functional-characterization-na-h-exchangers-intracellular

Functional Characterization of Na<sup>+</sup>/H<sup>+</sup> 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 ...

Université Nice-Sophia Antipolis, Laboratoire de Physiomédecine Moléculaire, CNRS UMR7370, and Laboratories of Excellence Ion Channel Science and Therapeutics

Variations in calcium concentration within the endoplasmic reticulum ([Ca(2+)](ER)) may play a role in cell growth. This study evaluates the regulation of calcium pools by growth modulators of prostate cancer (PC) cells, the insulin growth factor (IGF), and the tumor necrosis growth factor-alpha (TNFalpha) as well as evaluating the possible role of [Ca(2+)](ER) variations as signals for growth modulation. We show that IGF (5 ng/ml), which increases cell growth, induces an increase in [Ca(2+)](ER) whereas TNFalpha (1 ng/ml) which reduces cell proliferation and induces apoptosis, reduces [Ca(2+)](ER). IGF-induced [Ca(2+)](ER) increase is correlated to an overexpression of the sarcoendoplasmic calcium-ATPase 2B (SERCA2b), whereas TNFalpha-induced [Ca(2+)](ER) decrease is associated to a reduction in SERCA2b expression. Pretreatment with epidermal growth factors (EGF) or IGF does not prevent TNFalpha from affecting the induction of apoptosis, [Ca(2+)](ER) reduction and SERCA2b downregulation. Reduction in [Ca(2+)](ER) induced by thapsigargin (TG) (from 1 pM to 1 microM, 48 h) reduces LNCaP growth in a dose dependent manner and induces apoptosis when cells are treated with 1 microM TG. We also show that a transient TG application (1 pM, 1 nM, 1 microM 15 min) is insufficient to induce a long lasting decrease in [Ca(2+)](ER), since [Ca(2+)](ER) remains identical to the control for 48 h following TG application. These treatments (1 pM and 1 nM, 15 min) do not modify cell growth. However, TG (1 microM, 15 min) induces apoptosis. We thus identify [Ca(2+)](ER) and SERCA2b as a central targets for causing LNCaP PC cell life or death induced by growth modulators. Furthermore our results indicate that calcium pool contents can regulate cell growth.

Role of endoplasmic reticulum calcium content in prostate cancer cell growth regulation by IGF and TNFalpha.

In this study, we investigated ionic mechanisms involved in growth arrest induced by extracellular ATP in androgen-independent prostate cancer cells. Extracellular ATP reversibly induced a rapid and sustained intracellular pH (pH(i)) decrease from 7.41 to 7.11. Inhibition of Ca(2+) influx, lowering extracellular Ca(2+), and buffering cytoplasmic Ca(2+) inhibited ATP-induced acidification, thereby demonstrating that acidification is a consequence of Ca(2+) entry. We show that ATP induced reuptake of Ca(2+) by the mitochondria and a transient depolarization of the inner mitochondrial membrane. ATP-induced acidification was reduced after the dissipation of the mitochondrial proton gradient by rotenone and carbonyl cyanide p-trifluoromethoxyphenylhydrazone, after inhibition of Ca(2+) uptake into the mitochondria by ruthenium red, and after inhibition of the F(0)F(1)-ATPase with oligomycin. ATP-induced acidification was not induced by either stimulation of the Cl(-)/HCO(3)(-) exchanger or inhibition of the Na(+)/H(+) exchanger. In addition, intracellular acidification, induced by an ammonium prepulse method, reduced the amount of releasable Ca(2+) from the endoplasmic reticulum, assessed by measuring change in cytosolic Ca(2+) induced by thapsigargin or ATP in a Ca(2+)-free medium. This latter finding reveals cross talk between pH(i) and Ca(2+) homeostasis in which the Ca(2+)-induced intracellular acidification can in turn regulate the amount of Ca(2+) that can be released from the endoplasmic reticulum. Furthermore, pH(i) decrease was capable of reducing cell growth. Taken together, our results suggest that ATP-induced acidification in DU-145 cells results from specific effect of mitochondrial function and is one of the major mechanisms leading to growth arrest induced by ATP.

The role of intracellular pH in cell growth arrest induced by ATP.

Zellul&auml;re oder chemische Aktivatoren f&uuml;r die Transienten Rezeptor m&ouml;glichen Kan&auml;le der Vanilloid-Unterfamilie (TRPV) wurden in den letzten Jahren identifiziert. Eine bemerkenswerte Ausnahme ist TRPV2, f&uuml;r die zellul&auml;re Vorg&auml;nge um Kanal-Aktivierung sind noch Gegenstand der Diskussion. Vielf&auml;ltige Reize wie extreme Hitze oder Phosphatidylinositol-3-Kinase (PI3-Kinase) reguliert Membran Einf&uuml;gung sind gezeigt worden, um TRPV2-Channel-Aktivit&auml;ten zu f&ouml;rdern. Allerdings erwies es sich als schwierig zu reproduzieren, haben einige dieser Ergebnisse und kann zugrunde liegen verschiedene gating Mechanismen je nach Zelle in welche TRPV2-Kan&auml;le ausgedr&uuml;ckt werden. Hier zeigen wir, dass Ausdruck der rekombinanten TRPV2 Zytotoxizit&auml;t hervorrufen kann, die direkt mit Channel-Aktivit&auml;ten zusammenh&auml;ngt, da es durch die Einf&uuml;hrung kostenlos Ersatz in der Pore-forming-Dom&auml;ne des Kanals, oder durch eine Verringerung der extrazellul&auml;re Kalzium vorgebeugt werden kann. Stabil transfizierte Zellen f&uuml;hrt TRPV2-Ausdruck in einer &auml;u&szlig;erlich Gleichrichterdiode Str&ouml;mung, die an alle Potenziale aufgezeichnet werden kann und einem Anstieg der Ruhe intrazellul&auml;ren Calcium-Konzentration, die teilweise durch Serum Hunger verhindert werden kann. Zytotoxizit&auml;t als ein Auslesen des Channel-Aktivit&auml;ten und direkte Messungen der Zelle Oberfl&auml;che Ausdruck der TRPV2 verwendet wird, zeigen wir, dass die Hemmung der PI3-Kinase verringert TRPV2-Channel-Aktivit&auml;ten w&auml;hrend des Handels mit den Kanal an die Plasmamembran nicht betroffen. Wird der Schluss gezogen, dass PI3-Kinase induziert oder die Aktivit&auml;t der rekombinanten TRPV2-Kan&auml;le moduliert; im Gegensatz zu den bisher vorgeschlagenen Mechanismus ist keine Aktivierung TRPV2-Kan&auml;le von PI3-Kinase durch Translokation der Kanal an der Plasmamembran.

