Goldfarb D, Nord E P
Department of Medicine, University of California, School of Medicine, Los Angeles 90024.
Am J Physiol. 1987 Nov;253(5 Pt 2):F959-68. doi: 10.1152/ajprenal.1987.253.5.F959.
The affinity for Na+ of the cytoplasmic vs. external transport site of the amiloride-sensitive Na+-H+ antiporter was studied in confluent cultures of MDCK cells. Na+-H+ antiport activity was fluorometrically determined by monitoring changes in intracellular pH (pHi) using the pH-sensitive fluorescent probe, BCECF. Na+-dependent H+ fluxes were studied both in the functionally operative (H+ efflux/Na+ influx) and reverse (H+ influx/Na+ efflux) mode of antiport activity, under pH equilibrium, but Na+-gradient conditions. Thus the driving force for antiport activity was solely dependent on the transmembrane Na+ gradient. Independent experiments established that pHi and intracellular Na+ [Na+i] had been set at the desired values before the initiation of a particular experiment. Under conditions of pHi = pHo = 7.0, [Na+i] = 0 mM and varying extracellular Na+ concentration [Na+o], the apparent affinity for Na+ (KtNa) for the external transport site was 24 +/- 3 mM. When antiport activity was measured in the reverse mode of operation, but under identical pH conditions, KtNa at the internal site was 7 +/- 1 mM. When ambient pH was elevated to 7.5, KtNa at the internal site was 14 +/- 1 mM. Maximum H+ flux (JmaxH+) for the antiporter under all three conditions was not significantly different. In summary, the Na+-H+ antiporter displays asymmetric affinity for Na+ at the internal vs. external transport site. Under pH equilibrium conditions, the affinity of the Na+-H+ antiporter for Na+ is three- to four-fold greater at the internal vs. external locus, and the affinity for Na+ at the internal site is enhanced by lower pHi. The close similarity between values for KtNa (inside) and reported values for intracellular Na+ concentration suggests that regulation of the Na+-H+ antiporter may be affected by changes in intracellular Na+ concentration.
在MDCK细胞的汇合培养物中研究了氨氯地平敏感的Na⁺-H⁺反向转运体胞质侧与外侧转运位点对Na⁺的亲和力。使用pH敏感荧光探针BCECF通过监测细胞内pH（pHi）的变化，以荧光法测定Na⁺-H⁺反向转运活性。在pH平衡但存在Na⁺梯度的条件下，以反向转运活性的功能运作模式（H⁺外流/Na⁺内流）和反向模式（H⁺内流/Na⁺外流）研究了Na⁺依赖性H⁺通量。因此，反向转运活性的驱动力仅取决于跨膜Na⁺梯度。独立实验确定，在特定实验开始前，pHi和细胞内Na⁺[Na⁺i]已设定为所需值。在pHi = pHo = 7.0、[Na⁺i] = 0 mM且细胞外Na⁺浓度[Na⁺o]变化的条件下，外侧转运位点对Na⁺的表观亲和力（KtNa）为24±3 mM。当以反向运作模式测量反向转运活性，但在相同pH条件下时，内侧位点的KtNa为7±1 mM。当环境pH升高到7.5时，内侧位点的KtNa为14±1 mM。在所有三种条件下，反向转运体的最大H⁺通量（JmaxH⁺）无显著差异。总之，Na⁺-H⁺反向转运体在胞质侧与外侧转运位点对Na⁺表现出不对称亲和力。在pH平衡条件下，Na⁺-H⁺反向转运体对Na⁺的亲和力在胞质侧比对外侧位点大3至4倍，并且内侧位点对Na⁺的亲和力会因较低的pHi而增强。KtNa（内侧）的值与报道的细胞内Na⁺浓度值非常相似，这表明Na⁺-H⁺反向转运体的调节可能受细胞内Na⁺浓度变化的影响。
