AIM:Pancreatic ductal adenocarcinoma (PDAC) develops within a uniquely dynamic pH landscape shaped by substantial acid-base fluxes produced by the exocrine pancreas. Secretion of alkaline pancreatic juice, normally linked to digestion, produces intermittent acidifications of the pancreatic interstitium, which challenges epithelial and stromal cells. It was postulated that these unique pancreatic pH dynamics can facilitate PDAC initiation and progression through selection of a more aggressive phenotype emerging with PDAC driver mutations. METHODS:Here, we summarize evidence that pH-regulatory transport proteins have an important role in shaping the PDAC microenvironment. RESULTS:pH-regulatory transport proteins generate and sense their microenvironment and act as signaling hubs to regulate proliferation, migration, and metabolism, and immune evasion. In this way, transport proteins that are crucial for the normal physiology of the exocrine pancreas are misused and become coerced into playing a pro-cancer role in pancreatic tumor cells, pancreatic stellate cells, or infiltrating immune cells. Experiments with PDAC mouse models revealed a therapeutic potential of targeting pH dynamics, notably by inhibition or genetic ablation of pH-regulatory proteins. It is a consistent finding that these maneuvers have a marked impact on the tumor immune defense and the communication between cancer and immune cells. CONCLUSION:Collectively, we present a case for considering pH-regulating proteins as a therapeutic avenue.

Brugada syndrome (BrS) is a genetically determined cardiac arrhythmogenic syndrome with increased risk of sudden cardiac death. BrS is mostly caused by mutations in SCN5A gene encoding the primary ɑ-subunit of the cardiac sodium channel NaV1.5. We aimed at characterizing the functional alterations caused by the R893C mutation, identified in a proband diagnosed with BrS, and establishing whether the mutation is associated with BrS. Although several mutations have been reported in the close vicinity of R893, the functional role of this region remains unknown and, in addition, exploring SCN5A mutations in patients with inherited arrhythmogenic syndromes is critical for understanding the pathogenesis of arrhythmias. The mutations were introduced by site-directed mutagenesis. The variants were transiently expressed in CHO cells and potassium currents were measured using the whole-cell patch clamp technique. Patch clamp recordings have demonstrated that R893C almost completely abolished the sodium current, INa, though the mutation did not exert dominant-negative effect on wild-type NaV1.5 channels. We also observed significant decrease in channel activation and a depolarized shift of steady–state inactivation curve, however, the kinetics of inactivation and recovery from fast inactivation were not changed by the mutation. Moreover, the reducing agent Dithiotreitol partially restored the normal function of NaV1.5 in the R893C mutant highlighting a likely mechanism for loss of conduction via formation of disulphide bridges. We showed that R893H channels also failed to produce any detectable INa that confirms the importance of the highly conserved R893 in gating. Our study reveals R893C is a loss-of-function mutation with altered electrophysiological characteristics of NaV1.5. Thus, R893C may contribute to the BrS phenotype of the proband. Our findings may facilitate the understanding of the mechanisms of arrhythmogenesis in BrS, as it helps to identify mutational hotspots in BrS. Moreover, our work may improve novel gene therapy and new therapeutic drug design targeting NaV1.5 channelopathies.