PI3-Kinase f&ouml;rdert TRPV2 Aktivit&auml;t unabh&auml;ngig von Kanal-Translokation an der Plasmamembran.

Attivatori chimici o cellulari per transitori pi&ugrave; canali potenziali di recettore della sottofamiglia vanilloidi (TRV1) sono stati identificati negli ultimi anni. Una notevole eccezione a questa regola &egrave; TRPV2, per cui gli eventi cellulari che portano all&#39;attivazione del canale sono ancora materia di dibattito. Inserimento membrana diversi stimoli quali calore estremo o fosfatidilinositolo 3 chinasi (PI3 chinasi) regolamentati hanno dimostrato di promuovere le attivit&agrave; del canale TRPV2. Tuttavia, alcuni di questi risultati hanno dimostrato difficile da riprodurre e pu&ograve; essere alla base di diversi meccanismi di controllo a seconda del tipo di cellula in cui TRPV2 canali sono espressi. Mostriamo qui, che espressione di TRPV2 ricombinante pu&ograve; indurre citotossicit&agrave; che &egrave; direttamente collegata all&#39;attivit&agrave; del canale, poich&eacute; pu&ograve; essere evitata introducendo una sostituzione carica nel dominio che formano poro del canale, o riducendo il calcio extracellulare. In celle transfected stabile, espressione TRPV2 risultati in una corrente esteriormente rettifica che pu&ograve; essere registrata a tutti i potenziali e in un aumento della concentrazione di calcio intracellulare che pu&ograve; essere parzialmente prevenuta da fame siero di riposo. Utilizzando citotossicit&agrave; come un indicatore di attivit&agrave; del canale e misurazioni dirette di espressione di superficie delle cellule di TRPV2, mostriamo che l&#39;inibizione della PI3-chinasi diminuisce l&#39;attivit&agrave; del canale TRPV2 ma non influisce il traffico del canale di membrana del plasma. Si &egrave; concluso che PI3 chinasi induce o modula l&#39;attivit&agrave; dei canali TRPV2 ricombinante; in contrasto con il meccanismo proposto in precedenza, attivazione dei canali TRPV2 da PI3 chinasi non &egrave; a causa di traslocazione di canale di membrana del plasma.

PI3 chinasi promuove attivit&agrave; TRPV2 indipendentemente dalla traslocazione di canale di membrana del plasma.

