Hogan E M, Cohen M A, Boron W F
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
J Gen Physiol. 1995 Nov;106(5):821-44. doi: 10.1085/jgp.106.5.821.
We used microelectrodes to monitor the recovery (i.e., decrease) of intracellular pH (pHi) after using internal dialysis to load squid giant axons with alkali to pHi values of 7.7, 8.0, or 8.3. The dialysis fluid (DF) contained 400 mM K+ but was free of Na+ and Cl-. The artificial seawater (ASW) lacked Na+, K+, and Cl-, thereby eliminating effects of known acid-base transporters on pHi. Under these conditions, halting dialysis unmasked a slow pHi decrease caused at least in part by acid-base transport we refer to as "base efflux." Replacing K+ in the DF with either NMDG+ or TEA+ significantly reduced base efflux and made membrane voltage (Vm) more positive. Base efflux in K(+)-dialyzed axons was stimulated by decreasing the pH of the ASW (pHo) from 8 to 7, implicating transport of acid or base. Although postdialysis acidifications also occurred in axons in which we replaced the K+ in the DF with Li+, Na+, Rb+, or Cs+, only with Rb+ was base efflux stimulated by low pHo. Thus, the base effluxes supported by K+ and Rb+ appear to be unrelated mechanistically to those observed with Li+, Na+, or Cs+. The combination of 437 mM K+ and 12 mM HCO3- in the ASW, which eliminates the gradient favoring a hypothetical K+/HCO3- efflux, blocked pHi recovery in K(+)-dialyzed axons. However, the pHi recovery was not blocked by the combination of 437 mM Na+, veratridine, and CO2/HCO3- in the ASW, a treatment that inverts electrochemical gradients for H+ and HCO3- and would favor passive H+ and HCO3- fluxes that would have alkalinized the axon. Similarly, the recovery was not blocked by K+ alone or HCO3- alone in the ASW, nor was it inhibited by the K-H pump blocker Sch28080 nor by the Na-H exchange inhibitors amiloride and hexamethyleneamiloride. Our data suggest that a major component of base efflux in alkali-loaded axons cannot be explained by metabolism, a H+ or HCO3- conductance, or by a K-H exchanger. However, this component could be mediated by a novel K/HCO3- cotransporter.
我们使用微电极监测在用内部透析将乌贼巨大轴突加载碱至细胞内pH值（pHi）为7.7、8.0或8.3后细胞内pH值的恢复（即降低）情况。透析液（DF）含有400 mM K⁺，但不含Na⁺和Cl⁻。人工海水（ASW）缺乏Na⁺、K⁺和Cl⁻，从而消除了已知酸碱转运体对pHi的影响。在这些条件下，停止透析揭示了一种缓慢的pHi降低，这至少部分是由我们称为“碱外流”的酸碱转运引起的。用NMDG⁺或TEA⁺替代DF中的K⁺可显著减少碱外流，并使膜电压（Vm）更正。将ASW的pH值（pHo）从8降至7可刺激K⁺透析轴突中的碱外流，这暗示了酸或碱的转运。尽管在用Li⁺、Na⁺、Rb⁺或Cs⁺替代DF中的K⁺的轴突中也会发生透析后酸化，但只有用Rb⁺时，低pHo才会刺激碱外流。因此，由K⁺和Rb⁺支持的碱外流在机制上似乎与用Li⁺、Na⁺或Cs⁺观察到的碱外流无关。ASW中437 mM K⁺和12 mM HCO₃⁻的组合消除了有利于假设的K⁺/HCO₃⁻外流的梯度，从而阻止了K⁺透析轴突中pHi的恢复。然而，ASW中437 mM Na⁺、藜芦碱和CO₂/HCO₃⁻的组合并未阻止pHi的恢复，这种处理会反转H⁺和HCO₃⁻的电化学梯度，并有利于使轴突碱化的被动H⁺和HCO₃⁻通量。同样，ASW中单独的K⁺或单独的HCO₃⁻也未阻止恢复，K⁺-H⁺泵阻滞剂Sch28080、Na⁺-H⁺交换抑制剂阿米洛利和六甲烯阿米洛利也未抑制恢复。我们的数据表明，碱加载轴突中碱外流的主要成分不能用代谢、H⁺或HCO₃⁻电导或K⁺-H⁺交换体来解释。然而，这一成分可能由一种新型的K⁺/HCO₃⁻共转运体介导。
