SK2 channels set a signalling hub bolstering CAF-triggered tumourigenic processes in pancreatic cancer.

OBJECTIVE: Intercellular communication within pancreatic ductal adenocarcinoma (PDAC) dramatically contributes to metastatic processes. The underlying mechanisms are poorly understood, resulting in a lack of targeted therapy to counteract stromal-induced cancer cell aggressiveness. Here, we investigated whether ion channels, which remain understudied in cancer biology, contribute to intercellular communication in PDAC. DESIGN: We evaluated the effects of conditioned media from patient-derived cancer-associated fibroblasts (CAFs) on electrical features of pancreatic cancer cells (PCC). The molecular mechanisms were deciphered using a combination of electrophysiology, bioinformatics, molecular and biochemistry techniques in cell lines and human samples. An orthotropic mouse model where CAF and PCC were co-injected was used to evaluate tumour growth and metastasis dissemination. Pharmacological studies were carried out in the Pdx1-Cre, Ink4afl/fl LSL-KrasG12D (KICpdx1) mouse model. RESULTS: We report that the K+ channel SK2 expressed in PCC is stimulated by CAF-secreted cues (8.84 vs 2.49 pA/pF) promoting the phosphorylation of the channel through an integrin-epidermal growth factor receptor (EGFR)-AKT (Protein kinase B) axis. SK2 stimulation sets a positive feedback on the signalling pathway, increasing invasiveness in vitro (threefold) and metastasis formation in vivo. The CAF-dependent formation of the signalling hub associating SK2 and AKT requires the sigma-1 receptor chaperone. The pharmacological targeting of Sig-1R abolished CAF-induced activation of SK2, reduced tumour progression and extended the overall survival in mice (11.7 weeks vs 9.5 weeks). CONCLUSION: We establish a new paradigm in which an ion channel shifts the activation level of a signalling pathway in response to stromal cues, opening a new therapeutic window targeting the formation of ion channel-dependent signalling hubs.

Cell polarity and adherens junction formation inhibit epithelial Fas cell death receptor signaling.

Finely tuned regulation of epithelial cell death maintains tissue integrity and homeostasis. At the cellular level, life and death decisions are controlled by environmental stimuli such as the activation of death receptors. We show that cell polarity and adherens junction formation prevent proapoptotic signals emanating from the Fas death receptor. Fas is sequestered in E-cadherin actin-based adhesion structures that are less able to induce downstream apoptosis signaling. Using a proteomic-based approach, we find that the polarity molecule Dlg1 interacts with the C-terminal PDZ-binding site in Fas and that this interaction decreases formation of the death-inducing complex upon engagement with Fas ligand (FasL), thus acting as an additional cell death protection mechanism. We propose that E-cadherin and Dlg1 inhibit FasL-induced cell death by two complementary but partially independent mechanisms that help to maintain epithelial homeostasis by protecting normal polarized epithelia from apoptosis. When polarity is lost, the Fas-cadherin-Dlg1 antiapoptotic complex is disrupted, and FasL can promote the elimination of compromised nonpolarized cells.

Bidirectional KCNQ1:ß-catenin interaction drives colorectal cancer cell differentiation.

