Biological fractionation of lithium isotopes by cellular Na+/H+ exchangers unravels fundamental transport mechanisms.

Lithium (Li) has a wide range of uses in science, medicine, and industry, but its isotopy is underexplored, except in nuclear science and in geoscience. 6Li and 7Li isotopic ratio exhibits the second largest variation on earth's surface and constitutes a widely used tool for reconstructing past oceans and climates. As large variations have been measured in mammalian organs, plants or marine species, and as 6Li elicits stronger effects than natural Li (∼95% 7Li), a central issue is the identification and quantification of biological influence of Li isotopes distribution. We show that membrane ion channels and Na+-Li+/H+ exchangers (NHEs) fractionate Li isotopes. This systematic 6Li enrichment is driven by membrane potential for channels, and by intracellular pH for NHEs, where it displays cooperativity, a hallmark of dimeric transport. Evidencing that transport proteins discriminate between isotopes differing by one neutron opens new avenues for transport mechanisms, Li physiology, and paleoenvironments.

Correction: The Naturally Processed CD95L Elicits a c-Yes/Calcium/PI3K-Driven Cell Migration Pathway.

[This corrects the article DOI: 10.1371/journal.pbio.1001090.].

Renal Ischemia Tolerance Mediated by eIF5A Hypusination Inhibition Is Regulated by a Specific Modulation of the Endoplasmic Reticulum Stress.

Through kidney transplantation, ischemia/reperfusion is known to induce tissular injury due to cell energy shortage, oxidative stress, and endoplasmic reticulum (ER) stress. ER stress stems from an accumulation of unfolded or misfolded proteins in the lumen of ER, resulting in the unfolded protein response (UPR). Adaptive UPR pathways can either restore protein homeostasis or can turn into a stress pathway leading to apoptosis. We have demonstrated that N1-guanyl-1,7-diamineoheptane (GC7), a specific inhibitor of eukaryotic Initiation Factor 5A (eIF5A) hypusination, confers an ischemic protection of kidney cells by tuning their metabolism and decreasing oxidative stress, but its role on ER stress was unknown. To explore this, we used kidney cells pretreated with GC7 and submitted to either warm or cold anoxia. GC7 pretreatment promoted cell survival in an anoxic environment concomitantly to an increase in xbp1 splicing and BiP level while eiF2α phosphorylation and ATF6 nuclear level decreased. These demonstrated a specific modulation of UPR pathways. Interestingly, the pharmacological inhibition of xbp1 splicing reversed the protective effect of GC7 against anoxia. Our results demonstrated that eIF5A hypusination inhibition modulates distinctive UPR pathways, a crucial mechanism for the protection against anoxia/reoxygenation.

How Does Our Knowledge on the Na+/H+ Exchanger NHE1 Obtained by Biochemical and Molecular Analyses Keep up With Its Recent Structure Determination?

Na+/H+ exchangers are membrane transporters conserved in all living systems and therefore are assumed to be amongst the most ancestral molecular devices that equipped the first protocells. Following the cloning and sequencing of its gene, the mammalian NHE1, that regulates pH and volume in all cells, has been thoroughly scrutinized by molecular and biochemical analyses. Those gave a series of crucial clues concerning its topology, dimeric organization, pharmacological profile, regulation, and the role of key amino acids. Recently thanks to cryogenic Electron Microscopy (Cryo-EM) the long-awaited molecular structures have been revealed. With this information in mind we will challenge the robustness of the earlier conclusions and highlight how the new information enriches our understanding of this key cellular player. At the mechanistic level, we will pinpoint how the NHE1 3D structures reveal that the previously identified amino acids and regions are organized to coordinate transported cations, and shape the allosteric transition that makes NHE1 able to sense intracellular pH and be regulated by signaling pathways.

Intracellular pH Control by Membrane Transport in Mammalian Cells. Insights Into the Selective Advantages of Functional Redundancy.

Intracellular pH is a vital parameter that is maintained close to neutrality in all mammalian cells and tissues and acidic in most intracellular compartments. After presenting the main techniques used for intracellular an vesicular pH measurements we will briefly recall the main molecular mechanisms that affect and regulate intracellular pH. Following this we will discuss the large functional redundancy found in the transporters of H+ or acid-base equivalents. For this purpose, we will use mathematical modeling to simulate cellular response to persistent and/or transient acidification, in the presence of different transporters, single or in combination. We will also test the presence or absence of intracellular buffering. This latter section will highlight how modeling can yield fundamental insight into deep biological questions such as the utility of functional redundancy in natural selection.

Targeting oxidative stress, a crucial challenge in renal transplantation outcome.

