Boron W F, Knakal R C
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510.
J Gen Physiol. 1989 Jan;93(1):123-50. doi: 10.1085/jgp.93.1.123.
Intracellular pH (pHi) of the squid axon is regulated by a stilbenesensitive transporter that couples the influx of Na+ and HCO3- (or the equivalent) to the efflux of Cl-. According to one model, the extracellular ion pair NaCO3- exchanges for intracellular Cl-. In the present study, the ion-pair model was tested by examining the interaction of the reversible stilbene derivative 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) with extracellular Na+ and HCO3-. Axons (initial pHi approximately 7.4) were internally dialyzed with a pH 6.5 solution containing 400 mM Cl- but no Na+. After pHi, as measured with a glass microelectrode, had fallen to approximately 6.6, dialysis was halted. In the presence of both external Na+ and HCO3- (pHo = 8.0, 22 degrees C), pHi increased due to the pHi-regulating mechanism. At a fixed [Na+]o of 425 mM and [HCO3-]o of 12 mM, DNDS reversibly reduced the equivalent acid-extrusion rate (JH) calculated from the rate of pHi recovery. The best-fit value for maximal inhibition was 104%, and for the [DNDS]o at half-maximal inhibition, 0.3 mM. At a [Na+]o of 425 mM, the [HCO3-]o dependence of JH was examined at 0, 0.1, and 0.25 mM DNDS. Although Jmax was always approximately 20 pmol cm-2 s-1, Km(HCO3-) was 2.6, 5.7, and 12.7 mM, respectively. Thus, DNDS is competitive with HCO3-. At a [HCO3-]o of 12 mM, the [Na+]o dependence of JH was examined at 0 and 0.1 mM DNDS. Although Jmax was approximately 20 pmol cm-2 s-1 in both cases, Km(Na+) was 71 and 179 mM, respectively. At a [HCO3-]o of 48 mM, Jmax was approximately 20 pmol cm-2 s-1 at [DNDS]o levels of 0, 0.1, and 0.25 mM. However, Km(Na+) was 22, 45, and 90 mM, respectively. Thus, DNDS (an anion) is also competitive with Na+. The results are consistent with simple competition between DNDS and NaCO3-, and place severe restrictions on other kinetic models.
枪乌贼轴突的细胞内pH值（pHi）由一种对芪敏感的转运体调节，该转运体将Na⁺和HCO₃⁻（或等效物）的内流与Cl⁻的外流偶联。根据一种模型，细胞外离子对NaCO₃⁻与细胞内Cl⁻进行交换。在本研究中，通过研究可逆芪衍生物4,4'-二硝基芪-2,2'-二磺酸盐（DNDS）与细胞外Na⁺和HCO₃⁻的相互作用来测试离子对模型。轴突（初始pHi约为7.4）用含有400 mM Cl⁻但无Na⁺的pH 6.5溶液进行内部透析。在用玻璃微电极测量pHi降至约6.6后，停止透析。在同时存在外部Na⁺和HCO₃⁻（pHo = 8.0，22℃）的情况下，由于pHi调节机制，pHi升高。在固定的[Na⁺]o为425 mM和[HCO₃⁻]o为12 mM时，DNDS可逆地降低了根据pHi恢复速率计算的等效酸外排速率（JH）。最大抑制的最佳拟合值为104%，半最大抑制时的[DNDS]o为0.3 mM。在[Na⁺]o为425 mM时，在0、0.1和0.25 mM DNDS下研究了JH对[HCO₃⁻]o的依赖性。尽管Jmax始终约为20 pmol cm⁻² s⁻¹，但Km（HCO₃⁻）分别为2.6、5.7和12.7 mM。因此，DNDS与HCO₃⁻具有竞争性。在[HCO₃⁻]o为12 mM时，在0和0.1 mM DNDS下研究了JH对[Na⁺]o的依赖性。尽管在两种情况下Jmax均约为20 pmol cm⁻² s⁻¹，但Km（Na⁺）分别为71和179 mM。在[HCO₃⁻]o为48 mM时，在[DNDS]o水平为0、0.1和0.25 mM时Jmax约为20 pmol cm⁻² s⁻¹。然而，Km（Na⁺）分别为22、45和90 mM。因此，DNDS（一种阴离子）也与Na⁺具有竞争性。结果与DNDS和NaCO₃⁻之间的简单竞争一致，并对其他动力学模型施加了严格限制。