Ryanodine receptors (RyRs) are Ca2 +-release channels of the sarcoplasmic reticulum. Because uncontrolled Ca2 +-release underlies several muscle and cardiac disorders, pharmacological inhibition of RyRs represents an attractive therapeutic strategy. Natural peptide toxins have provided valuable molecular templates for inhibitor design, with calcins being a particularly promising group. Calcins bind RyRs with high specificity, but, instead of producing the desired full channel block, they stabilize subconductance states that promote Ca2 + leak. Given the mechanistic similarity between calcins' action and the well-known charybdotoxin (ChTX)-mediated K+ channel blockade, we examined whether ChTX showed functional cross-reactivity with RyR channels. Single-channel recordings were performed on skeletal muscle RyRs reconstituted into lipid bilayers. ChTX induced long-lasting (>0.5 s) closed events (LLCEs) in a voltage-dependent manner with an apparent Kd of 85 nm at +60 mV. Under these conditions, channels spent ∼10 s min-1 in the LLCE state. Notably, isosteric substitution of K27, which is essential for K+ channel block, retained RyR inhibition, indicating a pore interaction mechanistically distinct from that described in K+ channels. Cardiac RyRs were similarly affected by the K27N variant. Guided by in silico docking to the RyR cryo-electron microscopy structure, we mapped the ChTX-RyR interaction surface using five additional ChTX variants, focusing on basic residues. We found that the R25Q and R34Q substitutions abolished LLCE formation, whereas others had no effect or enhanced channel block. These results describe a novel ChTX-RyR interaction mode functionally convergent with calcin peptides, and they also identify K27N ChTX as a selective RyR inhibitor and a promising scaffold for future drug design. KEY POINTS: Charybdotoxin (ChTX) blocks ryanodine receptor (RyR) single channel currents in bilayer recordings. Amino acid residues R25 and R34 are essential for the block. K27, which is important for K+ channel block, is irrelevant in RyR block; thereby, the K27N ChTX variant represents a RyR-selective blocker. ChTX shares a common binding site with calcin peptides in the channel vestibule, outside the gate.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a desmoplastic stroma driven by pancreatic stellate cells (PSCs) and cancer-associated fibroblasts (CAFs). The Ca2+-permeable ion channel ORAI1 is known to influence PSC proliferation and activation, but its role in collagen secretion and tumor fibrosis remains unclear. Using the PSC cell line PS-1, we developed and validated a high-throughput fibrosis assay based on the collagen-binding peptide CNA-35-tdTomato. Collagen secretion was assessed following treatment with transforming growth factor β1 (TGF-β1) and/or vitamin C, with ORAI1 inhibition achieved via siRNA or Synta-66. In vivo, Orai1 expression was examined by immunohistochemistry in the genetically engineered murine PDAC model KPfC (Kraswt/LSL-G12D Tp53fl/+ PDX1Cre+). The intracellular Ca2+ concentration was assessed with the fluorescent Ca2+ indicator Fura-2. TGF-β1 and vitamin C synergistically enhanced collagen deposition in PSCs, a process regulated by ORAI1. Vitamin C induced a significant rise in intracellular Ca2+ in PSCs, which was attenuated by the ORAI inhibitor Synta-66. ORAI1 expression was detected in αSMA+ CAFs in KPfC-derived PDAC tissue, with a higher prevalence in collagen-rich fibrotic tumor regions compared to non-fibrotic areas or non-tumorous tissue. In addition, vessel-lining endothelial cells also express ORAI1. Our findings highlight ORAI1 as a regulator of collagen secretion in PSCs and reveal its enrichment in CAFs within fibrotic PDAC stroma, underscoring its potential role in PDAC-associated desmoplasia. Zoltan Pethö, Ilka Neumann, Andrea Oeckinghaus, Albrecht Schwab, Rieke Schleinhege. Orai1 regulates collagen secretion in pancreatic stellate cells and is enriched in fibrotic cancer-associated fibroblasts in PDAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 63.