The K+ channel KCNQ1 has been proposed as a tumor suppressor in colorectal cancer (CRC). We investigated the molecular mechanisms regulating KCNQ1:ß-catenin bidirectional interactions and their effects on CRC differentiation, proliferation, and invasion. Molecular and pharmacologic approaches were used to determine the influence of KCNQ1 expression on the Wnt/ß-catenin signaling and epithelial-to-mesenchymal transition (EMT) in human CRC cell lines of varying stages of differentiation. The expression of KCNQ1 was lost with increasing mesenchymal phenotype in poorly differentiated CRC cell lines as a consequence of repression of the KCNQ1 promoter by ß-catenin:T-cell factor (TCF)-4. In well-differentiated epithelial CRC cell lines, KCNQ1 was localized to the plasma membrane in a complex with ß-catenin and E-cadherin. The colocalization of KCNQ1 with adherens junction proteins was lost with increasing EMT phenotype. ShRNA knock-down of KCNQ1 caused a relocalization of ß-catenin from the plasma membrane and a loss of epithelial phenotype in CRC spheroids. Overexpression of KCNQ1 trapped ß-catenin at the plasma membrane, induced a patent lumen in CRC spheroids, and slowed CRC cell invasion. The KCNQ1 ion channel inhibitor chromanol 293B caused membrane depolarization, redistribution of ß-catenin into the cytosol, and a reduced transepithelial electrical resistance, and stimulated CRC cell proliferation. Analysis of human primary CRC tumor patient databases showed a positive correlation between KCNQ1:KCNE3 channel complex expression and disease-free survival. We conclude that the KCNQ1 ion channel is a target gene and regulator of the Wnt/ß-catenin pathway, and its repression leads to CRC cell proliferation, EMT, and tumorigenesis.

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/ß1-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.

A direct interaction between the sigma-1 receptor and the hERG voltage-gated K+ channel revealed by atomic force microscopy and homogeneous time-resolved fluorescence (HTRF®).

The sigma-1 receptor is an endoplasmic reticulum chaperone protein, widely expressed in central and peripheral tissues, which can translocate to the plasma membrane and modulate the function of various ion channels. The human ether-à-go-go-related gene encodes hERG, a cardiac voltage-gated K(+) channel that is abnormally expressed in many human cancers and is known to interact functionally with the sigma-1 receptor. Our aim was to investigate the nature of the interaction between the sigma-1 receptor and hERG. We show that the two proteins can be co-isolated from a detergent extract of stably transfected HEK-293 cells, consistent with a direct interaction between them. Atomic force microscopy imaging of the isolated protein confirmed the direct binding of the sigma-1 receptor to hERG monomers, dimers, and tetramers. hERG dimers and tetramers became both singly and doubly decorated by sigma-1 receptors; however, hERG monomers were only singly decorated. The distribution of angles between pairs of sigma-1 receptors bound to hERG tetramers had two peaks, at ∼90 and ∼180° in a ratio of ∼2:1, indicating that the sigma-1 receptor interacts with hERG with 4-fold symmetry. Homogeneous time-resolved fluorescence (HTRF®) allowed the detection of the interaction between the sigma-1 receptor and hERG within the plane of the plasma membrane. This interaction was resistant to sigma ligands, but was decreased in response to cholesterol depletion of the membrane. We suggest that the sigma-1 receptor may bind to hERG in the endoplasmic reticulum, aiding its assembly and trafficking to the plasma membrane.

The sigma-1 receptor: a regulator of cancer cell electrical plasticity?

Originally mistaken as an opioid receptor, the sigma-1 receptor (Sig1R) is a ubiquitous membrane protein that has been involved in many cellular processes. While the precise function of Sig1R has long remained mysterious, recent studies have shed light on its role and the molecular mechanisms triggered. Sig1R is in fact a stress-activated chaperone mainly associated with the ER-mitochondria interface that can regulate cell survival through the control of calcium homeostasis. Sig1R functionally regulates ion channels belonging to various molecular families and it has thus been involved in neuronal plasticity and central nervous system diseases. Interestingly, Sig1R is frequently expressed in tumors but its function in cancer has not been yet clarified. In this review, we discuss the current understanding of Sig1R. We suggest herein that Sig1R shapes cancer cell electrical signature upon environmental conditions. Thus, Sig1R may be used as a novel therapeutic target to specifically abrogate pro-invasive functions of ion channels in cancer tissue.

The sigma-1 receptor binds to the Nav1.5 voltage-gated Na+ channel with 4-fold symmetry.

