Mallorie Poët

A mechanism for the activation of the Na/H exchanger NHE‐1 by cytoplasmic acidification and mitogens

Eukaryotic cells constantly have to fight against internal acidification. In mammals, this task is mainly performed by the ubiquitously expressed electroneutral Na+/H+ exchanger NHE‐1, which activates in a cooperative manner when cells become acidic. Despite its biological importance, the mechanism of this activation is still poorly understood, the most commonly accepted hypothesis being the existence of a proton‐sensor site on the internal face of the transporter. This work uncovers mutations that lead to a nonallosteric form of the exchanger and demonstrates that NHE‐1 activation is best described by a Monod–Wyman–Changeux concerted mechanism for a dimeric transporter. During intracellular acidification, a low‐affinity form of NHE‐1 is converted into a form possessing a higher affinity for intracellular protons, with no requirement for an additional proton‐sensor site on the protein. This new mechanism also explains the activation of the exchanger by growth signals, which shift the equilibrium towards the high‐affinity form.

NaV1.5 Na+ channels allosterically regulate the NHE-1 exchanger and promote the activity of breast cancer cell invadopodia

Summary The degradation of the extracellular matrix by cancer cells represents an essential step in metastatic progression and this is performed by cancer cell structures called invadopodia. NaV1.5 (also known as SCN5A) Na+ channels are overexpressed in breast cancer tumours and are associated with metastatic occurrence. It has been previously shown that NaV1.5 activity enhances breast cancer cell invasiveness through perimembrane acidification and subsequent degradation of the extracellular matrix by cysteine cathepsins. Here, we show that NaV1.5 colocalises with Na+/H+ exchanger type 1 (NHE-1) and caveolin-1 at the sites of matrix remodelling in invadopodia of MDA-MB-231 breast cancer cells. NHE-1, NaV1.5 and caveolin-1 co-immunoprecipitated, which indicates a close association between these proteins. We found that the expression of NaV1.5 was responsible for the allosteric modulation of NHE-1, rendering it more active at the intracellular pH range of 6.4–7; thus, it potentially extrudes more protons into the extracellular space. Furthermore, NaV1.5 expression increased Src kinase activity and the phosphorylation (Y421) of the actin-nucleation-promoting factor cortactin, modified F-actin polymerisation and promoted the acquisition of an invasive morphology in these cells. Taken together, our study suggests that NaV1.5 is a central regulator of invadopodia formation and activity in breast cancer cells.

Nongenomic effects of cisplatin: acute inhibition of mechanosensitive transporters and channels without actin remodeling

Cisplatin is an antineoplastic drug, mostly documented to cause cell death through the formation of DNA adducts. In patients, it exhibits a range of short-term side effects that are unlikely to be related to its genomic action. As cisplatin has been shown to modify membrane properties in different cell systems, we investigated its effects on mechanosensitive ion transporters and channels. We show here that cisplatin is a noncompetitive inhibitor of the mechanosensitive Na(+)/H(+) exchanger NHE-1, with a half-inhibition concentration of 30 μg/mL associated with a decrease in V(max) and Hill coefficient. We also showed that it blocks the Cl(-) and K(+) mechanosensitive channels VSORC and TREK-1 at similar concentrations. In contrast, the nonmechanosensitive Cl(-) and K(+) channels CFTR and TASK-1 and the Na(+)-coupled glucose transport, which share functional features with VSORC, TREK-1, and NHE-1, respectively, were insensitive to cisplatin. We next investigated whether cisplatin action was due to a direct effect on membrane or to cortical actin remodeling that would affect mechanosensors. Using scanning electron microscopy, in vivo actin labeling, and atomic force microscopy, we did not observe any modification of the Youngs modulus and actin cytoskeleton for up to 60 and 120 μg/mL cisplatin, whereas these concentrations modified membrane morphology. Our results reveal a novel mechanism for cisplatin, which affects mechanosensitive channels and transporters involved in cell fate programs and/or expressed in mechanosensitive organs in which cisplatin elicits strong secondary effects, such as the inner ear or the peripheral nervous system. These results might constitute a common denominator to previously unrelated effects of this drug.