κB-Ras/RalGAP complexes limit the activity of Ral GTPases, which function in EGFR/Ras signaling. RalGAP expression is down-regulated in pancreatic cancer; however, the role of RalGAP and Ral GTPases in tumor development in vivo remained unclear. Here, we show that pancreatic RalGAPβ deficiency alone is sufficient to induce inflammation and neoplasia in vivo. We identify that this phenotype is triggered by disturbance of the secretory pathway and polarized exocytosis in acinar cells, demonstrating that RalGAP complexes uphold spatial control of Ral activity. We furthermore show that RALGAPβ deficiency results in defective primary cilium assembly, a process required for efficient acinar regeneration upon inflammation. Only primary cilium formation depends on κB-Ras proteins, suggesting that κB-Ras proteins are not essential for all RalGAP complex-controlled processes. In combination with an oncogenic KRAS G12D mutation, RalGAPβ deficiency leads to a dramatic shortening of tumor latency and median survival. Our results highlight an important role of RalGAP/Ral signaling in upholding acinar cell identity and preventing pancreatic cancer development.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer marked by extensive fibrosis and limited therapeutic options. The calcium-activated potassium channel KCa3.1 has been implicated in cancer progression. with Its elevated expression correlatesing with poor survival in PDAC patients. Here, we explored the in vivo therapeutic potential of targeting KCa3.1 channels using selective inhibitors in PDAC. Tumor-bearing KPfC (Kraswt/LSL-G12D Tp53fl/+ PDX1Cre+) mice were treated with TRAM-34 or maurotoxin, inhibitors of KCa3.1, either alone or in combination with gemcitabine. KCa3.1 expression, and markers of epithelial-to-mesenchymal transition (EMT) were assessed via immunohistochemistry. Tumor progression was evaluated by histological analysis, measuring tumor node size and Sirius Red staining to quantify fibrosis. Distinct effects of plasma membrane and mitochondrial KCa3.1 inhibition were determined by comparing maurotoxin with the membrane-permeable TRAM-34. KCa3.1 channels were expressed in CK18+ tumor cells and α-SMA+ cancer-associated fibroblasts. Maurotoxin reduced the tumor node size as compared to vehicle treatment, especially in combination with gemcitabine. In contrast, TRAM-34 and gemcitabine in monotherapy showed no significant impact on tumor node size. Gemcitabine alone induced a marked increase in fibrosis within tumor nodes, an effect not observed with other treatments. Notably, co-treatment with KCa3.1 inhibitors prevented gemcitabine-induced fibrosis and reversed EMT marker expression. Selective targeting of plasma membrane KCa3.1 channels with maurotoxin reduces tumor node size and mitigates gemcitabine-induced fibrosis in PDAC. These findings highlight the therapeutic potential of KCa3.1 inhibition as a strategy to enhance the efficacy of gemcitabine and limit desmoplasia. Zoltan Pethö, Ilka Neumann, Weronika Wilczak, Micol Rugi, Sarah Sargin, Sandra Schimmelpfennig, Elke Nass, Gonçalo Mesquita, Dominika Ciechanska, Andrea Oeckinghaus, Serena Pillozzi, Natalia Prevarskaya, V’yacheslav Lehen’kyi, Albrecht Schwab, Benjamin Soret. Targeting plasma membrane KCa3.1 channels reduces tumor size and mitigates gemcitabine-induced fibrosis in a PDAC mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6926.

Pancreatic stellate cells (PSCs) are primarily responsible for producing the stiff tumor tissue in pancreatic ductal adenocarcinoma (PDAC). Thereby, PSCs generate a stiffness gradient between the healthy pancreas and the tumor. This gradient induces durotaxis, a form of directional cell migration driven by differential stiffness. However, the molecular sensors behind durotaxis are still unclear. To investigate the role of mechanosensitive ion channels in PSC durotaxis, we established a two-dimensional stiffness gradient mimicking PDAC. Using pharmacological and genetic methods, we investigated the contribution of the ion channels Piezo1, TRPC1, and TRPV4 in PSC durotaxis. We found that PSC migration towards a stiffer substrate is diminished by altering Piezo1 activity. Moreover, disrupting TRPC1 along with TRPV4 abolishes PSC durotaxis even when Piezo1 is functional. Our results demonstrate that optimal PSC durotaxis requires an intermediary level of ion channel activity, which we simulated via a numerically discretized mathematical model. These findings suggest that mechanosensitive Piezo1 channels detect the differential stiffness microenvironment. The resulting intracellular signals are amplified by TRPV4 and TRPC1 channels to guide efficient PSC durotaxis.

