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1.
J Cell Sci ; 134(4)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33526715

RESUMO

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.


Assuntos
Processamento Alternativo , Fibronectinas , Células Cultivadas , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos
2.
J Cell Sci ; 126(Pt 21): 4835-42, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-23902689

RESUMO

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.


Assuntos
Neoplasias da Mama/metabolismo , Extensões da Superfície Celular/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Neoplasias da Mama/genética , Caveolina 1/genética , Caveolina 1/metabolismo , Linhagem Celular Tumoral , Extensões da Superfície Celular/genética , Cortactina/genética , Cortactina/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Humanos , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Fosforilação , Ligação Proteica , Trocadores de Sódio-Hidrogênio/genética
3.
iScience ; 26(6): 106887, 2023 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-37324528

RESUMO

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.

4.
Cells ; 12(3)2023 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-36766751

RESUMO

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.


Assuntos
Estresse do Retículo Endoplasmático , Isquemia , Rim , Fatores de Iniciação de Peptídeos , Traumatismo por Reperfusão , Humanos , Estresse do Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/fisiologia , Hipóxia/genética , Hipóxia/metabolismo , Isquemia/genética , Isquemia/metabolismo , Rim/irrigação sanguínea , Rim/metabolismo , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Resposta a Proteínas não Dobradas , Fator de Iniciação de Tradução Eucariótico 5A
5.
Front Physiol ; 13: 907587, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35910559

RESUMO

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.

6.
Front Mol Biosci ; 9: 825028, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35252350

RESUMO

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.

7.
Free Radic Biol Med ; 169: 258-270, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33892115

RESUMO

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.


Assuntos
Transplante de Rim , Traumatismo por Reperfusão , Antioxidantes/uso terapêutico , Humanos , Estresse Oxidativo , Espécies Reativas de Oxigênio
8.
Biochemistry ; 47(51): 13674-85, 2008 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-19035652

RESUMO

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.


Assuntos
Regulação da Expressão Gênica , Pressão Osmótica , Trocadores de Sódio-Hidrogênio/química , Animais , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular , Cricetinae , Citosol/metabolismo , Fibroblastos/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Microscopia de Fluorescência , Modelos Biológicos , Isoformas de Proteínas , Transdução de Sinais , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/metabolismo
9.
J Cell Physiol ; 216(1): 207-20, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18264982

RESUMO

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 L(0). In this study, influence of cholesterol- and caveolin-rich microdomains on NHE-1 activity was examined by using cholesterol depleting agents, including methyl-beta-cyclodextrin (MBCD). These agents activated NHE-1 by modulating its L(0) 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 ((625)KDKEEEIRK(635) 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.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Caveolinas/metabolismo , Colesterol/metabolismo , Microdomínios da Membrana , Trocadores de Sódio-Hidrogênio/metabolismo , Regulação Alostérica , Animais , Proteínas de Transporte de Cátions/genética , Linhagem Celular , Colesterol Oxidase/metabolismo , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Gangliosídeo G(M1)/metabolismo , Humanos , Hidrogênio/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Mutagênese Sítio-Dirigida , Sódio/metabolismo , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genética , beta-Ciclodextrinas/metabolismo
10.
Methods Mol Biol ; 1557: 95-102, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28078585

RESUMO

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.


Assuntos
Transporte de Íons , Prótons , Receptor fas/metabolismo , Células Cultivadas , Concentração de Íons de Hidrogênio , Espaço Intracelular/metabolismo , Cinética , Trocador 1 de Sódio-Hidrogênio/metabolismo
11.
Sci Rep ; 6: 28008, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27302366

RESUMO

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.


Assuntos
Proteína Ligante Fas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Quinases Associadas a rho/metabolismo , Linhagem Celular , Movimento Celular , Humanos , Fosforilação , Prótons , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismo
12.
J Vis Exp ; (97)2015 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-25867523

RESUMO

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.


Assuntos
Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Endossomos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Transporte de Íons , Prótons , Trocadores de Sódio-Hidrogênio/química
13.
Cell Rep ; 7(3): 689-96, 2014 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-24767989

RESUMO

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.


Assuntos
Endocitose/fisiologia , Potássio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Sódio/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular , Membrana Celular/metabolismo , Citoplasma/metabolismo , Endossomos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Transporte de Íons , Íons/química , Lítio/metabolismo , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Prótons , RNA Interferente Pequeno/metabolismo , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/genética
14.
Cell Biochem Biophys ; 63(1): 47-57, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22331497

RESUMO

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.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Membrana Celular/metabolismo , Lipídeos/química , Trocadores de Sódio-Hidrogênio/metabolismo , Regulação Alostérica , Membrana Celular/química , Humanos , Pressão Osmótica , Trocador 1 de Sódio-Hidrogênio , Tensão Superficial
15.
Recent Pat Anticancer Drug Discov ; 6(3): 334-46, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21756245

RESUMO

"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.


