Lacroix Jérôme, Poët Mallorie, Huc Laurence, Morello Vincent, Djerbi Nadir, Ragno Michel, Rissel Mary, Tekpli Xavier, Gounon Pierre, Lagadic-Gossmann Dominique, Counillon Laurent
Université de Nice-Sophia Antipolis, CNRS FRE3093 Transport Ionique aspects normaux et pathologiques, Faculté des Sciences Parc Valrose, 06108 Nice cedex 2, France.
Biochemistry. 2008 Dec 23;47(51):13674-85. doi: 10.1021/bi801368n.
NHE-1 is a ubiquitous, mitogen-activatable, mammalian Na+/H+ exchanger that maintains cytosolic pH and regulates cell volume. We have previously shown that the kinetics of NHE-1 positive cooperative activation by intracellular acidifications fit best with a Monod-Wyman-Changeux mechanism, in which a dimeric NHE-1 oscillates between a low- and a high-affinity conformation for intracellular protons. The ratio between these two forms, the allosteric equilibrium constant L0, is in favor of the low-affinity form, making the system inactive at physiological pH. Conversely the high-affinity form is stabilized by intracellular protons, resulting in the observed positive cooperativity. The aim of the present study was to investigate the kinetics and mechanism of NHE-1 regulation by osmotic shocks. We show that they modify the L0 parameter (865 +/- 95 and 3757 +/- 328 for 500 and 100 mOsM, respectively, vs 1549 +/- 57 in isotonic conditions).This results in an activation of NHE-1 by hypertonic shocks and, conversely, in an inhibition by hypotonic media. Quantitatively, this modulation of L0 follows an exponential distribution relative to osmolarity, that is, additive to the activation of NHE-1 by intracellular signaling pathways. These effects can be mimicked by the asymmetric insertion of amphiphilic molecules into the lipid bilayer. Finally, site-directed mutagenesis of NHE-1 shows that neither its association with membrane PIP2 nor its interaction with cortical actin are required for mechanosensation. In conclusion, NHE-1 allosteric equilibrium and, thus, its cooperative response to intracellular acidifications is extremely sensitive to modification of its membrane environment.
NHE-1是一种普遍存在的、有丝分裂原可激活的哺乳动物Na+/H+交换体，可维持细胞溶质pH值并调节细胞体积。我们之前已经表明，细胞内酸化对NHE-1的正协同激活动力学最符合莫诺德-怀曼-尚热机制，即二聚体NHE-1在对细胞内质子的低亲和力构象和高亲和力构象之间振荡。这两种形式之间的比例，即变构平衡常数L0，有利于低亲和力形式，使得该系统在生理pH值下无活性。相反，高亲和力形式被细胞内质子稳定，导致观察到的正协同性。本研究的目的是研究渗透压休克对NHE-1的调节动力学和机制。我们发现它们改变了L0参数（500和100 mOsM时分别为865±95和3757±328，等渗条件下为1549±57）。这导致高渗休克激活NHE-1，相反，低渗介质抑制NHE-1。定量地说，L0的这种调节相对于渗透压呈指数分布，也就是说，它与细胞内信号通路对NHE-1的激活是相加的。这些效应可以通过两亲性分子不对称插入脂质双层来模拟。最后，NHE-1的定点诱变表明，机械感受既不需要它与膜PIP2的结合，也不需要它与皮质肌动蛋白的相互作用。总之，NHE-1的变构平衡以及因此它对细胞内酸化的协同反应对其膜环境的改变极其敏感。
