Dimerization is crucial for the function of the Na+/H+ exchanger NHE1
Dimerization is crucial for the function of the Na+/H+ exchanger NHE1
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DOI：
10.1021/bi0608616
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发表时间：
2006-11-07
期刊：
影响因子：
2.9
通讯作者：
Wakabayashi, Shigeo
中科院分区：
文献类型：
--
作者：
Hisamitsu, Takashi;Ben Ammar, Youssef;Wakabayashi, Shigeo
Na+/H+交换器1（NHE 1）在质膜中以同源二聚体形式存在。在本研究中，我们已经调查了二聚化的功能意义，使用两个非功能性NHE 1突变体，表面表达缺陷型G309 V和转运缺陷型E262 I。生物化学和免疫细胞化学实验表明，这些NHE 1突变体能够与野生型NHE 1相互作用，从而形成异源二聚体。G309 V的表达将野生型NHE 1保留在ER膜上，表明NHE 1将首先在ER中形成二聚体。另一方面，E262 I的表达通过细胞内pH（pH（i））依赖性的酸性位移显著降低了野生型NHE 1的交换活性，表明在生理pH（i）范围内交换活性需要二聚化。然而，当在酸性pH（i）下测量交换活性时，未检测到E262 I的显性负效应，这意味着当细胞内H+浓度足够高时，一个活性亚基足以催化离子转运。此外，在细胞外位置Ser（375）处与双官能巯基试剂的分子间半胱氨酸交联主要通过诱导pH（i）依赖性的酸性转变来显著抑制交换活性，并消除细胞外刺激诱导的NHE 1活化，而不引起对细胞外Na+或抑制剂EIPA的亲和力的大的变化。由于单功能巯基试剂没有影响，它是可能的交联抑制NHE 1的活性，通过限制在运输过程中的两个亚基之间的耦合运动。总之，这些数据支持这样的观点，即NHE 1需要两个活性亚基的二聚化才能在中性pH（i）范围内具有交换活性，尽管每个亚基都能够在酸性pH（i）范围内催化转运。
The Na+/H+ exchanger 1 (NHE1) exists as a homo-dimer in the plasma membranes. In the present study, we have investigated the functional significance of the dimerization, using two nonfunctional NHE1 mutants, surface-expression-deficient G309V and transport-deficient E262I. Biochemical and immunocytochemical experiments revealed that these NHE1 mutants are capable of interacting with the wild-type NHE1 and, thus, forming a heterodimer. Expression of G309V retained the wild-type NHE1 to the ER membranes, suggesting that NHE1 would first form a dimer in the ER. On the other hand, expression of E262I markedly reduced the exchange activity of the wild-type NHE1 through an acidic shift in the intracellular pH (pH(i)) dependence, suggesting that dimerization is required for exchange activity in the physiological pH(i) range. However, a dominant-negative effect of E262I was not detected when exchange activity was measured at acidic pH(i), implying that one active subunit is sufficient to catalyze ion transport when the intracellular H+ concentration is sufficiently high. Furthermore, intermolecular cysteine cross-linking at extracellular position Ser(375) with a bifunctional sulfhydryl reagent dramatically inhibited exchange activity mainly by inducing the acidic shift of pH(i) dependence and abolished extracellular stimuli-induced activation of NHE1 without causing a large change in the affinities for extracellular Na+ or an inhibitor EIPA. Because monofunctional sulfhydryl regents had no effect, it is likely that cross-linking inhibited the activity of NHE1 by restricting a coupled motion between the two subunits during transport. Taken together, these data support the view that dimerization of two active subunits are required for NHE1 to possess the exchange activity in the neutral pH(i) range, although each subunit is capable of catalyzing transport in the acidic pH(i) range.