Ikeda T, Schmitt B, Pouysségur J, Wakabayashi S, Shigekawa M
Department of Molecular Physiolgy, National Cardiovascular Center Research Institute, Osaka.
J Biochem. 1997 Feb;121(2):295-303. doi: 10.1093/oxfordjournals.jbchem.a021586.
To precisely identify the cytoplasmic subdomains that are responsible for the intracellular pH (pHi)-sensitivity, ATP depletion-induced inhibition and Ca2+ activation of the Na+/H+ exchanger (NHE1), we generated a set of deletion mutants of carboxyl-terminated cytoplasmic domain and expressed them in the exchanger-deficient cell line PS120. We evaluated pHi-sensitivity of these mutants by measuring the resting pHi in cells placed in an acidic medium (pH 6.0) and pHi-dependence of 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na+ uptake. Detailed analysis revealed that the cytoplasmic domain of NHE1 is consists of at least four subdomains in terms of pHi-sensitivity of the unstimulated NHE1: I, aa 516-590/595; II, aa 596-635; III aa 636-659; and IV, aa 660-815. Subdomains II and IV were silent for pHi-sensitivity. Subdomain I had a pHi-maintenance function, preserving pHi-sensitivity in a physiological range, whereas subdomain III, overlapping with the high affinity calmodulin (CaM)-binding site, exhibited an autoinhibitory function. Deletion of subdomain I abolished the decrease of pHi-sensitivity induced by cell ATP depletion, indicating that domain I plays a crucial role in this phenomenon. Deletion of subdomain III rendered the inhibition by ATP depletion less efficient, suggesting the possible interaction between subdomains I and III. On the other hand, tandem elongation of subdomain II by insertion did not affect either the inhibitory function of domain III or the removal of this inhibition by ionomycin or thrombin. However, deletion of subdomain II partially abolished the inhibitory effect of subdomain III. Subdomain II thus seems to function as a mobile "flexible loop," permitting the CaM-binding subdomain III to exert its normal function. These findings, together with our previous data, support a concept that cell ATP, Ca2+, and growth factors regulate NHE1 via a mechanism involving direct or indirect interactions of specific cytoplasmic subdomains with the "H(+)-modifier site.".
为了精确识别负责细胞内pH（pHi）敏感性、ATP耗竭诱导的抑制作用以及Na⁺/H⁺交换体（NHE1）的Ca²⁺激活的细胞质亚结构域，我们构建了一组羧基末端细胞质结构域的缺失突变体，并在缺乏交换体的细胞系PS120中进行表达。我们通过测量置于酸性培养基（pH 6.0）中的细胞的静息pHi以及5-（N-乙基-N-异丙基）阿米洛利敏感的²²Na⁺摄取的pHi依赖性，来评估这些突变体的pHi敏感性。详细分析表明，就未受刺激的NHE1的pHi敏感性而言，NHE1的细胞质结构域至少由四个亚结构域组成：I，氨基酸516 - 590/595；II，氨基酸596 - 635；III，氨基酸636 - 659；以及IV，氨基酸660 - 815。亚结构域II和IV对pHi敏感性无影响。亚结构域I具有pHi维持功能，在生理范围内保持pHi敏感性，而与高亲和力钙调蛋白（CaM）结合位点重叠的亚结构域III表现出自抑制功能。亚结构域I的缺失消除了细胞ATP耗竭诱导的pHi敏感性降低，表明结构域I在这一现象中起关键作用。亚结构域III的缺失使ATP耗竭的抑制作用效率降低，提示亚结构域I和III之间可能存在相互作用。另一方面，通过插入使亚结构域II串联延长，既不影响结构域III的抑制功能，也不影响离子霉素或凝血酶对这种抑制作用的消除。然而，亚结构域II的缺失部分消除了亚结构域III的抑制作用。因此，亚结构域II似乎起到一个可移动的“柔性环”的作用，使CaM结合亚结构域III能够发挥其正常功能。这些发现与我们之前的数据一起支持了一个概念，即细胞ATP、Ca²⁺和生长因子通过一种涉及特定细胞质亚结构域与“H⁺修饰位点”直接或间接相互作用的机制来调节NHE1。
