Functional importance of charged residues within the putative intracellular loops in pH regulation by Na+/H+ exchanger NHE1
Functional importance of charged residues within the putative intracellular loops in pH regulation by Na+/H+ exchanger NHE1
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DOI：
10.1111/j.1742-4658.2007.05962.x
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发表时间：
2007-08-01
期刊：
影响因子：
5.4
通讯作者：
Wakabayashi, Shigeo
中科院分区：
文献类型：
--
作者：
Hisamitsu, Takashi;N'amada, Keiji;Wakabayashi, Shigeo
质膜Na+/H+交换器1响应于各种外在因素而被激活，并且该过程由细胞内pH敏感机制调节。为了确定负责细胞内pH调节的候选残基，我们分析了工程化Na+/H+交换器1突变体的功能特性，其具有位于细胞内环中的带电残基的电荷逆转突变。Na+/H+交换器1突变体在11个位置处在质膜中表达良好，而E247 R突变体在质膜中不表达，表明Glu 247对Na+/H+交换器1的功能表达很重要。Glu 131的电荷反转突变（E131 R，E131 K）和Arg 327（R327 E）使Na ~+摄取测定的交换活性的细胞内pH依赖性向酸性侧移动，并消除了对生长因子和高渗介质的反应;而Asp 448（D448 R）和Arg 500（R500 E）的突变使其略微向碱性侧移动。在E131 R中，除了细胞内pH依赖性的变化之外，对细胞外Na+、Li+和抑制剂5-（N-乙基-N-异丙基）阿米洛利的亲和力显著增加。此外，发现E131的电荷保守突变（E131 D）没有影响，而电荷中和（E131 Q）导致交换的轻微酸性位移。这些结果支持这样的观点，即在本研究中鉴定的多个带电残基，沿着先前报道的几个碱性残基，参与Na+/H+交换器1的细胞内pH传感的调节。此外，Glu 131可能对阳离子转运也很重要。
The plasma membrane Na+/H+ exchanger 1 is activated in response to various extrinsic factors, and this process is regulated by an intracellular pH-sensing mechanism. To identify the candidate residues responsible for intracellular pH regulation, we analyzed the functional properties of engineered Na+/H+ exchanger 1 mutants with charge-reversal mutations of charged residues located in the intracellular loops. Na+/H+ exchanger 1 mutants with mutations at 11 positions were well expressed in the plasma membrane, but that with E247R was not, suggesting that Glu247 is important for the functional expression of Na+/H+ exchanger 1. Charge-reversal mutations of Glu131 (E131R, E131K) and Arg327 (R327E) resulted in a shift in the intracellular pH dependence of the exchange activity measured by 22 Na+ uptake to the acidic side, and it abolished the response to growth factors and a hyperosmotic medium; however, mutations of Asp448 (D448R) and Arg500 (R500E) slightly shifted it to the alkaline side. In E131R, in addition to the change in intracellular pH dependence, the affinities for extracellular Na+, Li+ and the inhibitor 5-(N-ethyl-N-iso-propyl)amiloride significantly increased. Furthermore, charge-conserved mutation of E131 (E131D) was found to have no effect, whereas charge neutralization (E131Q) resulted in a slight acidic shift of exchange. These results support the view that the multiple charged residues identified in this study, along with several basic residues reported previously, participate in the regulation of the intracellular pH sensing of Na+/H+ exchanger 1. In addition, Glu131 may also be important for cation transport.