&#x8FD1;&#x51E0;&#x5E74;&#x5DF2;&#x786E;&#x5B9A;&#x7EC6;&#x80DE;&#x6216;&#x5316;&#x5B66;&#x6D3B;&#x5316;&#x5242;&#x8FA3;&#x6912;&#x7D20;&#x4E9A;&#x79D1; (TRPV) &#x6700;&#x77AC;&#x65F6;&#x53D7;&#x4F53;&#x6F5C;&#x5728;&#x6E20;&#x9053;&#x3002;&#x663E;&#x8457;&#x7684;&#x4F8B;&#x5916;&#xFF0C;&#x5C31;&#x662F; TRPV2&#xFF0C;&#x5BFC;&#x81F4;&#x901A;&#x9053;&#x6FC0;&#x6D3B;&#x7EC6;&#x80DE;&#x4E8B;&#x4EF6;&#x8FD8;&#x6709;&#x4E00;&#x4E2A;&#x8BA8;&#x8BBA;&#x7684;&#x95EE;&#x9898;&#x3002;&#x5404;&#x79CD;&#x523A;&#x6FC0;&#xFF0C;&#x5982;&#x6781;&#x7AEF;&#x70ED;&#x6216;&#x78F7;&#x8102;&#x9170;&#x808C;&#x9187;-3 &#x6FC0;&#x9176; (PI3 &#x6FC0;&#x9176;) &#x89C4;&#x7BA1;&#x819C;&#x63D2;&#x5165;&#x8868;&#x660E;&#x53EF;&#x4FC3;&#x8FDB; TRPV2 &#x901A;&#x9053;&#x6D3B;&#x52A8;&#x3002;&#x7136;&#x800C;&#xFF0C;&#x4E00;&#x4E9B;&#x8FD9;&#x4E9B;&#x7ED3;&#x679C;&#x5DF2;&#x8BC1;&#x660E;&#x96BE;&#x4EE5;&#x91CD;&#x73B0;&#x548C;&#x53EF;&#x80FD;&#x4F1A;&#x5F62;&#x6210;&#x4E0D;&#x540C;&#x7684;&#x95E8;&#x63A7;&#x673A;&#x5236;&#xFF0C;&#x53D6;&#x51B3;&#x4E8E;&#x5728;&#x54EA;&#x4E2A; TRPV2 &#x6E20;&#x9053;&#x90FD;&#x8868;&#x793A;&#x7684;&#x5355;&#x5143;&#x683C;&#x7C7B;&#x578B;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x663E;&#x793A;&#x8868;&#x8FBE;&#x7684;&#x91CD;&#x7EC4; TRPV2 &#x53EF;&#x4EE5;&#x8BF1;&#x4F7F;&#x7EC6;&#x80DE;&#x6BD2;&#x6027;&#x4F5C;&#x7528;&#xFF0C;&#x56E0;&#x4E3A;&#x5B83;&#x53EF;&#x4EE5;&#x9632;&#x6B62;&#x901A;&#x8FC7;&#x5F15;&#x5165;&#x6536;&#x8D39;&#x66FF;&#x6362;&#x6210;&#x5B54;&#x57DF;&#x4E2D;&#x7684;&#x901A;&#x9053;&#xFF0C;&#x6216;&#x51CF;&#x5C11;&#x80DE;&#x5916;&#x9499;&#x79BB;&#x5B50;&#x901A;&#x9053;&#x6D3B;&#x52A8;&#x76F4;&#x63A5;&#x76F8;&#x5173;&#x3002;&#x5728;&#x7A33;&#x5B9A;&#x8F6C;&#x67D3;&#x7EC6;&#x80DE;&#xFF0C;TRPV2 &#x8868;&#x8FBE;&#x5F0F;&#x7684;&#x7ED3;&#x679C;&#x53EF;&#x4EE5;&#x5728;&#x6240;&#x6709;&#x7684;&#x6F5C;&#x80FD;&#xFF0C;&#x5F55;&#x5F97;&#x7684;&#x5916;&#x5411;&#x6574;&#x6D41;&#x7535;&#x6D41;&#x548C;&#x9759;&#x606F;&#x53EF;&#x4EE5;&#x90E8;&#x5206;&#x56E0;&#x8840;&#x6E05;&#x9965;&#x997F;&#x7684;&#x7EC6;&#x80DE;&#x5185;&#x9499;&#x79BB;&#x5B50;&#x6D53;&#x5EA6;&#x7684;&#x589E;&#x52A0;&#x3002;&#x6211;&#x4EEC;&#x4F7F;&#x7528;&#x4F5C;&#x4E3A;&#x8BFB;&#x51FA;&#x7684;&#x901A;&#x9053;&#x6D3B;&#x52A8;&#x548C;&#x76F4;&#x63A5;&#x6D4B;&#x91CF;&#x7684;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#x8868;&#x8FBE;&#x7684; TRPV2 &#x7684;&#x7EC6;&#x80DE;&#x6BD2;&#x6027;&#x4F5C;&#x7528;&#xFF0C;&#x663E;&#x793A; PI3 &#x6FC0;&#x9176;&#x7684;&#x6291;&#x5236;&#x4F5C;&#x7528;&#x964D;&#x4F4E; TRPV2 &#x901A;&#x9053;&#x6D3B;&#x52A8;&#xFF0C;&#x4F46;&#x4E0D;&#x5F71;&#x54CD;&#x8D29;&#x8FD0;&#x5230;&#x7EC6;&#x80DE;&#x8D28;&#x819C;&#x7684;&#x901A;&#x9053;&#x3002;&#x5B83;&#x88AB;&#x7ED3;&#x675F; PI3 &#x6FC0;&#x9176;&#x8BF1;&#x4F7F;&#x6216;&#x8C03;&#x8282;&#x6D3B;&#x6027;&#x7684;&#x91CD;&#x7EC4; TRPV2 &#x6E20;&#x9053; &#xFF1B;&#x4E0E;&#x5148;&#x524D;&#x5EFA;&#x8BAE;&#x7684;&#x673A;&#x5236;&#xFF0C;&#x6FC0;&#x6D3B; PI3 &#x6FC0;&#x9176; TRPV2 &#x9891;&#x9053;&#x4E0D;&#x662F;&#x56E0;&#x4E3A;&#x5BF9;&#x7EC6;&#x80DE;&#x819C;&#x901A;&#x9053;&#x6613;&#x4F4D;&#x3002;

PI3 &#x6FC0;&#x9176;&#x4FC3;&#x8FDB;&#x72EC;&#x7ACB;&#x901A;&#x9053;&#x79FB;&#x4F4D;&#x5230;&#x7EC6;&#x80DE;&#x8D28;&#x819C;&#x7684; TRPV2 &#x6D3B;&#x52A8;&#x3002;

Cellular or chemical activators for most transient receptor potential channels of the vanilloid subfamily (TRPV) have been identified in recent years. A remarkable exception to this is TRPV2, for which cellular events leading to channel activation are still a matter of debate. Diverse stimuli such as extreme heat or phosphatidylinositol-3 kinase (PI3-kinase) regulated membrane insertion have been shown to promote TRPV2 channel activity. However, some of these results have proved difficult to reproduce and may underlie different gating mechanisms depending on the cell type in which TRPV2 channels are expressed. Here, we show that expression of recombinant TRPV2 can induce cytotoxicity that is directly related to channel activity since it can be prevented by introducing a charge substitution in the pore-forming domain of the channel, or by reducing extracellular calcium. In stably transfected cells, TRPV2 expression results in an outwardly rectifying current that can be recorded at all potentials, and in an increase of resting intracellular calcium concentration that can be partly prevented by serum starvation. Using cytotoxicity as a read-out of channel activity and direct measurements of cell surface expression of TRPV2, we show that inhibition of the PI3-kinase decreases TRPV2 channel activity but does not affect the trafficking of the channel to the plasma membrane. It is concluded that PI3-kinase induces or modulates the activity of recombinant TRPV2 channels; in contrast to the previously proposed mechanism, activation of TRPV2 channels by PI3-kinase is not due to channel translocation to the plasma membrane.