Role of TASK2 in the Control of Apoptotic Volume Decrease in Proximal Kidney Cells

Apoptotic volume decrease (AVD) is prerequisite to apoptotic events that lead to cell death. In a previous study, we demonstrated in kidney proximal cells that the TASK2 channel was involved in the K+ efflux that occurred during regulatory volume decrease. The aim of the present study was to determine the role of the TASK2 channel in the regulation of AVD and apoptosis phenomenon. For this purpose renal cells were immortalized from primary cultures of proximal convoluted tubules (PCT) from wild type and TASK2 knock-out mice (task2-/-). Apoptosis was induced by staurosporine, cyclosporin A, or tumor necrosis factor α. Cell volume, K+ conductance, caspase-3, and intracellular reactive oxygen species (ROS) levels were monitored during AVD. In wild type PCT cells the K+ conductance activated during AVD exhibited characteristics of TASK2 currents. In task2-/- PCT cells, AVD and caspase activation were reduced by 59%. Whole cell recordings indicated that large conductance calcium-activated K+ currents inhibited by iberiotoxin (BK channels) partially compensated for the deletion of TASK2 K+ currents in the task2-/- PCT cells. This result explained the residual AVD measured in these cells. In both cell lines, apoptosis was mediated via intracellular ROS increase. Moreover AVD, K+ conductances, and caspase-3 were strongly impaired by ROS scavenger N-acetylcysteine. In conclusion, the main K+ channels involved in staurosporine, cyclosporin A, and tumor necrosis factor-α-induced AVD are TASK2 K+ channels in proximal wild type cells and iberiotoxin-sensitive BK channels in proximal task2-/- cells. Both K+ channels could be activated by ROS production.

Exploration of the pore structure of a peptide-gated Na+ channel

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 positions 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.

Regulation of Na+/H+ exchanger 1 allosteric balance by its localization in cholesterol‐ and caveolin‐rich membrane microdomains

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.

Kinetic Analysis of the Regulation of the Na+/H+ Exchanger NHE-1 by Osmotic Shocks

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.

The cleaved FAS ligand activates the Na + /H + exchanger NHE1 through Akt/ROCK1 to stimulate cell motility

Transmembrane CD95L (Fas ligand) can be cleaved to release a promigratory soluble ligand, cl-CD95L, which can contribute to chronic inflammation and cancer cell dissemination. The motility signaling pathway elicited by cl-CD95L remains poorly defined. Here, we show that in the presence of cl-CD95L, CD95 activates the Akt and RhoA signaling pathways, which together orchestrate an allosteric activation of the Na+/H+ exchanger NHE1. Pharmacologic inhibition of Akt or ROCK1 independently blocks the cl-CD95L-induced migration. Confirming these pharmacologic data, disruption of the Akt and ROCK1 phosphorylation sites on NHE1 decreases cell migration in cells exposed to cl-CD95L. Together, these findings demonstrate that NHE1 is a novel molecular actor in the CD95 signaling pathway that drives the cl-CD95L-induced cell migration through both the Akt and RhoA signaling pathways.

On the role of the difference in surface tensions involved in the allosteric regulation of NHE-1 induced by low to mild osmotic pressure, membrane tension and lipid asymmetry

The sodium-proton exchanger 1 (NHE-1) is a membrane transporter that exchanges Na+ for H+ ion across the membrane of eukaryotic cells. It is cooperatively activated by intracellular protons, and this allosteric regulation is modulated by the biophysical properties of the plasma membrane and related lipid environment. Consequently, NHE-1 is a mechanosensitive transporter that responds to osmotic pressure, and changes in membrane composition. The purpose of this study was to develop the relationship between membrane surface tension, and the allosteric balance of a mechanosensitive transporter such as NHE-1. In eukaryotes, the asymmetric composition of membrane leaflets results in a difference in surface tensions that is involved in the creation of a reservoir of intracellular vesicles and membrane buds contributing to buffer mechanical constraints. Therefore, we took this phenomenon into account in this study and developed a set of relations between the mean surface tension, membrane asymmetry, fluid phase endocytosis and the allosteric equilibrium constant of the transporter. We then used the experimental data published on the effects of osmotic pressure and membrane modification on the NHE-1 allosteric constant to fit these equations. We show here that NHE-1 mechanosensitivity is more based on its high sensitivity towards the asymmetry between the bilayer leaflets compared to mean global membrane tension. This compliance to membrane asymmetry is physiologically relevant as with their slower transport rates than ion channels, transporters cannot respond as high pressure-high conductance fast-gating emergency valves.

Functional Characterization of Na + /H + Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.