RT-PCR to express the NHE-1 isoform of the antiporter, reverse (intracellular Na(+)-driven) Na+/H+ exchange caused a cytosolic acidification and activated an outward current, whereas forward (extracellular Na(+)-driven) exchange produced a cytosolic alkalinization and reduced a basal outward current. The currents mirrored the changes in pHi, were strictly dependent on the presence of a Na+ gradient and were reversibly blocked by amiloride. However, the currents were seemingly not carried by the Na+/H+ exchanger itself, but were instead due to a shift in the voltage dependence of a preexisting H+ conductance. This was supported by measurements of the reversal potential (Erev) of tail currents, which identified H+ (equivalents) as the charge carrier. During Na+/H+ exchange, Erev changed along with the measured changes in pHi (by 60-69 mV/pH). Moreover, the current and Na+/H+ exchange could be dissociated. Zn2+, which inhibits the H+ conductance, reversibly blocked the currents without altering Na+/H+ exchange. In Chinese hamster ovary (CHO) cells, which lack the H+ conductance, Na+/H+ exchange produced pHi changes that were not accompanied by transmembrane currents. Similar results were obtained in CHO cells transfected with either the NHE-1, NHE-2, or NHE-3 isoforms of the antiporter, indicating that exchange through these isoforms is electroneutral. In all the isoforms tested, the amplitude and time-course of the antiport-induced pHi changes were independent of the holding voltage. We conclude that mammalian NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent. In cells endowed with a pH-sensitive H+ conductance, such as macrophages, activation of Na(+)-H+ exchange can modulate a transmembrane H+ current. The currents reported in turtle colon might be due to a similar "cross-talk" between the antiporter and a H+ conductance.
脊椎动物中的钠氢交换被认为是电中性的，且对膜电压不敏感。这一基本概念受到了近期关于龟结肠上皮中反向转运相关电流的报道的挑战（波斯特和道森，1992年，1994年）。为了确定哺乳动物反向转运体的电生性质，我们使用了全细胞膜片钳技术，并结合细胞内pH值（pHi）的微量荧光测量。在通过逆转录聚合酶链反应发现表达反向转运体NHE - 1亚型的小鼠巨噬细胞中，反向（细胞内钠离子驱动）钠氢交换导致胞质酸化并激活外向电流，而正向（细胞外钠离子驱动）交换则产生胞质碱化并降低基础外向电流。这些电流反映了pHi的变化，严格依赖于钠离子梯度的存在，并被氨氯吡咪可逆性阻断。然而，这些电流似乎并非由钠氢交换体本身携带，而是由于预先存在的氢离子电导的电压依赖性发生了改变。这一点得到了尾电流反转电位（Erev）测量结果的支持，该测量结果确定氢离子（当量）为电荷载体。在钠氢交换过程中，Erev随着测量到的pHi变化而改变（每pH变化60 - 69毫伏）。此外，电流和钠氢交换可以分离。抑制氢离子电导的锌离子可逆性阻断电流，而不改变钠氢交换。在中国仓鼠卵巢（CHO）细胞中，由于缺乏氢离子电导，钠氢交换产生的pHi变化不伴随跨膜电流。在用反向转运体的NHE - 1、NHE - 2或NHE - 3亚型转染的CHO细胞中也得到了类似结果，表明通过这些亚型的交换是电中性 的。在所有测试的亚型中，反向转运诱导的pHi变化的幅度和时间进程与钳制电压无关。我们得出结论，哺乳动物的NHE - 1、NHE - 2和NHE - 3是电中性且与电压无关的。在具有pH敏感氢离子电导的细胞中，如巨噬细胞，钠氢交换的激活可以调节跨膜氢离子电流。龟结肠中报道的电流可能是由于反向转运体和氢离子电导之间类似的“串扰”所致。