PI3-kinase promotes TRPV2 activity independently of channel translocation to the plasma membrane.

Les activateurs cellulaires ou chimiques pour canaux potentiels plus transitoire des r&eacute;cepteurs de la sous-famille VANILLO&Iuml;DE (TRPV) ont &eacute;t&eacute; identifi&eacute;s au cours des derni&egrave;res ann&eacute;es. Une exception remarquable &agrave; cette r&egrave;gle est TRPV2, pour lesquelles les &eacute;v&eacute;nements cellulaires conduisant &agrave; l&#39;activation du canal sont encore sujette &agrave; d&eacute;bat. Insertion de membrane de divers stimuli tels que de la chaleur extr&ecirc;me ou de la phosphatidylinositol 3-kinase (pi 3-kinase) r&eacute;glement&eacute;s a &eacute;t&eacute; d&eacute;montr&eacute; pour promouvoir l&#39;activit&eacute; des canaux TRPV2. Cependant, certains de ces r&eacute;sultats ont &eacute;t&eacute; difficiles &agrave; reproduire et pourraient expliquer les diff&eacute;rents m&eacute;canismes de blocage selon le type de cellule dans laquelle TRPV2 canaux est exprim&eacute;es. Nous d&eacute;montrons ici que l&#39;expression de recombinaison TRPV2 peut induire cytotoxicit&eacute; qui est directement li&eacute;e &agrave; l&#39;activit&eacute; des canaux puisqu&#39;elle peut &ecirc;tre pr&eacute;venue par l&#39;introduction d&#39;une substitution de frais dans le domaine formant des pores du canal, ou en r&eacute;duisant le calcium extracellulaire. Dans les cellules transfect&eacute;es de fa&ccedil;on stable, TRPV2 expression traduit un courant ext&eacute;rieurement de rectification qui peut &ecirc;tre enregistr&eacute; &agrave; tous les potentiels et une augmentation de la concentration de calcium intracellulaire qui peut &ecirc;tre en partie &eacute;vit&eacute;e par privation de s&eacute;rum de repos. &Agrave; l&#39;aide de cytotoxicit&eacute; comme un affichage de l&#39;activit&eacute; des canaux et des mesures directes de l&#39;expression &agrave; la surface cellulaire de TRPV2, nous montrons que l&#39;inhibition de la PI 3-kinase diminue l&#39;activit&eacute; des canaux TRPV2, mais n&#39;affecte pas le trafic du canal vers la membrane plasmique. Il est conclu que pi 3-kinase induit ou module l&#39;activit&eacute; des recombinants TRPV2 canaux ; &agrave; la diff&eacute;rence du m&eacute;canisme propos&eacute; ant&eacute;rieurement, activation des canaux TRPV2 par pi 3-kinase n&#39;est pas &agrave; cause de la translocation de la cha&icirc;ne &agrave; la membrane plasmique.

Pi 3-kinase favorise l&#39;activit&eacute; TRPV2 ind&eacute;pendamment de la translocation vers la membrane plasmique de canal.

