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Summary: Vesicular H+-ATPases and ClC-chloride transporters are described to acidify intracellular compartments, which also express the highly conserved Na+/H+ exchangers NHE6, NHE7, and NHE9. Mutations of these exchangers cause autism-spectrum disorders and neurodegeneration. NHE6, NHE7, and NHE9 are hypothesized to exchange cytosolic K+ for H+ and alkalinize vesicles, but this notion has remained untested in K+ because their intracellular localization prevents functional measurements. Using proton-killing techniques, we selected a cell line that expresses wild-type NHE7 at the plasma membrane, enabling measurement of the exchanger’s transport parameters. We found that NHE7 transports Li+ and Na+, but not K+, is nonreversible in physiological conditions and is constitutively activated by cytosolic H+. Therefore, NHE7 acts as a proton-loading transporter rather than a proton leak. NHE7 mediates an acidification of intracellular vesicles that is additive to that of V-ATPases and that accelerates endocytosis. This study reveals an unexpected function for vesicular Na+/H+ exchangers and provides clues for understanding NHE-linked neurological disorders. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

The Na+/H+ exchanger 1, which plays an essential role in intracellular pH regulation in most tissues, is also known to be a key actor in both proliferative and apoptotic processes. Its activation by H+ is best described by the Monod–Wyman–Changeux model: the dimeric NHE-1 oscillates between a low and a high affinity conformation, the balance between the two forms being defined by the allosteric constant L0. In this study, influence of cholesterol- and caveolin-rich microdomains on NHE-1 activity was examined by using cholesterol depleting agents, including methyl-β-cyclodextrin (MBCD). These agents activated NHE-1 by modulating its L0 parameter, which was reverted by cholesterol repletion. This activation was associated with NHE-1 relocation outside microdomains, and was distinct from NHE-1 mitogenic and hormonal stimulation; indeed MBCD and serum treatments were additive, and serum alone did not change NHE-1 localization. Besides, MBCD activated a serum-insensitive, constitutively active mutated NHE-1 (625KDKEEEIRK635 into KNKQQQIRK). Finally, the membrane-dependent NHE-1 regulation occurred independently of Mitogen Activated Protein Kinases, especially Extracellular Regulated Kinase activation, although this kinase was activated by MBCD. In conclusion, localization of NHE-1 in membrane cholesterol- and caveolin-rich microdomains constitutes a novel physiological negative regulator of NHE-1 activity. J. Cell. Physiol. 216: 207–220, 2008. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The FMRF-amide-activated sodium channel (FaNaC), a member of the ENaC/Degenerin family, is a homotetramer, each subunit containing two transmembrane segments. We changed independently every residue of the first transmembrane segment (TM1) into a cysteine and tested each position's accessibility to the cysteine covalent reagents MTSET and MTSES. Eleven mutants were accessible to the cationic MTSET, showing that TM1 faces the ion translocation pathway. This was confirmed by the accessibility of cysteines present in the acid-sensing ion channels and other mutations introduced in FaNaC TM1. Modification of accessibilities for positions 69, 71 and 72 in the open state shows that the gating mechanism consists of the opening of a constriction close to the intracellular side. The anionic MTSES did not penetrate into the channel, indicating the presence of a charge selectivity filter in the outer vestibule. Furthermore, amiloride inhibition resulted in the channel occlusion in the middle of the pore. Summarizing, the ionic pore of FaNaC includes a large aqueous cavity, with a charge selectivity filter in the outer vestibule and the gate close to the interior. [ABSTRACT FROM AUTHOR]

The Na + /H + exchanger NHE1 is at the crossroads of a large diversity of signaling pathways, whose activation modifies the cooperative response of the transporter to intracellular H + ions. Here we show how the activation of the Na + /H + exchanger NHE1 by the cleaved ligand of CD95 can be measured. We demonstrate two different methods designed to set intracellular pH at precise values. Then we show how these can be coupled to fast kinetics of lithium transport, which will enable to measure the NHE1 activity like for an enzyme, because they will yield rates of transport.

The CLC gene family encodes nine different Cl() channels in mammals. These channels perform their functions in the plasma membrane or in intracellular organelles such as vesicles of the endosomal/lysosomal pathway or in synaptic vesicles. The elucidation of their cellular roles and their importance for the organism were greatly facilitated by mouse models and by human diseases caused by mutations in their respective genes. Human mutations in CLC channels are known to cause diseases as diverse as myotonia (muscle stiffness), Bartter syndrome (renal salt loss) with or without deafness, Dent's disease (proteinuria and kidney stones), osteopetrosis and neurodegeneration, and possibly epilepsy. Mouse models revealed blindness and infertility as further consequences of CLC gene disruptions. These phenotypes firmly established the roles CLC channels play in stabilizing the plasma membrane voltage in muscle and possibly in neurons, in the transport of salt and fluid across epithelia, in the acidification of endosomes and synaptic vesicles, and in the degradation of bone by osteoclasts.