Role of calcineurin B homologous protein in pH regulation by the Na+/H+ exchanger 1: tightly bound Ca2+ ions as important structural elements.
Role of calcineurin B homologous protein in pH regulation by the Na+/H+ exchanger 1: tightly bound Ca2+ ions as important structural elements.
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DOI：
10.1021/bi0360004
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发表时间：
2004-03
期刊：
影响因子：
2.9
通讯作者：
T. Pang;T. Hisamitsu;H. Mori;M. Shigekawa;S. Wakabayashi
中科院分区：
文献类型：
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作者：
T. Pang;T. Hisamitsu;H. Mori;M. Shigekawa;S. Wakabayashi
我们研究了钙调神经磷酸酶同源蛋白1（calcineurin homologous protein 1，CHP 1）与Na（+）/H（+）交换蛋白1（Na（+）/H（+）exchange 1，NHE 1）的相互作用，特别是其EF-手型Ca（2+）结合基序，在NHE 1的细胞内pH（pH（i））依赖性调节中的作用。我们发现（45）Ca（2+）以高亲和力（表观K（d）=约90 nM）与重组CHP 1蛋白的两个EF-手状基序（EF 3和4）结合。CHP 1和NHE 1的CHP 1结合结构域之间的复合物形成通过促进EF-手朝向紧密Ca（2+）结合形式的构象变化而导致Ca（2+）结合亲和力的显著增加（K（d）=约2 nM）。这表明，CHP 1总是包含两个Ca（2+）离子时，与NHE 1在细胞中。有趣的是，在EF 3或EF 4中突变的GFP标记的CHP 1的过表达显著降低了中性pH（i）范围内的交换活性，并部分损害了NHE 1响应于各种刺激（如生长因子和渗透胁迫）的活化。此外，我们发现，除了降低活性（V（max））之外，CHP 1结合缺陷型NHE 1突变体的pH（i）敏感性显著降低（约0.7 pH单位的酸性位移），从而消除了NHE 1的各种调节反应。这些观察结果表明，NHE 1与CHP 1的缔合对于维持NHE 1的pH（i）敏感性至关重要，并且紧密结合的Ca（2+）离子可能在NHE 1的“pH（i）传感器”中充当重要的结构元件。
We studied the role of the interaction of calcineurin homologous protein 1 (CHP1) with the Na(+)/H(+) exchanger 1 (NHE1), particularly its EF-hand Ca(2+) binding motifs, in the intracellular pH (pH(i))-dependent regulation of NHE1. We found that (45)Ca(2+) binds to two EF-hand motifs (EF3 and 4) of the recombinant CHP1 proteins with high affinity (apparent K(d) = approximately 90 nM). Complex formation between CHP1 and the CHP1 binding domain of NHE1 resulted in a marked increase in the Ca(2+) binding affinity (K(d) = approximately 2 nM) by promoting a conformational change of the EF-hands toward the tightly Ca(2+)-bound form. This suggests that CHP1 always contains two Ca(2+) ions when associated with NHE1 in cells. Interestingly, overexpression of GFP-tagged CHP1 with mutations in EF3 or EF4 significantly reduced the exchange activity in the neutral pH(i) range and partly impaired the activation of NHE1 in response to various stimuli, such as growth factors and osmotic stress. Furthermore, we found that, in addition to reducing the activity (V(max)), a CHP1 binding-defective NHE1 mutant had a marked reduction in pH(i) sensitivity ( approximately 0.7 pH unit acidic shift), which consequently abolished various regulatory responses of NHE1. These observations suggest that the association of NHE1 with CHP1 is crucial for maintenance of the pH(i) sensitivity of NHE1 and that tightly bound Ca(2+) ions may serve as important structural elements in the "pH(i) sensor" of NHE1.