Kinetic analysis of the regulation of the Na+/H+ exchanger NHE-1 by osmotic shocks.

Jérôme Lacroix (1) , Mallorie Poët (1, 2) , Laurence Huc (3) , Vincent Morello (2) , Nadir Djerbi (1, 2) , Michel Ragno (1, 4, 5) , Mary Rissel (6) , Xavier Tekpli (6) , Pierre Gounon (7) , Dominique Lagadic-Gossmann (6) , Laurent Counillon (2)

1 Transport ionique aspects normaux et pathologiques
2 IPMC - Institut de pharmacologie moléculaire et cellulaire
3 Experimental Medicine and Toxicology
4 BCI - Biologie du Cancer et de l'Infection
5 Pathogénie bactérienne et réponses cellulaires
6 SeRAIC - Signalisation et Réponses aux Agents Infectieux et Chimiques
7 Physiological Genomics of the Eukaryotes

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.

Domaines: Biochimie, Biologie Moléculaire

Jérôme Lacroix, Mallorie Poët, Laurence Huc, Vincent Morello, Nadir Djerbi, et al.. Kinetic analysis of the regulation of the Na+/H+ exchanger NHE-1 by osmotic shocks.. Biochemistry, 2008, 47 (51), pp.13674-85. ⟨10.1021/bi801368n⟩. ⟨hal-00674405⟩