The sigma-1 receptor (Sig1R) is up-regulated in many human tumors and plays a role in the control of cancer cell proliferation and invasiveness. At the molecular level, the Sig1R modulates the activity of various ion channels, apparently through a direct interaction. We have previously shown using atomic force microscopy imaging that the Sig1R binds to the trimeric acid-sensing ion channel 1A with 3-fold symmetry. Here, we investigated the interaction between the Sig1R and the Nav1.5 voltage-gated Na(+) channel, which has also been implicated in promoting the invasiveness of cancer cells. We show that the Sig1R and Nav1.5 can be co-isolated from co-transfected cells, consistent with an intimate association between the two proteins. Atomic force microscopy imaging of the co-isolated proteins revealed complexes in which Nav1.5 was decorated by Sig1Rs. Frequency distributions of angles between pairs of bound Sig1Rs had two peaks, at ∼90° and ∼180°, and the 90° peak was about twice the size of the 180° peak. These results demonstrate that the Sig1R binds to Nav1.5 with 4-fold symmetry. Hence, each set of six transmembrane regions in Nav1.5 likely constitutes a Sig1R binding site, suggesting that the Sig1R interacts with the transmembrane regions of its partners. Interestingly, two known Sig1R ligands, haloperidol and (+)-pentazocine, disrupted the Nav1.5/Sig1R interaction both in vitro and in living cells. Finally, we show that endogenously expressed Sig1R and Nav1.5 also functionally interact.

Band 3 missense mutations and stomatocytosis: insight into the molecular mechanism responsible for monovalent cation leak.

Missense mutations in the erythroid band 3 protein (Anion Exchanger 1) have been associated with hereditary stomatocytosis. Features of cation leaky red cells combined with functional expression of the mutated protein led to the conclusion that the AE1 point mutations were responsible for Na(+) and K(+) leak through a conductive mechanism. A molecular mechanism explaining mutated AE1-linked stomatocytosis involves changes in AE1 transport properties that become leaky to Na(+) and K(+). However, another explanation suggests that point-mutated AE1 could regulate a cation leak through other transporters. This short paper intends to discuss these two alternatives.

Stomatin-deficient cryohydrocytosis results from mutations in SLC2A1: a novel form of GLUT1 deficiency syndrome.

The hereditary stomatocytoses are a series of dominantly inherited hemolytic anemias in which the permeability of the erythrocyte membrane to monovalent cations is pathologically increased. The causative mutations for some forms of hereditary stomatocytosis have been found in the transporter protein genes, RHAG and SLC4A1. Glucose transporter 1 (glut1) deficiency syndromes (glut1DSs) result from mutations in SLC2A1, encoding glut1. Glut1 is the main glucose transporter in the mammalian blood-brain barrier, and glut1DSs are manifested by an array of neurologic symptoms. We have previously reported 2 cases of stomatin-deficient cryohydrocytosis (sdCHC), a rare form of stomatocytosis associated with a cold-induced cation leak, hemolytic anemia, and hepatosplenomegaly but also with cataracts, seizures, mental retardation, and movement disorder. We now show that sdCHC is associated with mutations in SLC2A1 that cause both loss of glucose transport and a cation leak, as shown by expression studies in Xenopus oocytes. On the basis of a 3-dimensional model of glut1, we propose potential mechanisms underlying the phenotypes of the 2 mutations found. We investigated the loss of stomatin during erythropoiesis and find this occurs during reticulocyte maturation and involves endocytosis. The molecular basis of the glut1DS, paroxysmal exercise-induced dyskinesia, and sdCHC phenotypes are compared and discussed.

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.

Characterization of the L683P mutation of SLC26A9 in Xenopus oocytes.

In the present study, we characterized a STAS-domain amino acid mutation of SLC26A9 having a significant impact on ion transport. We focused on the sole conserved L- leucine residue of the STAS domain identified among SLC26 members. We therefore characterized the L683P mutation of SLC26A9 in Xenopus oocytes by monitoring the protein functional expression (two-electrode technique for voltage-clamp analysis) and its presence at the cell membrane (surface protein biotinylation technique). This mutation was found to reduce Cl(-) transport through SLC26A9 as well as the positive interaction exerted by SLC26A9 on CFTR ion transport activity. The origin of this effect is discussed in the light of the presence of the SLC26A9-L683P mutant at the plasma membrane.

