Pang Tianxiang, Hisamitsu Takashi, Mori Hidezo, Shigekawa Munekazu, Wakabayashi Shigeo
Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan.
Biochemistry. 2004 Mar 30;43(12):3628-36. doi: 10.1021/bi0360004.
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.
我们研究了钙调神经磷酸酶同源蛋白1（CHP1）与钠/氢交换体1（NHE1）相互作用的作用，特别是其EF手型钙结合基序，在细胞内pH（pH(i)）依赖性调节NHE1中的作用。我们发现（45）钙以高亲和力（表观K(d) = 约90 nM）结合到重组CHP1蛋白的两个EF手型基序（EF3和4）上。CHP1与NHE1的CHP1结合结构域之间形成复合物，通过促进EF手型向紧密结合钙的形式构象变化，导致钙结合亲和力显著增加（K(d) = 约2 nM）。这表明当CHP1在细胞中与NHE1结合时总是含有两个钙离子。有趣的是，在EF3或EF4中带有突变的GFP标记的CHP1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NHE1对各种刺激（如生长因子和渗透应激）的激活。此外，我们发现，除了降低活性（V(max)）外，CHP1结合缺陷的NHE1突变体在pH(i)敏感性上有显著降低（约0.7个pH单位酸性偏移），从而消除了NHE1的各种调节反应。这些观察结果表明，NHE1与CHP1的结合对于维持NHE1的pH(i)敏感性至关重要，并且紧密结合的钙离子可能作为NHE1“pH(i)传感器”中的重要结构元件。
