Sonia Martial

Molecular characterization of a new urea transporter in the human kidney

A cDNA clone (HUT2) sharing 61.1% and 89.9% sequence identity with the human erythroid (HUT11) and the rabbit (UT2) urea transporters, respectively, was isolated by homology cloning from a human kidney library. HUT2 transcripts were restricted to the kidney and the HUT2 polypeptide was not immunoprecipitated with blood group Kidd‐related antibodies (anti‐Jk3) in coupled transcription‐translation assays. Functional expression studies in Xenopus oocytes demonstrated that HUT2‐mediated urea transport was not inhibited by p‐chloromercuribenzene sulfonate (pCMBS) which, however, inhibited the urea flux mediated by HUT11. These findings demonstrate that at least two distinct urea transporters are present in human tissues. By in situ hybridization, the gene encoding HUT2 has been assigned to chromosome 18q12.1‐q21‐1, as found previously for the Kidd/urea transporter HUT11, suggesting that both genes evolved from duplication of a common ancestor.

Sig1R Protein Regulates hERG Channel Expression through a Post-translational Mechanism in Leukemic Cells

Sig1R (Sigma-1receptor) is a 25-kDa protein structurally unrelated to other mammalian proteins. Sig1R is present in brain, liver, and heart and is overexpressed in cancer cells. Studies using exogenous sigma ligands have shown that Sig1R interacts with a variety of ion channels, but its intrinsic function and mechanism of action remain unclear. The human ether-à-gogo related gene (hERG) encodes a cardiac channel that is also abnormally expressed in many primary human cancers, potentiating tumor progression through the modulation of extracellular matrix adhesive interactions. We show herein that sigma ligands inhibit hERG current density and cell adhesion to fibronectin in K562 myeloid leukemia cells. Heterologous expression in Xenopus oocytes demonstrates that Sig1R potentiates hERG current by stimulating channel subunit biosynthesis. Silencing Sig1R in leukemic K562 cells depresses hERG current density and cell adhesion to fibronectin by reducing hERG membrane expression. In K562 cells, Sig1R silencing does not modify hERG mRNA contents but reduces hERG mature form densities. In HEK cells expressing hERG and Sig1R, both proteins co-immunoprecipitate, demonstrating a physical association. Finally, Sig1R expression enhances both channel protein maturation and stability. Altogether, these results demonstrate for the first time that Sig1R controls ion channel expression through the regulation of subunit trafficking activity.

Consequences of point mutations in trout anion exchanger 1 (tAE1) transmembrane domains: evidence that tAE1 can behave as a chloride channel.

In this study, we have shown that, when expressed in Xenopus oocytes, trout anion exchanger 1 (tAE1) was able to act as a bifunctional protein, either an anion exchanger or a chloride conductance. Point mutations of tAE1 were carried out and their effect on Cl− conductance and Cl− unidirectional flux were studied. We have shown that mutations made in transmembrane domain 7 had dramatic effects on tAE1 function. Indeed, when these residues were mutated, either individually or together (mutants E632K, D633G, and ED/KG), Cl− conductance was reduced to 28–44% that of wild‐type tAE1. Moreover, ion substitution experiments showed that anion selectivity was altered. However, the exchanger function was unchanged, as evidenced by the fact that Cl− influx and Km were identical for each of these mutants and similar to the wild‐type protein parameters. By contrast, mutations made in the C‐terminal domains of the protein (R819M, Q829K) affected both transport functions. Cl− conductance was increased by ∼200% with respect to tAE1 and anion selectivity was impaired. Likewise, Cl− influx was increased by ∼260% and was no longer saturable. These and other mutations carried out in transmembrane domains 7, 8, 12–14 of tAE1 allow us to demonstrate without doubt that, in addition to its anion exchanger activity, tAE1 can also function as a chloride channel. Above all, this work led us to identify amino acids involved in this double function organization. J. Cell. Physiol.

SigmaR1 regulates membrane electrical activity in response to extracellular matrix stimulation to drive cancer cell invasiveness

The sigma 1 receptor (Sig1R) is a stress-activated chaperone that regulates ion channels and is associated with pathologic conditions, such as stroke, neurodegenerative diseases, and addiction. Aberrant expression levels of ion channels and Sig1R have been detected in tumors and cancer cells, such as myeloid leukemia and colorectal cancer, but the link between ion channel regulation and Sig1R overexpression during malignancy has not been established. In this study, we found that Sig1R dynamically controls the membrane expression of the human voltage-dependent K(+) channel human ether-à-go-go-related gene (hERG) in myeloid leukemia and colorectal cancer cell lines. Sig1R promoted the formation of hERG/β1-integrin signaling complexes upon extracellular matrix stimulation, triggering the activation of the PI3K/AKT pathway. Consequently, the presence of Sig1R in cancer cells increased motility and VEGF secretion. In vivo, Sig1R expression enhanced the aggressiveness of tumor cells by potentiating invasion and angiogenesis, leading to poor survival. Collectively, our findings highlight a novel function for Sig1R in mediating cross-talk between cancer cells and their microenvironment, thus driving oncogenesis by shaping cellular electrical activity in response to extracellular signals. Given the involvement of ion channels in promoting several hallmarks of cancer, our study also offers a potential strategy to therapeutically target ion channel function through Sig1R inhibition.