South-east Asian ovalocytosis and the cryohydrocytosis form of hereditary stomatocytosis show virtually indistinguishable cation permeability defects.

The hereditary stomatocytoses are a group of dominantly inherited conditions in which the osmotic stability of the red cell is compromised by abnormally high cation permeability. This report demonstrates the very marked similarities between the cryohydrocytosis form of hereditary stomatocytosis and the common tropical condition south-east Asian ovalocytosis (SAO). We report two patients, one showing a novel cryohydrocytosis variant (Ser762Arg in SLC4A1) and a case of SAO. Both cases showed a mild haemolytic state with some stomatocytes on the blood film, abnormal intracellular sodium and potassium levels which were made markedly abnormal by storage of blood at 0°C, increased cation 'leak' fluxes at 37°C and increased Na(+) K(+) pump activity. In both cases, the anion exchange function of the mutant band 3 was destroyed. Extensive electrophysiological studies comparing the cation leak and conductance in Xenopus laevis oocytes expressing the two mutant genes showed identical patterns of abnormality. These data are consistent with the cryohydrocytosis form of hereditary stomatocytosis and we conclude that the cation leak in SAO is indistinguishable from that in cryohydrocytosis, and that SAO should be considered to be an example of hereditary stomatocytosis.

Dual transport properties of anion exchanger 1: the same transmembrane segment is involved in anion exchange and in a cation leak.

Previous results suggested that specific point mutations in human anion exchanger 1 (AE1) convert the electroneutral anion exchanger into a monovalent cation conductance. In the present study, the transport site for anion exchange and for the cation leak has been studied by cysteine scanning mutagenesis and sulfhydryl reagent chemistry. Moreover, the role of some highly conserved amino acids within members of the SLC4 family to which AE1 belongs has been assessed in AE1 transport properties. The results suggest that the same transport site within the AE1 spanning domain is involved in anion exchange or in cation transport. A functioning mechanism for this transport site is proposed according to transport properties of the different studied point mutations of AE1.

SLC26A9 stimulates CFTR expression and function in human bronchial cell lines.

We investigated the possible functional- and physical protein-interactions between two airway Cl(-) channels, SLC26A9 and CFTR. Bronchial CFBE41o- cell lines expressing CFTR(WT) or CFTR(ΔF508) were transduced with SLC26A9. Immunoblots identified a migrating band corresponding to SLC26A9 present in whole-cell lysates as on apical membrane of cells grown on polarized filters. CFTR levels were increased by the presence of SLC26A9 in both CFTR(WT) and CFTR(ΔF508) cell lines. In CFBE41o- cells and CFBE41o-/CFTR(WT) cells transduced with SLC26A9, currents associated to the protein expression were not detected. However, the forskolin (FK)-stimulated currents were enhanced in SLC26A9-transduced cells compared to control cells. Therefore, the presence of SLC26A9 resulted in an increase in CFTR activity (same % of CFTR((inh)-172) or GlyH-101 inhibition in both groups). In CFBE41o-/CFTR(ΔF508) cells transduced with SLC26A9 (at 27°C), a current associated to the protein expression was also lacking. FK-stimulated currents and level of CFTR((inh)-172) inhibition were not different in both groups. The presence of SLC26A9 in Xenopus oocytes expressing CFTR also enhanced the FK-stimulated currents as compared to oocytes expressing CFTR alone. This stimulation was mostly linked to CFTR. An enhancement of FK-stimulated currents was not found in oocytes co-expressing SLC26A9 and CFTR(ΔF508). In conclusion, in both protein expression systems used, SLC26A9 stimulates CFTR activity but not that of CFTR(ΔF508). Our co-immunoprecipitation studies demonstrate a physical interaction between both anion channels. We propose as an alternative hypothesis (not exclusive) to the known SLC26A9-STAS domain/CFTR interaction, that SLC26A9 favors the biogenesis and/or stabilization of CFTR, leading to stimulated currents.

