DeCoursey T E, Cherny V V
Department of Physiology, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612.
J Gen Physiol. 1994 May;103(5):755-85. doi: 10.1085/jgp.103.5.755.
Voltage-activated H(+)-selective currents were studied in cultured adult rat alveolar epithelial cells and in human neutrophils using the whole-cell configuration of the patch-clamp technique. The H+ conductance, gH, although highly selective for protons, was modulated by monovalent cations. In Na+ and to a smaller extent in Li+ solutions, H+ currents were depressed substantially and the voltage dependence of activation of the gH shifted to more positive potentials, when compared with the "inert" cation tetramethylammonium (TMA+). The reversal potential of the gH, Vrev, was more positive in Na+ solutions than in inert ion solutions. Amiloride at 100 microM inhibited H+ currents in the presence of all cations studied except Li+ and Na+, in which it increased H+ currents and shifted their voltage-dependence and Vrev to more negative potentials. The more specific Na(+)-H+ exchange inhibitor dimethylamiloride (DMA) at 10 microM similarly reversed most of the suppression of the gH by Na+ and Li+. Neither 500 microM amiloride nor 200 microM DMA added internally via the pipette solution were effective. Distinct inhibition of the gH was observed with 1% [Na+]o, indicating a mechanism with high sensitivity. Finally, the effects of Na+ and their reversal by amiloride were large when the proton gradient was outward (pHo parallel pHi 7 parallel 5.5), smaller when the proton gradient was abolished (pH 7 parallel 7), and absent when the proton gradient was inward (pH 6 parallel 7). We propose that the effects of Na+ and Li+ are due to their transport by the Na(+)-H+ antiporter, which is present in both cell types studied. Electrically silent H+ efflux through the antiporter would increase pHi and possibly decrease local pHo, both of which modulate the gH in a similar manner: reducing the H+ currents at a given potential and shifting their voltage-dependence to more positive potentials. A simple diffusion model suggests that Na(+)-H+ antiport could deplete intracellular protonated buffer to the extent observed. Evidently the Na(+)-H+ antiporter functions in perfused cells, and its operation results in pH changes which can be detected using the gH as a physiological sensor. Thus, the properties of the gH can be exploited to study Na(+)-H+ antiport in single cells under controlled conditions.
采用膜片钳技术的全细胞记录模式，研究了成年大鼠肺泡上皮细胞和成人心肌细胞中电压激活的H⁺选择性电流。H⁺电导gH虽然对质子具有高度选择性，但受单价阳离子调节。与“惰性”阳离子四甲基铵（TMA⁺）相比，在Na⁺溶液中以及在较小程度上在Li⁺溶液中，H⁺电流显著降低，并且gH激活的电压依赖性向更正的电位偏移。gH的反转电位Vrev在Na⁺溶液中比在惰性离子溶液中更正。100μM的氨氯地平在除Li⁺和Na⁺之外的所有研究阳离子存在下抑制H⁺电流，在Li⁺和Na⁺中它增加H⁺电流并将其电压依赖性和Vrev向更负的电位偏移。10μM更具特异性的Na⁺-H⁺交换抑制剂二甲基氨氯地平（DMA）同样逆转了Na⁺和Li⁺对gH的大部分抑制作用。通过移液管溶液内部添加500μM氨氯地平或200μM DMA均无效。观察到1%[Na⁺]o对gH有明显抑制作用，表明存在一种高灵敏度机制。最后，当质子梯度向外时（pHo平行pHi 7平行5.5），Na⁺及其被氨氯地平逆转的作用较大；当质子梯度消除时（pH 7平行7），作用较小；当质子梯度向内时（pH 6平行7），则无作用。我们认为Na⁺和Li⁺的作用是由于它们通过Na⁺-H⁺反向转运体进行转运，在所研究的两种细胞类型中均存在该转运体。通过反向转运体的电沉默H⁺外流会增加细胞内pH并可能降低局部pHo，两者均以类似方式调节gH：在给定电位下降低H⁺电流并将其电压依赖性向更正的电位偏移。一个简单的扩散模型表明，Na⁺-H⁺反向转运可能会使细胞内质子化缓冲剂耗尽至观察到的程度。显然，Na⁺-H⁺反向转运体在灌注细胞中起作用，其运作会导致pH变化，可使用gH作为生理传感器检测到这种变化。因此，gH的特性可用于在可控条件下研究单细胞中的Na⁺-H⁺反向转运。