Importance of several cysteine residues for the chloride conductance of trout anion exchanger 1 (tAE1).

In this study, we devised a cysteine‐focused point mutation analysis of the chloride channel function of trout anion exchanger 1 (tAE1) expressed in X. lævis oocytes. Seven cysteines, belonging to the transmembrane domain of tAE1, were mutated into serines (either individually or in groups) and the effects of these mutations on the chloride conductance of injected oocytes were measured. We showed that three cysteines were essential for the functional expression of tAE1. Namely, mutations C462S, C583S and C588S reduced Cl− conductance by 68%, 52% and 83%, respectively, when compared to wild type tAE1. These residual conductances were still inhibited by 0.5 mM niflumic acid. Western blot experiments demonstrated that C462 was involved in protein expression onto the plasma membrane. A mutant devoid of this residue was unable to express onto the plasma membrane, especially if several other cysteines were missing: consequently, the cysteine‐less mutant of tAE1 was not functional. C583 and C588 were involved in the channel function of tAE1 as shown by anion substitution experiments proving that selectivity of the mutated pore differs from the wild type one. On the contrary, they were not involved in the Cl−/HCO  3− exchange function of tAE1, as demonstrated by intracellular pH measurements. These and several complementary mutations allow us to conclude that a mutant of tAE1 containing the sole C462 can drive a marginal Cl− current; however, the minimal configuration necessary to get optimal functional expression of the tAE1 chloride channel is that of a mutant containing unaffected residues C462, C583 and C588. J. Cell. Physiol. 213: 70–78, 2007.

Involvement of ion channels and transporters in carcinoma angiogenesis and metastasis

Angiogenesis is a finely tuned process, which is the result of the equilibrium between pro- and antiangiogenic factors. In solid tumor angiogenesis, the balance is highly in favor of the production of new, but poorly functional blood vessels, initially intended to provide growing tumors with nutrients and oxygen. Among the numerous proteins involved in tumor development, several types of ion channels are overexpressed in tumor cells, as well as in stromal and endothelial cells. Ion channels thus actively participate in the different hallmarks of cancer, especially in tumor angiogenesis and metastasis. Indeed, from their strategic localization in the plasma membrane, ion channels are key operators of cell signaling, as they sense and respond to environmental changes. This review aims to decipher how ion channels of different families are intricately involved in the fundamental angiogenesis and metastasis hallmarks, which lead from a nascent tumor to systemic dissemination. An overview of the possible use of ion channels as therapeutic targets will also be given, showing that ion channel inhibitors or specific antibodies may provide effective tools, in the near future, in the treatment of carcinomas.

[Unexpected properties of red blood cell anion exchanger: The fish lesson].

Conclusions Les microglies provenant de la moelle osseuse offrent un potentiel formidable en vue du traitement de la maladie d’Alzheimer, puisqu’elles sont attirees vers les proteines toxiques et sont en mesure de les eliminer. Il est interessant de noter que des proteines toxiques sont presentes dans d’autres maladies neurodegeneratives, telle que l’alpha-synucleine dans la maladie de Parkinson et la superoxyde dismutase dans la sclerose laterale amyotrophique. Les cellules souches de la moelle osseuse pourront ainsi se diriger vers les differents foyers inflammatoires provoques par ces proteines toxiques. Elles ont aussi l’avantage, contrairement aux autres types de cellules souches, d’infiltrer de facon naturelle les regions endommagees et de s’adapter aux conditions inflammatoires. De plus, le prelevement des cellules souches hematopoietiques evitera le rejet des cellules genetiquement modifiees, puisque le patient servira a la fois de donneur et de receveur. Il nous reste maintenant a trouver la facon de rendre ces cellules plus resistantes, a selectionner de meilleurs phagocytes par exemple, plus specifiques a la βA (ou a d’autres proteines toxiques), tout en evitant l’emballement de la reponse inflammatoire. Un tres beau defi scientifique et medical a relever ! ‡ Bone marrow stem cells to the rescue of Alzheimer’s disease REFERENCES