Band 3 Edmonton I, a novel mutant of the anion exchanger 1 causing spherocytosis and distal renal tubular acidosis.

dRTA (distal renal tubular acidosis) and HS (hereditary spherocytosis) are two diseases that can be caused by mutations in the gene encoding the AE1 (anion exchanger 1; Band 3). dRTA is characterized by defective urinary acidification, leading to metabolic acidosis, renal stones and failure to thrive. HS results in anaemia, which may require regular blood transfusions and splenectomy. Mutations in the gene encoding AE1 rarely cause both HS and dRTA. In the present paper, we describe a novel AE1 mutation, Band 3 Edmonton I, which causes dominant HS and recessive dRTA. The patient is a compound heterozygote with the new mutation C479W and the previously described mutation G701D. Red blood cells from the patient presented a reduced amount of AE1. Expression in a kidney cell line showed that kAE1 (kidney AE1) C479W is retained intracellularly. As kAE1 is a dimer, we performed co-expression studies and found that, in kidney cells, kAE1 C479W and G701D proteins traffic independently from each other despite their ability to form heterodimers. Therefore the patient carries one kAE1 mutant that is retained in the Golgi (G701D) and another kAE1 mutant (C479W) located in the endoplasmic reticulum of kidney cells, and is thus probably unable to reabsorb bicarbonate into the blood. We conclude that the C479W mutant is a novel trafficking mutant of AE1, which causes HS due to a decreased cell-surface AE1 protein and results in dRTA due to its intracellular retention in kidney.

A novel erythroid anion exchange variant (Gly796Arg) of hereditary stomatocytosis associated with dyserythropoiesis.

BACKGROUND: Stomatocytoses are a group of inherited autosomal dominant hemolytic anemias and include overhydrated hereditary stomatocytosis, dehydrated hereditary stomatocytosis, hereditary cryohydrocytosis and familial pseudohyperkalemia. DESIGN AND METHODS: We report a novel variant of hereditary stomatocytosis due to a de novo band 3 mutation (p. G796R-band3 CEINGE) associated with a dyserythropoietic phenotype. Band 3 genomic analysis, measurement at of hematologic parameters and red cell indices and morphological analysis of bone marrow were carried out. We then evaluated the red cell membrane permeability and ion transport systems by functional studies of the patient's erythrocytes and Xenopus oocytes transfected with mutated band 3. We analyzed the red cell membrane tyrosine phosphorylation profile and the membrane association of the tyrosine kinases Syk and Lyn from the Src-family-kinase group, since the activity of the membrane cation transport pathways is related to cyclic phosphorylation-dephosphorylation events. RESULTS: The patient showed mild hemolytic anemia with circulating stomatocytes together with signs of dyserythropoiesis. Her red cells displayed increased Na(+) content with decreased K(+)content and abnormal membrane cation transport activities. Functional characterization of band 3 CEINGE in Xenopus oocytes showed that the mutated band 3 is converted from being an anion exchanger (Cl(-), HCO(3)(-)) to being a cation pathway for Na(+) and K(+). Increased tyrosine phosphorylation of some red cell membrane proteins was observed in diseased erythrocytes. Syk and Lyn membrane association was increased in the patient's red cells compared to in normal controls, indicating perturbation of phospho-signaling pathways involved in cell volume regulation events. CONCLUSIONS: Band 3 CEINGE alters function from that of anion exchange to cation transport, affects the membrane tyrosine phosphorylation profile, in particular of band 3 and stomatin, and its presence during red cell development likely contributes to dyserythropiesis.