&#x6700;&#x3082;&#x904E;&#x6E21;&#x53D7;&#x5BB9;&#x4F53;&#x306E;&#x6F5C;&#x5728;&#x7684;&#x306A;&#x30C1;&#x30E3;&#x30CD;&#x30EB; &#x30D0;&#x30CB;&#x30ED;&#x30A4;&#x30C9;&#x4E9C;&#x79D1; (TRPV) &#x306E;&#x7D30;&#x80DE;&#x3084;&#x5316;&#x5B66;&#x6D3B;&#x6027;&#x5264;&#x306F;&#x3001;&#x8FD1;&#x5E74;&#x3067;&#x8B58;&#x5225;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x9855;&#x8457;&#x306A;&#x4F8B;&#x5916; TRPV2 &#x306E;&#x305F;&#x3081;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306E;&#x6D3B;&#x6027;&#x5316;&#x306B;&#x3064;&#x306A;&#x304C;&#x308B;&#x7D30;&#x80DE;&#x306E;&#x30A4;&#x30D9;&#x30F3;&#x30C8;&#x304C;&#x8B70;&#x8AD6;&#x306E;&#x554F;&#x984C;&#x3092;&#x307E;&#x3060;&#x3067;&#x3059;&#x3067;&#x3059;&#x3002;&#x6975;&#x7AEF;&#x306A;&#x6691;&#x3055;&#x3084;&#x30DB;&#x30B9;&#x30D5;&#x30A1;&#x30C1;&#x30B8;&#x30EB;&#x30A4;&#x30CE;&#x30B7;&#x30C8;&#x30FC;&#x30EB; 3 &#x30AD;&#x30CA;&#x30FC;&#x30BC; (PI3-&#x30AD;&#x30CA;&#x30FC;&#x30BC;) &#x306A;&#x3069;&#x306E;&#x591A;&#x69D8;&#x306A;&#x523A;&#x6FC0;&#x306E;&#x898F;&#x5236;&#x819C;&#x633F;&#x5165; TRPV2 &#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306E;&#x6D3B;&#x52D5;&#x3092;&#x4FC3;&#x9032;&#x3059;&#x308B;&#x793A;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;.&#x305F;&#x3060;&#x3057;&#x3001;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D50;&#x679C;&#x3092;&#x518D;&#x73FE;&#x3059;&#x308B;&#x3053;&#x3068;&#x306F;&#x56F0;&#x96E3;&#x8A3C;&#x660E;&#x3057;&#x3066;&#x304A;&#x308A;&#x3001;&#x3069;&#x306E; TRPV2 &#x306E;&#x30C1;&#x30E3;&#x30F3;&#x30CD;&#x30EB;&#x3092;&#x8868;&#x73FE;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x30BB;&#x30EB;&#x306E;&#x7A2E;&#x985E;&#x306B;&#x5FDC;&#x3058;&#x3066;&#x7570;&#x306A;&#x308B;&#x30B2;&#x30FC;&#x30C6;&#x30A3;&#x30F3;&#x30B0; &#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x304C;&#x6839;&#x5E95;&#x306B;&#x3042;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x907A;&#x4F1D;&#x5B50;&#x7D44;&#x63DB;&#x3048; TRPV2 &#x306E;&#x767A;&#x73FE;&#x7D30;&#x80DE;&#x6BD2;&#x6027;&#x3001;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306E;&#x96FB;&#x8377;&#x7F6E;&#x63DB;&#x7D30;&#x5B54;&#x5F62;&#x6210;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x306E;&#x5C0E;&#x5165;&#x306B;&#x3088;&#x308B;&#x307E;&#x305F;&#x306F;&#x7D30;&#x80DE;&#x5916;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x3092;&#x6E1B;&#x3089;&#x3059;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x9632;&#x3050;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x306E;&#x3067;&#x3001;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306E;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D3;&#x30C6;&#x30A3;&#x306B;&#x76F4;&#x63A5;&#x95A2;&#x9023;&#x3057;&#x3066;&#x3044;&#x308B;&#x3092;&#x8A98;&#x767A;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x56FA;&#x5B9A;&#x3057;&#x3066; transfected &#x30BB;&#x30EB;&#x3067;&#x3001;TRPV2 &#x5F0F;&#x3059;&#x3079;&#x3066;&#x306E;&#x96FB;&#x4F4D;&#x3067;&#x8A18;&#x9332;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x306F;&#x3001;&#x5916;&#x898B;&#x4E0A;&#x6574;&#x6D41;&#x96FB;&#x6D41;&#x3068;&#x5B89;&#x9759;&#x90E8;&#x5206;&#x7684;&#x8840;&#x6E05;&#x98E2;&#x9913;&#x306B;&#x3088;&#x3063;&#x3066;&#x9632;&#x3050;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x7D30;&#x80DE;&#x5185;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x6FC3;&#x5EA6;&#x306E;&#x5897;&#x52A0;&#x306E;&#x7D50;&#x679C;&#x3057;&#x307E;&#x3059;&#x3002;&#x7D30;&#x80DE;&#x6BD2;&#x6027;&#x306E;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306E;&#x5229;&#x7528;&#x72B6;&#x6CC1;&#x306E;&#x8AAD;&#x307F;&#x51FA;&#x3057;&#x3068; TRPV2 &#x306E;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x767A;&#x73FE;&#x306E;&#x76F4;&#x63A5;&#x6E2C;&#x5B9A;&#x3068;&#x3057;&#x3066;&#x3092;&#x7528;&#x3044;&#x3066;, PI3 &#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x306E;&#x963B;&#x5BB3;&#x306F; TRPV2 &#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x6D3B;&#x52D5;&#x306E;&#x6E1B;&#x5C11;&#x304C;&#x7D30;&#x80DE;&#x819C;&#x306B;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306E;&#x4EBA;&#x8EAB;&#x58F2;&#x8CB7;&#x306B;&#x306F;&#x5F71;&#x97FF;&#x3057;&#x307E;&#x305B;&#x3093;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;PI3 &#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x8A98;&#x5C0E;&#x307E;&#x305F;&#x306F;&#x7D44;&#x63DB;&#x3048; TRPV2 &#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306E;&#x6D3B;&#x6027;&#x3092;&#x8ABF;&#x7BC0;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x7DE0;&#x7D50;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x4EE5;&#x524D;&#x63D0;&#x6848;&#x306E;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3068;&#x306F;&#x5BFE;&#x7167;&#x7684; TRPV2 &#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306B;&#x3088;&#x3063;&#x3066; PI3 &#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x306E;&#x6D3B;&#x6027;&#x5316;&#x30D7;&#x30E9;&#x30BA;&#x30DE;&#x819C;&#x3078;&#x306E;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x8EE2;&#x3067;&#x306F;&#x306A;&#x3044;&#x3067;&#x3059;&#x3002;

PI3 &#x30AD;&#x30CA;&#x30FC;&#x30BC; &#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x8EE2;&#x5EA7;&#x539F;&#x5F62;&#x8CEA;&#x819C;&#x306B;&#x7121;&#x95A2;&#x4FC2;&#x306B; TRPV2 &#x6D3B;&#x52D5;&#x3092;&#x4FC3;&#x9032;&#x3057;&#x307E;&#x3059;&#x3002;

