Morgan K, Canessa M
Endocrine-Hypertension Division, Brigham and Women's Hospital, Boston, Massachusetts.
J Membr Biol. 1990 Dec;118(3):193-214. doi: 10.1007/BF01868604.
We have studied the kinetic properties of rabbit red cell (RRBC) Na+/Na+ and Na+/H+ exchanges (EXC) in order to define whether or not both transport functions are conducted by the same molecule. The strategy has been to determine the interactions of Na+ and H+ at the internal (i) and external (o) sites for both exchanges modes. RRBC containing varying Nai and Hi were prepared by nystatin and DIDS treatment of acid-loaded cells. Na+/Na+ EXC was measured as Nao-stimulated Na+ efflux and Na+/H+ EXC as Nao-stimulated H+ efflux and delta pHo-stimulated Na+ influx into acid-loaded cells. The activation of Na+/Na+ EXC by Nao at pHi 7.4 did not follow simple hyperbolic kinetics. Testing of different kinetic models to obtain the best fit for the experimental data indicated the presence of high (Km 2.2 mM) and low affinity (Km 108 mM) sites for a single- or two-carrier system. The activation of Na+/H+ EXC by Nao (pHi 6.6, Nai less than 1 mM) also showed high (Km 11 mM) and low (Km 248 mM) affinity sites. External H+ competitively inhibited Na+/Na+ EXC at the low affinity Nao site (KH 52 nM) while internally H+ were competitive inhibitors (pK 6.7) at low Nai and allosteric activators (pK 7.0) at high Nai. Na+/H+ EXC was also inhibited by acid pHo and allosterically activated by Hi (pK 6.4). We also established the presence of a Nai regulatory site which activates Na+/H+ and Na+/Na+ EXC modifying the affinity for Nao of both pathways. At low Nai, Na+/Na+ EXC was inhibited by acid pHi and Na+/H+ stimulated but at high Nai, Na+/Na+ EXC was stimulated and Na+/H+ inhibited being the sum of both pathways kept constant. Both exchange modes were activated by two classes of Nao sites, cis-inhibited by external Ho, allosterically modified by the binding of H+ to a Hi regulatory site and regulated by Nai. These findings are consistent with Na+/Na+ EXC being a mode of operation of the Na+/H+ exchanger. Na+/H+ EXC was partially inhibited (80-100%) by dimethyl-amiloride (DMA) but basal or pHi-stimulated Na+/Na+ EXC (pHi 6.5, Nai 80 mM) was completely insensitive indicating that Na+/Na+ EXC is an amiloride-insensitive component of Na+/H+ EXC. However, Na+ and H+ efflux into Na-free media were stimulated by cell acidification and also partially (10 to 40%) inhibited by DMA; this also indicates that the Na+/H+ EXC might operate in reverse or uncoupled modes in the absence of Na+/Na+ EXC.(ABSTRACT TRUNCATED AT 400 WORDS)
我们研究了兔红细胞（RRBC）的Na⁺/Na⁺和Na⁺/H⁺交换（EXC）的动力学特性，以确定这两种转运功能是否由同一分子介导。我们采用的策略是确定两种交换模式下Na⁺和H⁺在内部（i）和外部（o）位点的相互作用。通过制霉菌素和DIDS处理酸负载细胞制备了具有不同细胞内Na⁺（Nai）和H⁺（Hi）浓度的RRBC。Na⁺/Na⁺ EXC通过测量细胞外Na⁺（Nao）刺激的Na⁺外流来测定，而Na⁺/H⁺ EXC则通过测量Nao刺激的H⁺外流以及细胞外pH值变化（ΔpHo）刺激的Na⁺流入酸负载细胞来测定。在细胞内pH值（pHi）为7.4时，Nao对Na⁺/Na⁺ EXC的激活并不遵循简单的双曲线动力学。对不同动力学模型进行测试以获得与实验数据的最佳拟合，结果表明在单载体或双载体系统中存在高亲和力（Km为2.2 mM）和低亲和力（Km为108 mM）位点。Nao对Na⁺/H⁺ EXC的激活（pHi为6.6，Nai小于1 mM）也显示出高亲和力（Km为11 mM）和低亲和力（Km为248 mM）位点。外部H⁺在低亲和力的Nao位点竞争性抑制Na⁺/Na⁺ EXC（抑制常数KH为52 nM），而内部H⁺在低Nai时是竞争性抑制剂（解离常数pK为6.7），在高Nai时是变构激活剂（pK为7.0）。Na⁺/H⁺ EXC也受到酸性细胞外pH值的抑制，并被Hi变构激活（pK为6.4）。我们还确定了存在一个细胞内调节位点，该位点通过改变两条途径对Nao的亲和力来激活Na⁺/H⁺和Na⁺/Na⁺ EXC。在低Nai时，Na⁺/Na⁺ EXC受到酸性pHi的抑制，而Na⁺/H⁺ EXC受到刺激；但在高Nai时，Na⁺/Na⁺ EXC受到刺激，Na⁺/H⁺ EXC受到抑制，而两条途径的总和保持不变。两种交换模式均由两类Nao位点激活，受到外部H⁺的顺式抑制，通过H⁺与细胞内调节位点的结合进行变构调节，并受Nai调控。这些发现表明Na⁺/Na⁺ EXC是Na⁺/H⁺交换器的一种运作模式。Na⁺/H⁺ EXC受到二甲基氨氯吡脒（DMA）的部分抑制（80 - 100%），但基础或pHi刺激的Na⁺/Na⁺ EXC（pHi为6.5，Nai为80 mM）对DMA完全不敏感，这表明Na⁺/Na⁺ EXC是Na⁺/H⁺ EXC中对氨氯吡脒不敏感的成分。然而，细胞酸化会刺激Na⁺和H⁺向无Na⁺培养基中的外流，并且也会受到DMA的部分抑制（10%至40%）；这也表明在不存在Na⁺/Na⁺ EXC的情况下，Na⁺/H⁺ EXC可能以反向或解偶联模式运作。（摘要截取自400字）
