Identification of Key Signaling Molecules Involved in the Activation of the Swelling-Activated Chloride Current in Human Glioblastoma Cells

Abstract

The swelling-activated chloride current (I Cl,Vol) is abundantly expressed in glioblastoma (GBM) cells, where it controls cell volume and invasive migration. The transduction pathway mediating I Cl,Vol activation in GBM cells is, however, poorly understood. By means of pharmacological and electrophysiological approaches, on GL-15 human GBM cells we found that I Cl,Vol activation by hypotonic swelling required the activity of a U73122-sensitive phospholipase C (PLC). I Cl,Vol activation could also be induced by the membrane-permeable diacylglycerol (DAG) analog OAG. In contrast, neither calcium (Ca(2+)) chelation by BAPTA-AM nor changes in PKC activity were able to affect I Cl,Vol activation by hypotonic swelling. We further found that R59022, an inhibitor of diacylglycerol kinase (DGK), reverted I Cl,Vol activation, suggesting the involvement of phosphatidic acid. In addition, I Cl,Vol activation required the activity of a EHT1864-sensitive Rac1 small GTPase and the resulting actin polymerization, as I Cl,Vol activation was prevented by cytochalasin B. We finally show that I Cl,Vol can be activated by the promigratory fetal calf serum in a PLC- and DGK-dependent manner. This observation is potentially relevant because blood serum can likely come in contact with glioblastoma cells in vivo as a result of the tumor-related partial breakdown of the blood-brain barrier. Given the relevance of I Cl,Vol in GBM cell volume regulation and invasiveness, the several key signaling molecules found in this study to be involved in the activation of the I Cl,Vol may represent potential therapeutic targets against this lethal cancer.

Hypoxia Modulates the Swelling-Activated Cl Current in Human Glioblastoma Cells: Role in Volume Regulation and Cell Survival

“…In this case, we achieved our goal by displacing O 2 from our extracellular solution using N 2 bubbling for variable times, and monitored the O 2 level obtained using an O 2 sensor. Our results show that acute hypoxia activates a Cl current displaying the same biophysical and pharmacological features of the I Cl,swell , namely, instantaneous outward rectification in response to depolarizing current steps, inactivation at highly depolarized potentials, voltage‐dependent block by DIDS, but not by NPPB, properties congruent with the I Cl,swell found in the same cells (Catacuzzeno et al, ). The hypoxia‐activated Cl current was blocked by the I Cl,swell selective inhibitor DCPIB.…”

“…In addition, the application of the DGK inhibitor R59022 (50 μM) strongly inhibited the hypoxia‐activated Cl current. Since in GBM cells DAG phosphorilation by DGK is required for the activation of I Cl,swell following cell swelling (Catacuzzeno et al, ), these results confirm that cell swelling and the activation of the Cl current are sequential events. In this work we further demonstrated that, similar to what has been previously shown for GBM cells using hypotonic extracellular solution, also the hypoxia‐induced cell swelling was followed by an RVD mediated mainly by I Cl,swell .…”

“…C–E). These results indicate that the hypoxia‐activated Cl current has the biophysical and pharmacological features of the I Cl,swell expressed by these cells (Catacuzzeno et al, ).…”

“…D). We finally tested the effect of the DGK inhibitor, R59022 (50 μM), on the hypoxia‐activated Cl current, as in these cells the I Cl,swell activation induced by cell swelling requires DAG phosphorilation by DGK (Catacuzzeno et al, ). As shown in Figure C the application of R59022 markedly reduced the hypoxia‐activated Cl current (residual current was 4.2 ± 1.7%, n = 4; Fig.…”

Overexpression of Large‐Conductance Calcium‐Activated Potassium Channels in Human Glioblastoma Stem‐Like Cells and Their Role in Cell Migration

“…Several properties of cancers, including GBM, are influenced by the dysregulation of ion channel expression and function (Sontheimer, ; Catacuzzeno et al, , ; Sforna et al, ). One of the most studied dysregulated ion channels in GBM cells is the large‐conductance, calcium‐activated K (BK) channel, gated by the concerted action of both voltage and intracellular calcium (Turner and Sontheimer, ).…”