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Glioblastoma (GBM) is a deadly brain tumor, with fast recurrence even after surgical intervention, radio- and chemotherapies. One of the reasons for relapse is the early invasion of surrounding brain parenchyma by GBM, rendering tumor eradication difficult. Recent studies demonstrate that, in addition to eliminate possible residual tumoral cells after surgery, radiation stimulates the infiltrative behavior of GBM cells. The intermediate conductance of Ca2+-activated potassium channels (KCa3.1) play an important role in regulating the migration of GBM. Here, we show that high dose radiation of patient-derived GBM cells increases their invasion, and induces the transcription of key genes related to these functions, including the IL-4/IL-4R pair. In addition, we demonstrate that radiation increases the expression of KCa3.1 channels, and that their pharmacological inhibition counteracts the pro-invasive phenotype induced by radiation in tumor cells. Our data describe a possible approach to treat tumor resistance that follows radiation therapy in GBM patients.

Glioblastoma (GBM) is a deadly brain tumor, with fast recurrence even after surgical intervention, radio- and chemotherapies. One of the reasons for relapse is the early invasion of surrounding brain parenchyma by GBM, rendering tumor eradication difficult. Recent studies demonstrate that, in addition to eliminate possible residual tumoral cells after surgery, radiation stimulates the infiltrative behavior of GBM cells. The intermediate conductance of Ca2+-activated potassium channels (KCa3.1) play an important role in regulating the migration of GBM. Here, we show that high dose radiation of patient-derived GBM cells increases their invasion, and induces the transcription of key genes related to these functions, including the IL-4/IL-4R pair. In addition, we demonstrate that radiation increases the expression of KCa3.1 channels, and that their pharmacological inhibition counteracts the pro-invasive phenotype induced by radiation in tumor cells. Our data describe a possible approach to treat tumor resistance that follows radiation therapy in GBM patients.

Glial cells actively maintain the homeostasis of brain parenchyma, regulating neuronal excitability and preserving the physiological composition of the extracellular milieu. Under pathological conditions, some functions of glial cells could be compromised, exacerbating the neurotoxic processes. We investigated if the homeostatic activities of astrocytes and microglia could be modulated by the voltage-gated K+ channel Kv1.3. To this end we used in vitro and in vivo systems to model cell-to-cell interactions in tumoral conditions, using a specific inhibitor of Kv1.3 channels, 5-(4-phenoxybutoxy) psoralen (PAP-1). We demonstrated that PAP-1 increases astrocytic glutamate uptake, reduces glioma-induced neurotoxicity, and decreases microglial migration and phagocytosis. We also found in a tumor blood brain barrier model that Kv1.3 activity is required for its integrity. The crucial role of Kv1.3 channels as modulators of glial cell activity was confirmed in a mouse model of glioma, where PAP-1 treatment reduces tumor volume only in the presence of active glutamate transporters GLT-1. In the same mouse model, PAP-1 reduces astrogliosis and microglial infiltration. PAP-1 also reduces tumor cell invasion. All these findings point to Kv1.3 channels as potential targets to re-instruct glial cells toward their homeostatic functions, in the context of brain tumors.

This retrospective begins with Galvani's experiments on frogs at the end of the 18th century and his discovery of 'animal electricity'. It goes on to illustrate the numerous contributions to the field of physical chemistry in the second half of the 19th century (Nernst's equilibrium potential, based on the work of Wilhelm Ostwald, Max Planck's ion electrodiffusion, Einstein's studies of Brownian motion) which led Bernstein to propose his membrane theory in the early 1900s as an explanation of Galvani's findings and cell excitability. These processes were fully elucidated by Hodgkin and Huxley in 1952 who detailed the ionic basis of resting and action potentials, but without addressing the question of where these ions passed. The emerging question of the existence of ion channels, widely debated over the next two decades, was finally accepted and, a decade later, many of them began to be cloned. This led to the possibility of modelling the activity of individual neurons in the brain and then that of simple circuits. Taking advantage of the remarkable advances in computer science in the new millennium, together with a much deeper understanding of brain architecture, more ambitious scientific goals were dreamed of to understand the brain and how it works. The retrospective concludes by reviewing the main efforts in this direction, namely the construction of a digital brain, an in silico copy of the brain that would run on supercomputers and behave just like a real brain. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM), the most lethal form of brain tumors, bases its malignancy on the strong ability of its cells to migrate and invade the narrow spaces of healthy brain parenchyma. Cell migration and invasion are both critically dependent on changes in cell volume and shape driven by the transmembrane transport of osmotically important ions such as K+ and Cl−. However, while the Cl− channels participating in cell volume regulation have been clearly identified, the precise nature of the K+ channels involved is still uncertain. Using a combination of electrophysiological and imaging approaches in GBM U87‐MG cells, we found that hypotonic‐induced cell swelling triggered the opening of Ca2+‐activated K+ (KCa) channels of large and intermediate conductance (BKCa and IKCa, respectively), both highly expressed in GBM cells. The influx of Ca2+ mediated by the hypotonic‐induced activation of mechanosensitive channels was found to be a key step for opening both the BKCa and the IKCa channels. Finally, the activation of both KCa channels mediated by mechanosensitive channels was found to be essential for the development of the regulatory volume decrease following hypotonic shock. Taken together, these data indicate that KCa channels are the main K+ channels responsible for the volume regulation in U87‐MG cells. [ABSTRACT FROM AUTHOR]