Southeast Asian AE1 associated renal tubular acidosis: cation leak is a class effect.

Anion Exchanger 1 (AE1) is present in the erythrocyte and also in the alpha-intercalated cell; different mutations can cause either red cell disease or distal renal tubular acidosis (dRTA). Recently, we described a cation leak property in four dRTA-causing AE1 mutants, three autosomal dominant (AD) European mutants, one autosomal recessive (AR) from Southeast Asia, G701D. G701D had a very large leak property and is unusually common in SE Asia. We hypothesized that this property might confer a survival advantage. We characterized three other AR dRTA-associated AE1 mutants found in SE Asia, S773P, Delta850 and A858D via transport experiments in AE1-expressing Xenopus oocytes. These three SE Asian mutants also had cation leaks of similar magnitude to that seen in G701D, a property that distinguishes them as a discrete group. The clustering of these cation-leaky AE1 mutations to malarious areas of SE Asia suggests that they may confer malaria resistance.

The monovalent cation leak in overhydrated stomatocytic red blood cells results from amino acid substitutions in the Rh-associated glycoprotein.

Overhydrated hereditary stomatocytosis (OHSt) is a rare dominantly inherited hemolytic anemia characterized by a profuse membrane leak to monovalent cations. Here, we show that OHSt red cell membranes contain slightly reduced amounts of Rh-associated glycoprotein (RhAG), a putative gas channel protein. DNA analysis revealed that the OHSt patients have 1 of 2 heterozygous mutations (t182g, t194c) in RHAG that lead to substitutions of 2 highly conserved amino acids (Ile61Arg, Phe65Ser). Unexpectedly, expression of wild-type RhAG in Xenopus laevis oocytes induced a monovalent cation leak; expression of the mutant RhAG proteins induced a leak about 6 times greater than that in wild type. RhAG belongs to the ammonium transporter family of proteins that form pore-like structures. We have modeled RhAG on the homologous Nitrosomonas europaea Rh50 protein and shown that these mutations are likely to lead to an opening of the pore. Although the function of RhAG remains controversial, this first report of functional RhAG mutations supports a role for RhAG as a cation pore.

Characterization of SLC26A9, facilitation of Cl(-) transport by bicarbonate.

SLC26 family members are anionic transporters involved in Cl(-) and HCO(3)(-) absorption or secretion in epithelia. SLC26A9, preferentially expressed in the lung, is a poorly characterized member of this family. In this study, we investigated the transport properties of human SLC26A9 to determine its functional and pharmacological characteristics. SLC26A9 protein expression results in the appearance of an anionic current exhibiting an apparently linear current/voltage relationship and increases in (36)Cl influxes and effluxes. The sequences of conductivity, Cl(-) >I(-) > NO(3)(-) >/= gluconate > SO(4) (2-) and selectivity (P(x)/P(CI)), I(-) > NO(3)(-) > Cl(-) > gluconate > SO(4)(2-) are found. Cl(-) channel inhibitors DIDS and NS 3623 inhibit SLC26A9 associated currents while the specific CFTR inhibitor (CFTR(inh)-172) or glybenclamide has little effect. Elevation of intracellular cAMP (a CFTR activator) is also ineffective whereas increasing intracellular calcium blocks the SLC26A9 associated currents. The HCO(3)(-) conductance mediated by the SLC26A9 protein expression is low and no intracellular pHi changes are detectable under conditions favoring a Cl(-)/HCO(3)(-) exchange. However, the presence of HCO(3)(-)/CO(2) stimulates the Cl(-)-transporting activity of SLC26A9 in Xenopus laevis oocytes or SLC26A9-transduced COS-7 cells. As an important initial step in characterizing SLC26A9 function, we conclude that SLC26A9 is a Cl(-) channel and we suggest that HCO(3)(-) acts as a modulator of the channel. SLC26A9 physiological role in airway epithelia and its potential interaction with CFTR remain to be elucidated.