Fibrosis plays a crucial role in a range of chronic diseases, including cancer. Emerging evidence suggests that ion channels, transporters, and pumps-the transportome-have an essential share in fibrogenesis and fibrosis by regulating fibroblast and myofibroblast activity. This review bridges current knowledge gaps by integrating insights from multiple diseases affecting the heart, lungs, pancreas, kidney, and liver, as well as cancer. Thereby, we reveal shared molecular mechanisms of how the transportome modulates fibroblast activation, extracellular matrix deposition, tissue stiffness, and remodeling. We focus on the roles of various ion transport proteins, including PIEZO1, transient receptor potential (TRP), K+, and cystic fibrosis transmembrane regulator (CFTR) channels; the Na+/H+ exchanger NHE1; and the Na+/K+-ATPase. By comparing analogous pathways across different fibrotic diseases such as Ca2+ signaling and transforming growth factor β1 (TGF-β1) and Wnt/β-catenin pathways, we highlight the druggable potential of these ion transport proteins and suggest novel concepts for therapeutic intervention.

Pancreatic stellate cells (PSCs) are central in the development of acute pancreatitis and tumor fibrosis in pancreatic ductal adenocarcinoma (PDAC). Fibrosis and a unique pH landscape represent characteristic properties of the PDAC microenvironment. Mechanosensitive ion channels are involved in the activation of PSCs. Among these channels, K2P2.1 has not yet been studied in PSCs. K2P2.1 channels are pH- and mechanosensitive. We confirmed K2P2.1 expression in PSCs by RT-qPCR and immunofluorescence. PSCs from K2P2.1+/+ and K2P2.1−/− mice were studied under conditions mimicking properties of the PDAC microenvironment (acidic extracellular pH (pHe), ambient pressure elevated by + 100 mmHg). Migration and the cell area were taken as surrogates for PSC activation and evaluated with live cell imaging. pHe-dependent changes of the membrane potential of PSCs were investigated with DiBAC4(3), a voltage-sensitive fluorescent dye. We observed a correlation between morphological activation and progressive hyperpolarization of the cells in response to changes in pHe and pressure. The effect was in part dependent on the expression of K2P2.1 channels because the membrane potential of K2P2.1+/+ PSCs was always more hyperpolarized than that of K2P2.1−/− PSCs. Cell migration velocity of K2P2.1+/+ cells decreased upon pressure application when cells were kept in an acidic medium (pHe 6.6). This was not the case in K2P2.1−/− PSCs. Taken together, our study highlights the critical role of K2P2.1 channels in the combined sensing of environmental pressure and pHe by PSCs and in coordinating cellular morphology with membrane potential dynamics. Thus, K2P2.1 channels are important mechano-sensors in murine PSCs.

Voltage‐clamp fluorometry (VCF) enables the study of voltage‐sensitive proteins through fluorescent labeling accompanied by ionic current measurements for voltage‐gated ion channels. The heterogeneity of the fluorescent signal represents a significant challenge in VCF. The VCF signal depends on where the cysteine mutation is incorporated, making it difficult to compare data among different mutations and different studies and standardize their interpretation. We have recently shown that the VCF signal originates from quenching amino acids in the vicinity of the attached fluorophores, together with the effect of the lipid microenvironment. Based on these, we performed experiments to test the hypothesis that the VCF signal could be altered by amphiphilic quenching molecules in the cell membrane. Here we show that a phenylalanine‐conjugated flavonoid (4‐oxo‐2‐phenyl‐4H‐chromene‐7‐yl)‐phenylalanine, (later Oxophench) has potent effects on the VCF signals of the Ciona intestinalis H V 1 (CiHv1) proton channel. Using spectrofluorimetry, we showed that Oxophench quenches TAMRA (5(6)‐carboxytetramethylrhodamine‐(methane thiosulfonate)) fluorescence. Moreover, Oxophench reduces the baseline fluorescence in oocytes and incorporates into the cell membrane while reducing the membrane fluidity of HEK293 cells. Our model calculations confirmed that Oxophench, a potent membrane‐bound quencher, modifies the VCF signal during conformational changes. These results support our previously published model of VCF signal generation and point out that a change in the VCF signal may not necessarily indicate an altered conformational transition of the investigated protein.