&#xCD5C;&#xADFC; &#xBA87; &#xB144; &#xB3D9;&#xC548;&#xC5D0;&#xC11C; &#xAC00;&#xC7A5; &#xC77C;&#xC2DC;&#xC801; &#xC218;&#xC6A9; &#xCCB4; &#xC7A0;&#xC7AC;&#xC801;&#xC778; &#xCC44;&#xB110; vanilloid &#xC544; (TRPV)&#xC5D0; &#xB300; &#xD55C; &#xC140;&#xB8F0;&#xB7EC; &#xB610;&#xB294; &#xD654;&#xD559; &#xD65C;&#xC131; &#xD655;&#xC778; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774;&#xC5D0; &#xC8FC;&#xBAA9;&#xD560; &#xB9CC;&#xD55C; &#xC608;&#xC678;&#xB294; TRPV2&#xB294; &#xCC44;&#xB110; &#xD65C;&#xC131;&#xD654;&#xB97C; &#xC120;&#xB3C4; &#xD558;&#xB294; &#xC140;&#xB8F0;&#xB7EC; &#xC774;&#xBCA4;&#xD2B8;&#xB294; &#xC5EC;&#xC804;&#xD788; &#xB17C;&#xC7C1;&#xC758; &#xBB38;&#xC81C;. &#xB808; &#xADE4; &#xB808;&#xC774;&#xD2B8; &#xB41C; &#xADF9;&#xB2E8;&#xC801;&#xC778; &#xC5F4; &#xB610;&#xB294; phosphatidylinositol 3 &#xD0A4; (PI3 kinase) &#xB4F1; &#xB2E4;&#xC591; &#xD55C; &#xC790;&#xADF9; &#xB9C9; &#xC0BD;&#xC785; TRPV2 &#xCC44;&#xB110; &#xD65C;&#xB3D9;&#xC744; &#xCD09;&#xC9C4; &#xD45C;&#xC2DC; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xC774;&#xB7EC;&#xD55C; &#xACB0;&#xACFC;&#xC758; &#xC77C;&#xBD80;&#xB97C; &#xC7AC;&#xD604; &#xD558;&#xAE30; &#xC5B4;&#xB824;&#xC6B4; &#xC785;&#xC99D; &#xD558; &#xACE0;&#xB294; TRPV2 &#xCC44;&#xB110; &#xD45C;&#xD604; &#xB429;&#xB2C8;&#xB2E4; &#xC140; &#xC720;&#xD615;&#xC5D0; &#xB530;&#xB77C; &#xC11C;&#xB85C; &#xB2E4;&#xB978; &#xC81C;&#xC5B4; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC758; &#xAE30;&#xCD08;&#xAC00; &#xB420; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC;&#xAC00; &#xC7AC;&#xC870;&#xD569; trpv2&#xC758; &#xD45C;&#xD604; &#xB54C;&#xBB38;&#xC5D0; &#xC18C;&#xAC1C; &#xD558;&#xB294; &#xCC44;&#xB110;&#xC758; &#xAE30; &#xACF5; &#xD615;&#xC131; &#xB3C4;&#xBA54;&#xC778;&#xC5D0;&#xC11C; &#xCDA9;&#xC804; &#xB300;&#xCCB4; &#xD558;&#xAC70;&#xB098; &#xC138;&#xD3EC; &#xC678; &#xCE7C;&#xC298;&#xC744; &#xC904;&#xC5EC; &#xC608;&#xBC29;&#xD560; &#xC218; &#xC788;&#xB294; &#xCC44;&#xB110; &#xD65C;&#xB3D9;&#xACFC; &#xC9C1;&#xC811; &#xAD00;&#xB828; &#xB41C; &#xC138;&#xD3EC; &#xB3C5;&#xC131;&#xC744; &#xC77C;&#xC73C;&#xD0AC; &#xC218; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. &#xC548;&#xC815;&#xC801; transfected &#xC138;&#xD3EC;&#xC5D0; TRPV2 &#xC2DD; &#xBAA8;&#xB4E0; &#xC7A0;&#xC7AC;&#xB825;&#xC5D0; &#xAE30;&#xB85D; &#xB420; &#xC218; &#xC788;&#xB294; &#xAC89;&#xC73C;&#xB85C; &#xC815;&#xB958; &#xC804;&#xB958; &#xBC0F; &#xD734;&#xC2DD; &#xD608; &#xCCAD; &#xAE30;&#xC544;&#xC5D0; &#xC758;&#xD574; &#xBD80;&#xBD84;&#xC801;&#xC73C;&#xB85C; &#xBC29;&#xC9C0;&#xD560; &#xC218; &#xC788;&#xB294; &#xC138;&#xD3EC;&#xB0B4; &#xCE7C;&#xC298; &#xB18D;&#xB3C4;&#xC758; &#xC99D;&#xAC00; &#xACB0;&#xACFC;. &#xCC44;&#xB110; &#xC791;&#xC5C5;&#xC758; &#xC77D;&#xAE30; &#xBC0F; trpv2&#xC758; &#xC138;&#xD3EC; &#xD45C;&#xBA74; &#xC2DD;&#xC758; &#xC9C1;&#xC811; &#xCE21;&#xC815;&#xC73C;&#xB85C; &#xC138;&#xD3EC; &#xB3C5;&#xC131;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC0B4;&#xD3B4;&#xBCF4;&#xACA0;&#xC2B5;&#xB2C8;&#xB2E4; PI3 kinase &#xC800;&#xD574; TRPV2 &#xCC44;&#xB110; &#xD65C;&#xB3D9; &#xAC10;&#xC18C; &#xD558;&#xC9C0;&#xB9CC; &#xD50C;&#xB77C;&#xC988;&#xB9C8; &#xBA64;&#xBE0C;&#xB808;&#xC778; &#xCC44;&#xB110;&#xC758; &#xC778;&#xC2E0; &#xB9E4;&#xB9E4;&#xC5D0; &#xC601;&#xD5A5;&#xC744; &#xC8FC;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4;. PI3 kinase &#xC720;&#xB3C4; &#xB610;&#xB294; &#xC7AC;&#xC870;&#xD569; TRPV2 &#xCC44;&#xB110;; &#xD65C;&#xB3D9; &#xBCC0;&#xC870; &#xACB0;&#xB860; &#xC774;&#xB2E4; &#xC774;&#xC804;&#xC5D0; &#xC81C;&#xC548; &#xB41C; &#xBA54;&#xCEE4;&#xB2C8;&#xC998; &#xB2EC;&#xB9AC; PI3 kinase, TRPV2 &#xCC44;&#xB110;&#xC758; &#xD65C;&#xC131;&#xD654; &#xB54C;&#xBB38;&#xC5D0; &#xD50C;&#xB77C;&#xC988;&#xB9C8; &#xBA64;&#xBE0C;&#xB808;&#xC778; &#xCC44;&#xB110; &#xC804; &#xC544;&#xB2C8;&#xB2E4;.

