Department of Physiology and Membrane Biology, School of Medicine, University of California Davis, Davis, California, USA.
PLoS One. 2011;6(12):e29210. doi: 10.1371/journal.pone.0029210. Epub 2011 Dec 28.
The Na(+)/H(+)Exchanger isoform 1 (NHE1) is a highly versatile, broadly distributed and precisely controlled transport protein that mediates volume and pH regulation in most cell types. NHE1 phosphorylation contributes to Na(+)/H(+) exchange activity in response to phorbol esters, growth factors or protein phosphatase inhibitors, but has not been observed during activation by osmotic cell shrinkage (OCS). We examined the role of NHE1 phosphorylation during activation by OCS, using an ideal model system, the Amphiuma tridactylum red blood cell (atRBC). Na(+)/H(+) exchange in atRBCs is mediated by an NHE1 homolog (atNHE1) that is 79% identical to human NHE1 at the amino acid level. NHE1 activity in atRBCs is exceptionally robust in that transport activity can increase more than 2 orders of magnitude from rest to full activation. Michaelis-Menten transport kinetics indicates that either OCS or treatment with the phosphatase inhibitor calyculin-A (CLA) increase Na(+) transport capacity without affecting transport affinity (K(m)=44 mM) in atRBCs. CLA and OCS act non-additively to activate atNHE1, indicating convergent, phosphorylation-dependent signaling in atNHE1 activation. In situ(32)P labeling and immunoprecipitation demonstrates that the net phosphorylation of atNHE1 is increased 4-fold during OCS coinciding with a more than 2-order increase in Na(+) transport activity. This is the first reported evidence of increased NHE1 phosphorylation during OCS in any vertebrate cell type. Finally, liquid chromatography and mass spectrometry (LC-MS/MS) analysis of atNHE1 immunoprecipitated from atRBC membranes reveals 9 phosphorylated serine/threonine residues, suggesting that activation of atNHE1 involves multiple phosphorylation and/or dephosphorylation events.
钠氢交换体亚型 1（NHE1）是一种多功能、广泛分布且精确调控的转运蛋白，可调节大多数细胞类型的体积和 pH 值。NHE1 的磷酸化作用有助于响应佛波酯、生长因子或蛋白磷酸酶抑制剂的钠氢交换活性，但在渗透细胞收缩（OCS）激活时并未观察到。我们使用理想的模型系统，即 Amphiuma tridactylum 红细胞（atRBC），研究了 OCS 激活时 NHE1 磷酸化的作用。atRBC 中的钠氢交换由 NHE1 同源物（atNHE1）介导，atNHE1 与人类 NHE1 在氨基酸水平上具有 79%的同源性。atRBC 中的 NHE1 活性非常强，其转运活性可以从静止状态增加 2 个数量级以上至完全激活。米氏动力学分析表明，OCS 或使用磷酸酶抑制剂 calyculin-A（CLA）处理均可增加 atRBC 中的 Na+转运能力，而不影响转运亲和力（K(m)=44 mM）。CLA 和 OCS 非加性地作用于激活 atNHE1，表明在 atNHE1 激活中存在收敛的、依赖磷酸化的信号转导。原位（32）P 标记和免疫沉淀表明，OCS 期间 atNHE1 的净磷酸化增加了 4 倍，同时 Na+转运活性增加了 2 个数量级以上。这是在任何脊椎动物细胞类型中首次报道的 OCS 期间 NHE1 磷酸化增加的证据。最后，通过液相色谱和质谱（LC-MS/MS）分析从 atRBC 膜中免疫沉淀的 atNHE1，发现 9 个磷酸化丝氨酸/苏氨酸残基，表明 atNHE1 的激活涉及多个磷酸化和/或去磷酸化事件。