Rationale Pancreatic stellate cells (PSCs) produce a collagen-rich connective tissue in chronic pancreatitis and pancreatic ductal adenocarcinoma (PDAC). Ca2+-permeable ion channels such as ORAI1 are known to affect PSC proliferation and myofibroblastic phenotype. However, it is unknown whether these channels play a role in collagen secretion. Methods Using the PSC cell line PS-1, we characterized their cell-derived matrices using staining, mass spectroscopy, and cell migration assays. We developed and validated a high-throughput in vitro fibrosis assay to rapidly determine collagen quantity either with Sirius Red or, in the optimized version, with the collagen-binding peptide CNA-35-tdTomato. We assessed collagen deposition upon stimulating cells with transforming growth factor β1 (TGF-β1) and/or vitamin C without or with ORAI1 modulation. Orai1 expression was assessed by immunohistochemistry in the fibrotic tumor tissue of a murine PDAC model (KPfC). Results We found that TGF-β1 and vitamin C promote collagen deposition from PSCs. We used small interfering RNA (siRNA) and the inhibitor Synta-66 to demonstrate that ORAI1 regulates collagen secretion of PSCs but not NIH-3T3 fibroblasts. Physiological levels of vitamin C induce a drastic increase of the intracellular [Ca2+] in PSCs, with Synta-66 inhibiting Ca2+ influx. Lastly, we revealed Orai1 expression in cancer-associated fibroblasts (CAFs) in murine PDAC (KPfC) samples. Conclusion In conclusion, our study introduces a robust in vitro assay for fibrosis and identifies ORAI1 as being engaged in PSC-driven fibrosis.

Pancreatic ductal adenocarcinoma (PDAC) progresses in an organ with a unique pH landscape, where the stroma acidifies after each meal. We hypothesized that disrupting this pH landscape during PDAC progression triggers pancreatic stellate cells (PSCs) and cancer-associated fibroblasts (CAFs) to induce PDAC fibrosis. We revealed that alkaline environmental pH was sufficient to induce PSC differentiation to a myofibroblastic phenotype. We then mechanistically dissected this finding, focusing on the involvement of the Na+/H+ exchanger NHE1. Perturbing cellular pH homeostasis by inhibiting NHE1 with cariporide partially altered the myofibroblastic PSC phenotype. To show the relevance of this finding in vivo, we targeted NHE1 in murine PDAC (KPfC). Indeed, tumor fibrosis decreased when mice received the NHE1-inhibitor cariporide in addition to gemcitabine treatment. Moreover, the tumor immune infiltrate shifted from granulocyte rich to more lymphocytic. Taken together, our study provides mechanistic evidence on how the pancreatic pH landscape shapes pancreatic cancer through tuning PSC differentiation.