PI3 kinase &#xD50C;&#xB77C;&#xC988;&#xB9C8; &#xBA64;&#xBE0C;&#xB808;&#xC778; &#xCC44;&#xB110; &#xC804; &#xBCC4;&#xB3C4;&#xB85C; TRPV2 &#xD65C;&#xB3D9;&#xC744; &#xCD09;&#xC9C4;&#xD569;&#xB2C8;&#xB2E4;.

Activadores celulares o qu&iacute;micos para m&aacute;s transitorios canales potencial del receptor de la subfamilia vaniloides (detectan) han sido identificados en los &uacute;ltimos a&ntilde;os. Una notable excepci&oacute;n a esto es TRPV2, para que conduce a la activaci&oacute;n del canal de eventos celulares son todav&iacute;a un tema de debate. Inserci&oacute;n de membrana de diversos est&iacute;mulos como el calor extremo o fosfatidilinositol 3 quinasa (PI3) regulados han demostrado para promover la actividad del canal de TRPV2. Sin embargo, algunos de estos resultados han sido dif&iacute;ciles de reproducir y pueden ser la base de mecanismos bloquea diferentes dependiendo del tipo de c&eacute;lula en que TRPV2 se expresan canales. Aqu&iacute;, demostramos que la expresi&oacute;n de TRPV2 recombinante puede inducir citotoxicidad que est&aacute; directamente relacionada a la actividad del canal ya que se puede prevenir mediante la introducci&oacute;n de una substituci&oacute;n de la carga en el dominio de la formaci&oacute;n del poro del canal, o disminuyendo el calcio extracelular. En c&eacute;lulas transfected estable, TRPV2 expresi&oacute;n resulta en una corriente exterior rectificaci&oacute;n que se puede grabar en todos los potenciales y en un aumento de la concentraci&oacute;n de calcio intracelular que puede evitarse en parte por inanici&oacute;n de suero en reposo. Utilizando citotoxicidad como datos sobre la actividad del canal y mediciones directas de la expresi&oacute;n superficial de la c&eacute;lula de TRPV2, nos muestran que la inhibici&oacute;n de la cinasa PI3 disminuye la actividad del canal de TRPV2 pero no afecta el tr&aacute;fico del canal a la membrana plasm&aacute;tica. Se concluye que la cinasa PI3 induce o modula la actividad de los canales de TRPV2 recombinantes; en contraste con el mecanismo propuesto previamente, activaci&oacute;n de canales de TRPV2 por PI3-quinasa no es debido al desplazamiento del canal a la membrana plasm&aacute;tica.

Cinasa PI3 promueve actividad TRPV2 independientemente de desplazamiento del canal a la membrana plasm&aacute;tica.

The physiological role, the mechanisms of activation, as well as the endogenous regulators for the non-selective cationic channel TRPV2 are not known so far. In the present work we report that endogenous lysophospholipids such as lysophosphatidylcholine (LPC) and lysophosphatidylinositol (LPI) induce a calcium influx via TRPV2 channel. This activation is dependent on the length of the side-chain and the nature of the lysophospholipid head-group. TRPV2-mediated calcium uptake stimulated by LPC and LPI occurred via Gq/Go-protein and phosphatidylinositol-3,4 kinase (PI3,4K) signalling. We have shown that the mechanism of TRPV2 activation induced by LPC and LPI is due to the TRPV2 channel translocation to the plasma membrane. The activation of TRPV2 channel by LPC and LPI leads to an increase in the cell migration of the prostate cancer cell line PC3. We have demonstrated that TRPV2 is directly involved in both steady-state and lysophospholipid-stimulated cancer cell migration. Thus, for the first time, we have identified one of the natural regulators of TRPV2 channel, one of the mechanisms of TRPV2 activation and regulation, as well as its pathophysiological role in cancer.

Lysophospholipids stimulate prostate cancer cell migration via TRPV2 channel activation.

Castration resistance in prostate cancer (PCa) constitutes an advanced, aggressive disease with poor prognosis, associated with uncontrolled cell proliferation, resistance to apoptosis, and enhanced invasive potential. The molecular mechanisms involved in the transition of PCa to castration resistance are obscure. Here, we report that the nonselective cationic channel transient receptor potential vanilloid 2 (TRPV2) is a distinctive feature of castration-resistant PCa. TRPV2 transcript levels were higher in patients with metastatic cancer (stage M1) compared with primary solid tumors (stages T2a and T2b). Previous studies of the TRPV2 channel indicated that it is primarily involved in cancer cell migration and not in cell growth. Introducing TRPV2 into androgen-dependent LNCaP cells enhanced cell migration along with expression of invasion markers matrix metalloproteinase (MMP) 9 and cathepsin B. Consistent with the likelihood that TRPV2 may affect cancer cell aggressiveness by influencing basal intracellular calcium levels, small interfering RNA-mediated silencing of TRPV2 reduced the growth and invasive properties of PC3 prostate tumors established in nude mice xenografts, and diminished expression of invasive enzymes MMP2, MMP9, and cathepsin B. Our findings establish a role for TRPV2 in PCa progression to the aggressive castration-resistant stage, prompting evaluation of TRPV2 as a potential prognostic marker and therapeutic target in the setting of advanced PCa.

Role of cationic channel TRPV2 in promoting prostate cancer migration and progression to androgen resistance.