Assuntos
Antineoplásicos/administração & dosagem , Membrana Celular/fisiologia , Sistemas de Liberação de Medicamentos/métodos , Resistência a Múltiplos Medicamentos/fisiologia , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/farmacocinética , Biofísica , Membrana Celular/metabolismo , Cães , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Humanos , Modelos Biológicos , Neoplasias/metabolismo , Propriedades de Superfície , Senso de Humor e Humor como Assunto
16.
Channels (Austin) ; 5(4): 382-5, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21654198

RESUMO

On September 12-15, 2010 the French Ion Channels Association organized its annual scientific meeting on the French coast of Mediterranean Sea. This meeting takes place in an attractive location and provides a great opportunity for principal investigators as well as young researchers to present and discuss their recent advances and future challenges in the field of ion channels and transporters. The French Ion Channels Association was created more than 20 years ago and its goal is to organize an annual meeting and more recently to promote interactions (through the website www.canaux-ioniques.fr) between active members of the international scientific community in the field of ion channels. In this report of the 21(st) edition of the meeting, we are summarizing the five main symposia that reflect original works and relevant developments in the domain of ions channels and transporters.


Assuntos
Canais Iônicos , Sociedades Científicas , Animais , França , Humanos
17.
Cancer Res ; 70(19): 7514-22, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20841472

RESUMO

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 Young's 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.


Assuntos
Actinas/metabolismo , Cisplatino/farmacologia , Canais Iônicos/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Fenômenos Biomecânicos , Células COS , Forma Celular/efeitos dos fármacos , Chlorocebus aethiops , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Microscopia de Força Atômica , Proteínas do Tecido Nervoso/antagonistas & inibidores , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores
18.
J Biol Chem ; 282(50): 36692-703, 2007 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-17947235

RESUMO

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 alpha. 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-alpha-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.


Assuntos
Apoptose/fisiologia , Tamanho Celular , Túbulos Renais Proximais/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Animais , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Caspase 3/metabolismo , Linhagem Celular Transformada , Tamanho Celular/efeitos dos fármacos , Ciclosporina/farmacologia , Inibidores Enzimáticos/farmacologia , Sequestradores de Radicais Livres/farmacologia , Túbulos Renais Proximais/citologia , Camundongos , Camundongos Knockout , Peptídeos/farmacologia , Potássio/metabolismo , Estaurosporina/farmacologia , Fator de Necrose Tumoral alfa/farmacologia
19.
Proc Natl Acad Sci U S A ; 103(37): 13854-9, 2006 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-16950870

RESUMO

Mammalian CLC proteins function as Cl(-) channels or as electrogenic Cl(-)/H(+) exchangers and are present in the plasma membrane and intracellular vesicles. We now show that the ClC-6 protein is almost exclusively expressed in neurons of the central and peripheral nervous systems, with a particularly high expression in dorsal root ganglia. ClC-6 colocalized with markers for late endosomes in neuronal cell bodies. The disruption of ClC-6 in mice reduced their pain sensitivity and caused moderate behavioral abnormalities. Neuronal tissues showed autofluorescence at initial axon segments. At these sites, electron microscopy revealed electron-dense storage material that caused a pathological enlargement of proximal axons. These deposits were positive for several lysosomal proteins and other marker proteins typical for neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. However, the lysosomal pH of Clcn6(-/-) neurons appeared normal. CLCN6 is a candidate gene for mild forms of human NCL. Analysis of 75 NCL patients identified ClC-6 amino acid exchanges in two patients but failed to prove a causative role of CLCN6 in that disease.


Assuntos
Canais de Cloreto/genética , Canais de Cloreto/fisiologia , Hipocampo/ultraestrutura , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/patologia , Animais , Axônios/metabolismo , Axônios/ultraestrutura , Comportamento Animal , Biomarcadores/análise , Canais de Cloreto/análise , Análise Mutacional de DNA , Endossomos/química , Endossomos/metabolismo , Endossomos/ultraestrutura , Perfilação da Expressão Gênica , Hipocampo/fisiopatologia , Humanos , Doenças por Armazenamento dos Lisossomos/fisiopatologia , Lisossomos/química , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Camundongos , Camundongos Knockout , Lipofuscinoses Ceroides Neuronais/genética , Neurônios/química , Neurônios/metabolismo , Neurônios/ultraestrutura , Dor/genética , Dor/fisiopatologia
20.
Annu Rev Physiol ; 67: 779-807, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15709978

RESUMO

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.


Assuntos
Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Modelos Animais de Doenças , Doenças Genéticas Inatas/genética , Animais , Humanos , Camundongos , Família Multigênica
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