Pancreatic ductal adenocarcinoma (PDAC) stands as a highly aggressive and lethal cancer, characterized by a grim prognosis and scarce treatment alternatives. Within this context, TRPV6, a calcium-permeable channel, emerges as a noteworthy candidate due to its overexpression in various cancers, capable of influencing the cell behavior in different cancer entities. Nonetheless, the exact expression pattern and functional significance of TRPV6 in the context of PDAC remains enigmatic. This study scrutinizes the expression of TRPV6 in tissue specimens obtained from 46 PDAC patients across distinct stages and grades. We manipulated TRPV6 expression (knockdown, overexpression) in the human PDAC cell lines Panc-1 and Capan-1. Subsequently, we analyzed its impact on multiple facets, encompassing Ca2+ influx, proliferation, apoptosis, migration, chemoresistance, and tumor growth, both in vitro and in vivo. Notably, the data indicate a direct correlation between TRPV6 expression levels, tumor stage, and grade, establishing a link between TRPV6 and PDAC proliferation in tissue samples. Decreasing TRPV6 expression via knockdown hampered Ca2+ influx, resulting in diminished proliferation and viability in both cell lines, and cell cycle progression in Panc-1. The knockdown simultaneously led to an increase in apoptotic rates and increased the susceptibility of cells to 5-FU and gemcitabine treatments. Moreover, it accelerated migration and promoted collective movement among Panc-1 cells. Conversely, TRPV6 overexpression yielded opposing outcomes in terms of proliferation in Panc-1 and Capan-1, and the migration of Panc-1 cells. Intriguingly, both TRPV6 knockdown and overexpression diminished the process of tumor formation in vivo. This intricate interplay suggests that PDAC aggressiveness relies on a fine-tuned TRPV6 expression, raising its profile as a putative therapeutic target.

Pancreatic stellate cells (PSCs) that can co-metastasize with cancer cells shape the tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) by producing an excessive amount of extracellular matrix. This leads to a TME characterized by increased tissue pressure, hypoxia, and acidity. Moreover, cells within the tumor secrete growth factors. The stimuli of the TME trigger Ca 2+ signaling and cellular Na + loading. The Na + /Ca 2+ exchanger (NCX) connects the cellular Ca 2+ and Na + homeostasis. The NCX is an electrogenic transporter, which shuffles 1 Ca 2+ against 3 Na + ions over the plasma membrane in a forward or reverse mode. Here, we studied how the impact of NCX activity on PSC migration is modulated by cues from the TME. NCX expression was revealed with qPCR and Western blot. [Ca 2+ ] i , [Na + ] i , and the cell membrane potential were determined with the fluorescent indicators Fura-2, Asante NaTRIUM Green-2, and DiBAC 4 (3), respectively. PSC migration was quantified with live-cell imaging. To mimic the TME, PSCs were exposed to hypoxia, pressure, acidic pH (pH 6.6), and PDGF. NCX-dependent signaling was determined with Western blot analyses. PSCs express NCX1.3 and NCX1.9. [Ca 2+ ] i , [Na + ] i , and the cell membrane potential are 94.4 nmol/l, 7.4 mmol/l, and − 39.8 mV, respectively. Thus, NCX1 usually operates in the forward (Ca 2+ export) mode. NCX1 plays a differential role in translating cues from the TME into an altered migratory behavior. When NCX1 is operating in the forward mode, its inhibition accelerates PSC migration. Thus, NCX1-mediated extrusion of Ca 2+ contributes to a slow mode of migration of PSCs.

Pancreatic stellate cell (PSC) activation is a major event occurring during pancreatic ductal adenocarcinoma (PDAC) development. Up to now mechanisms underlying their activation by mechanical cues such as the elevated tissue pressure in PDAC remain poorly understood. Here we investigate the role of one potential mechano-transducer, TRPC1 ion channel, in PSC activation. Using pre-activated human siTRPC1 and murine TRPC1-KO PSCs, we show that TRPC1 promotes αSMA (α-smooth muscle actin) expression, the main activation marker, in cooperation with the phosphorylated SMAD2, under normal and elevated pressure. Functional studies following TRPC1 silencing demonstrate the dual role of TRPC1 in the modulation of PSC proliferation and IL-6 secretion through the activation of ERK1/2 and SMAD2 pathways. Moreover, pressurization changes the mechanical behavior of PSCs by increasing their cellular stiffness and emitted traction forces in a TRPC1-dependent manner. In summary, these results point to a role of TRPC1 channels in sensing and transducing the characteristic mechanical properties of the PDAC microenvironment in PSCs.