TRPC proteins become involved in Ca2+ entry following the activation of Gq-protein coupled receptors. TRPC6 is inserted into the plasma membrane upon stimulation and remains in the plasma membrane as long as the stimulus is present. However, the mechanism that regulates the trafficking of TRPC6 is unclear. In the present study, we highlighted the involvement of two Rab GTPases in the trafficking of TRPC6. Rab9 co-localized in vesicular structures with TRPC6 in HeLa cells and co-immunoprecipitated with TRPC6. When co-expressed with TRPC6, Rab9(S21N), a dominant negative mutant, caused an increase in the level of TRPC6 at the plasma membrane and in TRPC6-mediated Ca2+ entry upon activation by a muscarinic receptor agonist. Similarly, the expression of Rab11 also caused an increase in TRPC6 expression at the cell surface and an increase in TRPC6-mediated Ca2+ entry. The co-expression of TRPC6 with the dominant negative mutant Rab11(S25N) abolished CCh-induced TRPC6 activation and reduced the level of TRPC6 at the plasma membrane. This study demonstrates that the trans-Golgi network and recycling endosomes are involved in the intracellular trafficking of TRPC6 by regulating channel density at the cell surface.

Involvement of Rab9 and Rab11 in the intracellular trafficking of TRPC6.

TRPC6 is a cation channel in the plasma membrane that plays a role in Ca(2+) entry following the stimulation of a G(q)-protein coupled or tyrosine kinase receptor. A dysregulation of TRPC6 activity causes abnormal proliferation of smooth muscle cells and glomerulosclerosis. In the present study, we investigated the regulation of TRPC6 activity by protein kinase C (PKC). We showed that inhibiting PKC with GF1 or activating it with phorbol 12-myristate 13-acetate potentiated and inhibited agonist-induced Ca(2+) entry, respectively, into cells expressing TRPC6. Similar results were obtained when TRPC6 was directly activated with 1-oleyl-2-acetyl-sn-glycerol. Activation of the cells with carbachol increased the phosphorylation of TRPC6, an effect that was prevented by the inhibition of PKC. The target residue of PKC was identified by an alanine screen of all canonical PKC sites on TRPC6. Unexpectedly, all the mutants, including TRPC6(S768A) (a residue previously proposed to be a target for PKC), displayed PKC-dependent inhibition of channel activity. Phosphorylation prediction software suggested that Ser(448), in a non-canonical PKC consensus sequence, was a potential target for PKCδ. Ba(2+) and Ca(2+) entry experiments revealed that GF1 did not potentiate TRPC6(S448A) activity. Moreover, activation of PKC did not enhance the phosphorylation state of TRPC6(S448A). Using A7r5 vascular smooth muscle cells, which endogenously express TRPC6, we observed that a novel PKC isoform is involved in the inhibition of the vasopressin-induced Ca(2+) entry. Furthermore, knocking down PKCδ in A7r5 cells potentiated vasopressin-induced Ca(2+) entry. In summary, we provide evidence that PKCδ exerts a negative feedback effect on TRPC6 through the phosphorylation of Ser(448).

Protein kinase C-dependent phosphorylation of transient receptor potential canonical 6 (TRPC6) on serine 448 causes channel inhibition.

TRPC are nonselective cation channels involved in calcium entry. Their regulation by phosphorylation has been shown to modulate their routing and activity. TRPC6 activity increases following phosphorylation by Fyn, and is inhibited by protein kinase G and protein kinase C. A previous study by our group showed that TRPC6 is phosphorylated under unstimulated conditions in a human embryonic kidney cells line (HEK293). To investigate the mechanism responsible for this phosphorylation, we used a MS/MS approach combined with metabolic labeling and showed that the serine at position 814 is phosphorylated in unstimulated cells. The mutation of Ser(814) into Ala decreased basal phosphorylation but did not modify TRPC6 activity. Even though Ser(814) is within a consensus site for casein kinase II (CK2), we showed that CK2 is not involved in the phosphorylation of TRPC6 and does not modify its activity. In summary, we identified a new basal phosphorylation site (Ser(814)) on TRPC6 and showed that CK2 is not responsible for the phosphorylation of this site.

The serine 814 of TRPC6 is phosphorylated under unstimulated conditions.

TRPC6 is a cation channel in the plasma membrane that plays a role in Ca(2+) entry after the stimulation of a G(q)-protein-coupled or tyrosine-kinase receptor. TRPC6 translocates to the plasma membrane upon stimulation and remains there as long as the stimulus is present. However, the mechanism that regulates the trafficking and activation of TRPC6 are unclear. In this study we showed phosphoinositide 3-kinase and its antagonistic phosphatase, PTEN, are involved in the activation of TRPC6. The inhibition of PI3K by PIK-93, LY294002, or wortmannin decreased carbachol-induced translocation of TRPC6 to the plasma membrane and carbachol-induced net Ca(2+) entry into T6.11 cells. Conversely, a reduction of PTEN expression did not affect carbachol-induced externalization of TRPC6 but increased Ca(2+) entry through TRPC6 in T6.11 cells. We also showed that the PI3K/PTEN pathway regulates vasopressin-induced translocation of TRPC6 to the plasma membrane and vasopressin-induced Ca(2+) entry into A7r5 cells, which endogenously express TRPC6. In summary, we provided evidence that the PI3K/PTEN pathway plays an important role in the translocation of TRPC6 to the plasma membrane and may thus have a significant impact on Ca(2+) signaling in cells that endogenously express TRPC6.

Involvement of phosphoinositide 3-kinase and PTEN protein in mechanism of activation of TRPC6 protein in vascular smooth muscle cells.

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.

Michael Monet

Université Nice-Sophia Antipolis, Laboratoire de Physiomédecine Moléculaire, CNRS UMR7370, and Laboratories of Excellence Ion Channel Science and Therapeutics

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

Michael Monet

Université Nice-Sophia Antipolis, Laboratoire de Physiomédecine Moléculaire, CNRS UMR7370, and Laboratories of Excellence Ion Channel Science and Therapeutics

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

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