Yu F H, Shull G E, Orlowski J
Department of Physiology, McGill University, Montréal, Québec, Canada.
J Biol Chem. 1993 Dec 5;268(34):25536-41.
The primary structure and functional expression of the rat Na/H exchanger (NHE) NHE-2 isoform has recently been reported (Wang, Z., Orlowski, J., and Shull, G. E. (1993) J. Biol. Chem. 268, 11925-11928). To further characterize some of its functional properties, biochemical and pharmacological analyses were performed on exchanger-deficient Chinese hamster ovary cells (AP-1) that had been stably transfected with a full-length NHE-2 cDNA. Transport activity for NHE-2 was assayed by measuring amiloride-inhibitable 22Na+ influx following an acute intracellular acid load. Pharmacological analyses revealed that NHE-2 had a relatively high affinity for amiloride and some of its analogues. The most potent analogue was 5-(N-ethyl-N-isopropyl)amiloride (EIPA) (K0.5 = 79 nM), followed by 5-(N,N-dimethyl)amiloride (DMA) (K0.5 = 250 nM), amiloride (K0.5 = 1.4 microM), and benzamil (K0.5 = 320 microM). Nonamiloride compounds known to inhibit the activity of other Na/H exchanger isoforms also inhibited NHE-2 with the following order of potency: clonidine (K0.5 = 42 microM) > harmaline (K0.5 = 330 microM) approximately cimetidine (K0.5 = 330 microM). Biochemical analyses showed that the extracellular Na+ dependence of NHE-2 followed simple, saturating Michaelis-Menten kinetics with an apparent affinity constant for Na+ (KNa) of 50 mM. In contrast, intracellular H+ appeared to activate NHE-2 by a positive cooperative mechanism with an apparent half-maximal activation value of pK 6.90. Other cations, such as extracellular Li+ and H+, acted as competitive inhibitors of 22Na+ influx by NHE-2, with apparent Ki values of 3.0 mM and 10 nM, respectively. In contrast, extracellular K+ had no effect on the transport activity of NHE-2. These results indicated that the rat NHE-2 cDNA encodes a functional Na/H exchanger isoform with distinct properties compared to rat NHE-1 and -3.
大鼠钠/氢交换体（NHE）NHE - 2亚型的一级结构和功能表达最近已有报道（Wang, Z., Orlowski, J., and Shull, G. E. (1993) J. Biol. Chem. 268, 11925 - 11928）。为了进一步表征其一些功能特性，对已用全长NHE - 2 cDNA稳定转染的缺乏交换体的中国仓鼠卵巢细胞（AP - 1）进行了生化和药理学分析。通过在急性细胞内酸负荷后测量阿米洛利抑制的22Na+内流来测定NHE - 2的转运活性。药理学分析表明，NHE - 2对阿米洛利及其一些类似物具有相对较高的亲和力。最有效的类似物是5 -（N - 乙基 - N - 异丙基）阿米洛利（EIPA）（K0.5 = 79 nM），其次是5 -（N，N - 二甲基）阿米洛利（DMA）（K0.5 = 250 nM）、阿米洛利（K0.5 = 1.4 microM）和苄甲利（K0.5 = 320 microM）。已知抑制其他钠/氢交换体亚型活性的非阿米洛利化合物也抑制NHE - 2，其效力顺序如下：可乐定（K0.5 = 42 microM）> 骆驼蓬碱（K0.5 = 330 microM）≈西咪替丁（K0.5 = 330 microM）。生化分析表明，NHE - 2对细胞外Na+的依赖性遵循简单的饱和米氏动力学，对Na+的表观亲和常数（KNa）为50 mM。相比之下，细胞内H+似乎通过正协同机制激活NHE - 2，表观半最大激活值的pK为6.90。其他阳离子，如细胞外Li+和H+，作为NHE - 2介导的22Na+内流的竞争性抑制剂，表观Ki值分别为3.0 mM和10 nM。相比之下，细胞外K+对NHE - 2的转运活性没有影响。这些结果表明，大鼠NHE - 2 cDNA编码一种功能性钠/氢交换体亚型，与大鼠NHE - 1和 - 3相比具有不同的特性。
