Department of Molecular Physiology, National Cerebral and Cardiovascular Center Research Institute, Osaka, Japan (N.S.-S., T.H., T.Y.N., S.W.); and Basic Medical Science & Molecular Medicine, Tokai University School of Medicine, Kanagawa, Japan (N.H.).
Mol Pharmacol. 2014 Jan;85(1):18-28. doi: 10.1124/mol.113.089268. Epub 2013 Oct 17.
The plasma membrane Na(+)/H(+) exchanger 1 (NHE1) is rapidly activated in response to various stimuli. The membrane-proximal cytoplasmic region (∼60 residues), termed the lipid-interacting domain (LID), is an important regulatory domain of NHE1. Here, we used a pharmacological approach to further characterize the role of LID in the regulation of NHE1. Pharmacological analysis using staurosporine-like indolocarbazole and bisindolylmaleimide compounds suggested that the phorbol ester- and receptor agonist-induced activation of NHE1 occurs through a protein kinase C-independent mechanism. In particular, only indolocarbazole compounds that inhibited NHE1 activation were able to interact with the LID, suggesting that the inhibition of NHE1 activation is achieved through the direct action of these compounds on the LID. Furthermore, in addition to phorbol esters and a receptor agonist, okadaic acid and hyperosmotic stress, which are known to activate NHE1 through unknown mechanisms, were found to promote membrane association of the LID concomitant with NHE1 activation; these effects were inhibited by staurosporine, as well as by a mutation in the LID. Binding experiments using the fluorescent ATP analog trinitrophenyl ATP revealed that ATP and the NHE1 activator phosphatidylinositol 4,5-bisphosphate bind competitively to the LID. These findings suggest that modulation of NHE1 activity by various activators and inhibitors occurs through the direct binding of these molecules to the LID, which alters the association of the LID with the plasma membrane.
质膜 Na(+)/H(+) 交换体 1（NHE1）可迅速响应各种刺激而被激活。靠近细胞膜的细胞质区域（约 60 个残基），称为脂质相互作用域（LID），是 NHE1 的一个重要调节域。在这里，我们使用药理学方法进一步研究了 LID 在 NHE1 调节中的作用。使用 staurosporine 样吲哚并咔唑和双吲哚基马来酰亚胺化合物的药理学分析表明，佛波酯和受体激动剂诱导的 NHE1 激活是通过蛋白激酶 C 非依赖性机制发生的。特别是，只有能够与 LID 相互作用的抑制 NHE1 激活的吲哚并咔唑化合物，提示抑制 NHE1 激活是通过这些化合物对 LID 的直接作用实现的。此外，除了佛波酯和受体激动剂之外，已知通过未知机制激活 NHE1 的 okadaic 酸和高渗应激也能促进 LID 与 NHE1 激活的膜结合；这些效应被 staurosporine 以及 LID 突变所抑制。使用荧光 ATP 类似物三硝基苯膦酸 ATP 的结合实验表明，ATP 和 NHE1 激活剂磷脂酰肌醇 4,5-二磷酸可竞争性地与 LID 结合。这些发现表明，各种激活剂和抑制剂通过这些分子与 LID 的直接结合来调节 NHE1 活性，从而改变 LID 与质膜的结合。