Malignant neoplasms, such as pancreatic ductal adenocarcinoma (PDAC), show pathologically increased rigidity, which results in a stiffness gradient between tumorous and healthy tissue. Cells are able to detect such differences in rigidity and to migrate into the direction of higher stiffness which is referred to as durotaxis. In PDAC, pancreatic stellate cells (PSCs) are mainly responsible for the production of the rigid tumor environment. Thereby they indirectly attract more PSCs, and ultimately, promote the invasiveness of cancer cells.

Voltage-clamp fluorometry (VCF) supplies information about the conformational changes of voltage-gated proteins. Changes in the fluorescence intensity of the dye attached to a part of the protein that undergoes a conformational rearrangement upon the alteration of the membrane potential by electrodes constitute the signal. The VCF signal is generated by quenching and dequenching of the fluorescence as the dye traverses various local environments. Here we studied the VCF signal generation, using the Hv1 voltage-gated proton channel as a tool, which shares a similar voltage-sensor structure with voltage-gated ion channels but lacks an ion-conducting pore. Using mutagenesis and lipids added to the extracellular solution we found that the signal is generated by the combined effects of lipids during movement of the dye relative to the plane of the membrane and by quenching amino acids. Our 3-state model recapitulates the VCF signals of the various mutants and is compatible with the accepted model of two major voltage-sensor movements.

Ewing sarcoma (EwS) is a rare and highly malignant bone tumor occurring mainly in childhood and adolescence. Physiologically, the bone is a central hub for Ca2+ homeostasis, which is severely disturbed by osteolytic processes in EwS. Therefore, we aimed to investigate how ion transport proteins involved in Ca2+ homeostasis affect EwS pathophysiology. We characterized the expression of 22 candidate genes of Ca2+-permeable or Ca2+-regulated ion channels in three EwS cell lines and found the Ca2+-activated K+ channel KCa2.1 (KCNN1) to be exceptionally highly expressed. We revealed that KCNN1 expression is directly regulated by the disease-driving oncoprotein EWSR1-FL1. Due to its consistent overexpression in EwS, KCNN1 mRNA could be a prognostic marker in EwS. In a large cohort of EwS patients, however, KCNN1 mRNA quantity does not correlate with clinical parameters. Several functional studies including patch clamp electrophysiology revealed no evidence for KCa2.1 function in EwS cells. Thus, elevated KCNN1 expression is not translated to KCa2.1 channel activity in EwS cells. However, we found that the low K+ conductance of EwS cells renders them susceptible to hypoosmotic solutions. The absence of a relevant K+ conductance in EwS thereby provides an opportunity for hypoosmotic therapy that can be exploited during tumor surgery.

Glioblastoma (GBM) is the most aggressive glial tumor, where ion channels, including K Ca 1.1, are candidates for new therapeutic options. Since the auxiliary subunits linked to K Ca 1.1 in GBM are largely unknown we used electrophysiology combined with pharmacology and gene silencing to address the functional expression of K Ca 1.1/ β subunits complexes in both primary tumor cells and in the glioblastoma cell line U-87 MG. The pattern of the sensitivity (activation/inhibition) of the whole-cell currents to paxilline, lithocholic acid, arachidonic acid, and iberiotoxin; the presence of inactivation of the whole-cell current along with the loss of the outward rectification upon exposure to the reducing agent DTT collectively argue that K Ca 1.1/β3 complex is expressed in U-87 MG. Similar results were found using human primary glioblastoma cells isolated from patient samples. Silencing the β3 subunit expression inhibited carbachol-induced Ca 2+ transients in U-87 MG thereby indicating the role of the K Ca 1.1/β3 in the Ca 2+ signaling of glioblastoma cells. Functional expression of the K Ca 1.1/β3 complex, on the other hand, lacks cell cycle dependence. We suggest that the K Ca 1.1/β3 complex may have diagnostic and therapeutic potential in glioblastoma in the future.