Wakabayashi S, Ikeda T, Iwamoto T, Pouysségur J, Shigekawa M
Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka 565, Japan.
Biochemistry. 1997 Oct 21;36(42):12854-61. doi: 10.1021/bi9715472.
The calmodulin (CaM)-binding domain reduces the affinity of the Na+/H+ exchanger NHE1 for intracellular H+ by exerting an autoinhibitory function in quiescent cells. We replaced this domain (aa 637-656) with homologous segments from other NHE isoforms (NHE2 and 4) or functionally similar regions from other sources (Na+/Ca2+ exchanger, CaM-dependent protein kinase II, plasma membrane Ca2+-pump, or CaM-binding peptide Trp3). The NHE-1-, NHE2-, and NHE4-segments bound CaM with Kds of 16, 130, and 27 nM, respectively. These chimeric molecules were expressed in the exchanger-deficient cell PS120. NHE1 with incorporated NHE2-segment was activated in response to Ca2+-mobilizing agents ionomycin and thrombin resulting in an alkaline shift of the intracellular pH (pHi)-dependence of 22Na+ uptake, as was the case with the intact rat NHE2. In contrast, incorporation of the NHE4-segment or other CaM-binding segments induced a constitutive alkaline shift of pHi-dependence with concomitant abolishment of Ca2+-dependent activation, indicating that these segments could not function as an autoinhibitory domain in NHE1. Detailed analyses revealed that Leu639, Lys651 and Tyr652, conserved in the NHE1- and NHE2-segments, but not in the NHE4-segment, are important for the autoinhibition. Furthermore, 125I-labeled CaM-binding peptide from NHE1 was efficiently crosslinked to the NHE1 protein, suggesting that the inhibitory domain physically interacts with part(s) of the molecule. Together, these findings support the notion that the reduction of H+ affinity in Na+/H+ exchange occurs through a mechanism involving a highly sequence-specific interaction of the inhibitory domain with its putative acceptor in NHE1.
钙调蛋白（CaM）结合结构域通过在静息细胞中发挥自抑制功能，降低了Na⁺/H⁺交换蛋白NHE1对细胞内H⁺的亲和力。我们用其他NHE亚型（NHE2和4）的同源片段或其他来源（Na⁺/Ca²⁺交换蛋白、CaM依赖性蛋白激酶II、质膜Ca²⁺泵或CaM结合肽Trp3）的功能相似区域替换了该结构域（氨基酸637 - 656）。NHE-1、NHE2和NHE4片段与CaM结合的解离常数（Kds）分别为16、130和27 nM。这些嵌合分子在缺乏交换蛋白的细胞PS120中表达。掺入NHE2片段的NHE1对钙离子载体离子霉素和凝血酶有反应而被激活，导致22Na⁺摄取的细胞内pH（pHi）依赖性发生碱性偏移，完整的大鼠NHE2也是如此。相反，掺入NHE4片段或其他CaM结合片段会导致pHi依赖性的组成性碱性偏移，同时消除钙离子依赖性激活，这表明这些片段在NHE1中不能作为自抑制结构域发挥作用。详细分析表明，NHE1和NHE2片段中保守但NHE4片段中不存在的亮氨酸639、赖氨酸651和酪氨酸652对自抑制很重要。此外，来自NHE1的125I标记的CaM结合肽与NHE1蛋白有效交联，表明抑制结构域与分子的部分区域发生物理相互作用。总之，这些发现支持了这样一种观点，即Na⁺/H⁺交换中H⁺亲和力的降低是通过一种机制发生的，该机制涉及抑制结构域与其在NHE1中的假定受体的高度序列特异性相互作用。