Disorders characterized by ischemia/reperfusion (I/R) are the most common causes of debilitating diseases and death in stroke, cardiovascular ischemia, acute kidney injury or organ transplantation. In the latter example the I/R step defines both the amplitude of the damages to the graft and the functional recovery outcome. During transplantation the kidney is subjected to blood flow arrest followed by a sudden increase in oxygen supply at the time of reperfusion. This essential clinical protocol causes massive oxidative stress which is at the basis of cell death and tissue damage. The involvement of both reactive oxygen species (ROS) and nitric oxides (NO) has been shown to be a major cause of these cellular damages. In fact, in non-physiological situations, these species escape endogenous antioxidant control and dangerously accumulate in cells. In recent years, the objective has been to find clinical and pharmacological treatments to reduce or prevent the appearance of oxidative stress in ischemic pathologies. This is very relevant because, due to the increasing success of organ transplantation, clinicians are required to use limit organs, the preservation of which against oxidative stress is crucial for a better outcome. This review highlights the key actors in oxidative stress which could represent new pharmacological targets.

Fibronectin Extra Domains tune cellular responses and confer topographically distinct features to fibril networks.

Cellular fibronectin (FN; also known as FN1) variants harboring one or two alternatively spliced so-called extra domains (EDB and EDA) play a central bioregulatory role during development, repair processes and fibrosis. Yet, how the extra domains impact fibrillar assembly and function of the molecule remains unclear. Leveraging a unique biological toolset and image analysis pipeline for direct comparison of the variants, we demonstrate that the presence of one or both extra domains impacts FN assembly, function and physical properties of the matrix. When presented to FN-null fibroblasts, extra domain-containing variants differentially regulate pH homeostasis, survival and TGF-ß signaling by tuning the magnitude of cellular responses, rather than triggering independent molecular switches. Numerical analyses of fiber topologies highlight significant differences in variant-specific structural features and provide a first step for the development of a generative model of FN networks to unravel assembly mechanisms and investigate the physical and functional versatility of extracellular matrix landscapes.This article has an associated First Person interview with the first author of the paper.

Transcriptomic and Ultrastructural Signatures of K+-Induced Aggregation in Phytophthora parasitica Zoospores.

Most pathogenic oomycetes of the genus Phytophthora spread in water films as flagellated zoospores. Zoospores perceive and produce signals attracting other zoospores, resulting in autoaggregation in vitro or biofilm formation on plant surface. The mechanisms underlying intercellular communication and consequent attraction, adhesion and aggregation are largely unknown. In Phytophthora parasitica, the perception of a K+ gradient induces coordinated motion and aggregation. To define cellular and molecular events associated with oomycete aggregation, we combined transcriptomic and ultrastructural analyses. Results indicate involvement of electroception in K+ sensing. They establish that the transcriptome repertoire required for swimming and aggregation is already fully functional at zoospore release. At the time points analyzed, aggregates are mainly constituted of zoospores. They produce vesicular and fibrillary material discharged at cell-to-cell contacts. Consistently, the signature of transcriptome dynamics during transition to aggregates is an upregulation of genes potentially related to vesicular trafficking. Moreover, transcriptomic and functional analyses show a strong enhancement of carbonic anhydrase activity, indicating that pH homeostasis may contribute to aggregation by acting on both zoospore movement and adhesion. This study poses the molecular and cellular bases of aggregative behavior within oomycetes and expands the current knowledge of ion perception-mediated dissemination of propagules in the rhizosphere.

Pyrazine ring-based Na+/H+ exchanger (NHE) inhibitors potently inhibit cancer cell growth in 3D culture, independent of NHE1.

The Na+/H+ exchanger-1 (NHE1) supports tumour growth, making NHE1 inhibitors of interest in anticancer therapy, yet their molecular effects are incompletely characterized. Here, we demonstrate that widely used pyrazinoylguanidine-type NHE1 inhibitors potently inhibit growth and survival of cancer cell spheroids, in a manner unrelated to NHE1 inhibition. Cancer and non-cancer cells were grown as 3-dimensional (3D) spheroids and treated with pyrazinoylguanidine-type (amiloride, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), 5-(N,N-dimethyl)-amiloride (DMA), and 5-(N,N-hexamethylene)-amiloride (HMA)) or benzoylguanidine-type (eniporide, cariporide) NHE1 inhibitors for 2-7 days, followed by analyses of viability, compound accumulation, and stress- and death-associated signalling. EIPA, DMA and HMA dose-dependently reduced breast cancer spheroid viability while cariporide and eniporide had no effect. Although both compound types inhibited NHE1, the toxic effects were NHE1-independent, as inhibitor-induced viability loss was unaffected by NHE1 CRISPR/Cas9 knockout. EIPA and HMA accumulated extensively in spheroids, and this was associated with marked vacuolization, apparent autophagic arrest, ER stress, mitochondrial- and DNA damage and poly-ADP-ribose-polymerase (PARP) cleavage, indicative of severe stress and paraptosis-like cell death. Pyrazinoylguanidine-induced cell death was partially additive to that induced by conventional anticancer therapies and strongly additive to extracellular-signal-regulated-kinase (ERK) pathway inhibition. Thus, in addition to inhibiting NHE1, pyrazinoylguanidines exert potent, NHE1-independent cancer cell death, pointing to a novel relevance for these compounds in anticancer therapy.

