Evidence from simultaneous intracellular- and surface- pH transients that carbonic anhydrase IV enhances CO2 fluxes across Xenopus oocyte plasma membranes
Evidence from simultaneous intracellular- and surface- pH transients that carbonic anhydrase IV enhances CO2 fluxes across Xenopus oocyte plasma membranes
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DOI：
10.1152/ajpcell.00050.2014
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发表时间：
2014-11-01
影响因子：
5.5
通讯作者：
Boron, Walter F.
中科院分区：
文献类型：
--
作者：
Musa-Aziz, Raif;Occhipinti, Rossana;Boron, Walter F.
人碳酸酐酶 IV (CA IV) 以 GPI 锚定于外膜表面，催化 CO2/HCO3- 水合-脱水。我们检查了异源表达的 CA IV 对卵母细胞暴露于 CO2/HCO3-/pH 7.50 引起的细胞内 pH (pH(i)) 和表面 pH (pH(S)) 瞬变的影响。 CO2 流入导致 pH(i) 持续下降和 pH(S) 短暂上升； CO2 流出则相反。在 CO2 添加和去除过程中，CA IV 都会增加 pHi 变化的最大速率 (dpH(i)/dt)(max) 和最大 pH(S) 变化 (Delta pH(S))，并降低 pH(i) 变化 (tau(pHi)) 和 pHS 弛豫 (tau(pHS)) 的时间常数。时间常数的减小表明 CA IV 增强了 CO2 通量。细胞外乙酰唑胺可阻断所有 CA IV 效应，但不能阻断注射的 CA II 的效应。注射乙酰唑胺可部分降低 CA IV 效应。因此，细胞外CA是CA IV作用所必需的，并且细胞质可接触的CA的等价物增强了CA IV的作用。在细胞外 CA 存在或高 [CO2] 存在的情况下，增加细胞外非 CO2/HCO3 缓冲液（即 HEPES）的浓度，可加速 CO2 流入。使用两个 pHS 电极同时测量，一个位于垂直于流动轴的卵母细胞子午线上，一个位于细胞外溶液流动方向的下游，表明下游电极具有较大（即较慢）的 tau(pHS)，表明卵母细胞表面的 [CO2] 不对称。反应扩散数学模型（系列第三篇论文）解释了上述一般特征，并支持这样的结论：细胞外 CA 补充进入的 CO2 或消耗细胞外表面排出的 CO2，增强了驱动 CO2 穿过细胞膜流入的梯度。
Human carbonic anhydrase IV (CA IV) is GPI-anchored to the outer membrane surface, catalyzing CO2/HCO3- hydration-dehydration. We examined effects of heterologously expressed CA IV on intracellular-pH (pH(i)) and surface-pH (pH(S)) transients caused by exposing oocytes to CO2/HCO3-/pH 7.50. CO2 influx causes a sustained pH(i) fall and a transient pH(S) rise; CO2 efflux does the opposite. Both during CO2 addition and removal, CA IV increases magnitudes of maximal rate of pHi change (dpH(i)/dt)(max), and maximal pH(S) change (Delta pH(S)) and decreases time constants for pH(i) changes (tau(pHi)) and pHS relaxations (tau(pHS)). Decreases in time constants indicate that CA IV enhances CO2 fluxes. Extracellular acetazolamide blocks all CA IV effects, but not those of injected CA II. Injected acetazolamide partially reduces CA IV effects. Thus, extracellular CA is required for, and the equivalent of cytosol-accessible CA augments, the effects of CA IV. Increasing the concentration of the extracellular non-CO2/HCO3- buffer (i.e., HEPES), in the presence of extracellular CA or at high [CO2], accelerates CO2 influx. Simultaneous measurements with two pHS electrodes, one on the oocyte meridian perpendicular to the axis of flow and one downstream from the direction of extracellular-solution flow, reveal that the downstream electrode has a larger (i.e., slower) tau(pHS), indicating [CO2] asymmetry over the oocyte surface. A reaction-diffusion mathematical model (third paper in series) accounts for the above general features, and supports the conclusion that extracellular CA, which replenishes entering CO2 or consumes exiting CO2 at the extracellular surface, enhances the gradient driving CO2 influx across the cell membrane.