Correction: The Naturally Processed CD95L Elicits a c-Yes/Calcium/PI3K-Driven Cell Migration Pathway.

[This corrects the article DOI: 10.1371/journal.pbio.1001090.].

CD95-Mediated Proton Regulation.

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 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.

Na(+)/H(+) antiporter (NHE1) and lactate/H(+) symporters (MCTs) in pH homeostasis and cancer metabolism.

The Na(+)/H(+)-exchanger NHE1 and the monocarboxylate transporters MCT1 and MCT4 are crucial for intracellular pH regulation, particularly under active metabolism. NHE1, a reversible antiporter, uses the energy provided by the Na(+) gradient to expel H(+) ions generated in the cytosol. The reversible H(+)/lactate(-) symporters MCT1 and 4 cotransport lactate and proton, leading to the net extrusion of lactic acid in glycolytic tumors. In the first two sections of this article we review important features and remaining questions on the structure, biochemical function and cellular roles of these transporters. We then use a fully-coupled mathematical model to simulate their relative contribution to pH regulation in response to lactate production, as it occurs in highly hypoxic and glycolytic tumor cells. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.

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.

The intracellular Na(+)/H(+) exchanger NHE7 effects a Na(+)-coupled, but not K(+)-coupled proton-loading mechanism in endocytosis.

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.

Capturing intracellular pH dynamics by coupling its molecular mechanisms within a fully tractable mathematical model.

We describe the construction of a fully tractable mathematical model for intracellular pH. This work is based on coupling the kinetic equations depicting the molecular mechanisms for pumps, transporters and chemical reactions, which determine this parameter in eukaryotic cells. Thus, our system also calculates the membrane potential and the cytosolic ionic composition. Such a model required the development of a novel algebraic method that couples differential equations for slow relaxation processes to steady-state equations for fast chemical reactions. Compared to classical heuristic approaches based on fitted curves and ad hoc constants, this yields significant improvements. This model is mathematically self-consistent and allows for the first time to establish analytical solutions for steady-state pH and a reduced differential equation for pH regulation. Because of its modular structure, it can integrate any additional mechanism that will directly or indirectly affect pH. In addition, it provides mathematical clarifications for widely observed biological phenomena such as overshooting in regulatory loops. Finally, instead of including a limited set of experimental results to fit our model, we show examples of numerical calculations that are extremely consistent with the wide body of intracellular pH experimental measurements gathered by different groups in many different cellular systems.

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

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.

Hypoxia promotes tumor cell survival in acidic conditions by preserving ATP levels.

The efficacy of targeting pH disruption to induce cell death in the acidic and hypoxic tumor microenvironment continues to be assessed. Here we analyzed the impact of varying levels of hypoxia in acidic conditions on fibroblast and tumor cell survival. Across all cell lines tested, hypoxia (1% O(2)) provided protection against acidosis induced cell death compared to normoxia. Meanwhile severe hypoxia (0.1% O(2)) removed this protection and in some cases exacerbated acidosis-induced cell death. Differential survival between cell types during external acidosis correlated with their respective intracellular pH regulating capabilities. Cellular ATP measurements were conducted to determine their contribution to cell survival under these combined stresses. In general, hypoxia (1% O(2)) maintained elevated ATP levels in acidic conditions while severe hypoxia did not. To further explore this interaction we combined acidosis with ATP depletion using 2-deoxyglucose and observed an enhanced rate of cell mortality. Striking results were also observed with hypoxia providing protection against cell death in spite of a severe metabolic stress induced by a combination of acidosis and oligomycin. Finally, we demonstrated that both HIF1α and HIF2α expression were drastically reduced in hypoxic and acidic conditions indicating a sensitivity of this protein to cellular pH conditions. This knockdown of HIF expression by acidosis has implications for the development of therapies targeting the disruption of cellular pH regulation. Our results reinforce the proof of concept that acidosis and metabolic disruption affecting ATP levels could be exploited as a tumor cell killing strategy.

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.

Teaching new dogs old tricks: membrane biophysical properties in drug delivery and resistance.

"How do drugs cross the plasma membrane?" this may seem like a trivial question. This question is often overlooked to focus primarily on the different complex macro-molecular aspects involved in drug delivery or drug resistance. However, recent studies have highlighted the theme that to be fully understood, more knowledge of the underlying biology of the most complex biological processes involved in the delivery and resistance to drugs is needed. After all, why would a drug interact with a transporter then subsequently be excluded from P-glycoprotein (P-gp) expressing drug resistant cells? What are the determinants of this transition in behavior? Full consideration of the physical biology of drug delivery has allowed a better understanding of the reasons why specific membrane proteins are upregulated or overexpressed in drug resistant cells. This, in turn, allows us to identify new targets for drug chemicals. Better yet, it increases the significance of recents patents and underlines their importance in multi drug resistance.