Loss-of-function mutations in the KCNA1(Kv1.1) gene cause episodic ataxia type 1 (EA1), a neurological disease characterized by cerebellar dysfunction, ataxic attacks, persistent myokymia with painful cramps in skeletal muscles, and epilepsy. Precision medicine for EA1 treatment is currently unfeasible, as no drug that can enhance the activity of Kv1.1-containing channels and offset the functional defects caused by KCNA1 mutations has been clinically approved. Here, we uncovered that niflumic acid (NFA), a currently prescribed analgesic and anti-inflammatory drug with an excellent safety profile in the clinic, potentiates the activity of Kv1.1 channels. NFA increased Kv1.1 current amplitudes by enhancing the channel open probability, causing a hyperpolarizing shift in the voltage dependence of both channel opening and gating charge movement, slowing the OFF-gating current decay. NFA exerted similar actions on both homomeric Kv1.2 and heteromeric Kv1.1/Kv1.2 channels, which are formed in most brain structures. We show that through its potentiating action, NFA mitigated the EA1 mutation-induced functional defects in Kv1.1 and restored cerebellar synaptic transmission, Purkinje cell availability, and precision of firing. In addition, NFA ameliorated the motor performance of a knock-in mouse model of EA1 and restored the neuromuscular transmission and climbing ability in Shaker (Kv1.1) mutant Drosophila melanogaster flies (Sh5). By virtue of its multiple actions, NFA has strong potential as an efficacious single-molecule-based therapeutic agent for EA1 and serves as a valuable model for drug discovery. [ABSTRACT FROM AUTHOR]

Astrocytes, the main glial cells of the central nervous system, play a key role in brain volume control due to their intimate contacts with cerebral blood vessels and the expression of a distinctive equipment of proteins involved in solute/water transport. Among these is MLC1, a protein highly expressed in perivascular astrocytes and whose mutations cause megalencephalic leukoencephalopathy with subcortical cysts (MLC), an incurable leukodystrophy characterized by macrocephaly, chronic brain edema, cysts, myelin vacuolation, and astrocyte swelling. Although, in astrocytes, MLC1 mutations are known to affect the swelling-activated chloride currents (ICl,swell) mediated by the volume-regulated anion channel (VRAC), and the regulatory volume decrease, MLC1′s proper function is still unknown. By combining molecular, biochemical, proteomic, electrophysiological, and imaging techniques, we here show that MLC1 is a Ca2+/Calmodulin-dependent protein kinase II (CaMKII) target protein, whose phosphorylation, occurring in response to intracellular Ca2+ release, potentiates VRAC-mediated ICl,swell. Overall, these findings reveal that MLC1 is a Ca2+-regulated protein, linking volume regulation to Ca2+ signaling in astrocytes. This knowledge provides new insight into the MLC1 protein function and into the mechanisms controlling ion/water exchanges in the brain, which may help identify possible molecular targets for the treatment of MLC and other pathological conditions caused by astrocyte swelling and brain edema. [ABSTRACT FROM AUTHOR]

The activation of ion channels is crucial during cell movement, including glioblastoma cell invasion in the brain parenchyma. In this context, we describe for the first time the contribution of intermediate conductance [Ca.sup.2+]-activated K ([IK.sub.ca]) channel activity in the chemotactic response of human glioblastoma cell lines, primary cultures, and freshly dissociated tissues to CXC chemokine ligand 12 (CXCL12), a chemokine whose expression in glioblastoma has been correlated with its invasive capacity. We show that blockade of the [IK.sub.Ca] channel with its specific inhibitor 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) or [IK.sub.Ca] channel silencing by short hairpin RNA (shRNA) completely abolished CXCL12-induced cell migration. We further demonstrate that this is not a general mechanism in glioblastoma cell migration since epidermal growth factor (EGF), which also activates [IK.sub.Ca] channels in the glioblastoma-derived cell line GL15, stimulate cell chemotaxis even if the [IK.sub.Ca] channels have been blocked or silenced. Furthermore, we demonstrate that both CXCL12 and EGF induce [Ca.sup.2+] mobilization and [IK.sub.Ca] channel activation but only CXCL12 induces a long-term upregulation of the [IK.sub.Ca] channel activity. Furthermore, the [Ca.sup.2+]-chelating agent BAPTA-AM abolished the CXCL12-induced, but not the EGF-induced, glioblastoma cell chemotaxis. In addition, we demonstrate that the extracellular signal-regulated kinase (ERK)1/2 pathway is only partially implicated in the modulation of CXCL12-induced glioblastoma cell movement, whereas the phosphoinositol-3 kinase (PI3K) pathway is not involved. In contrast, EGF-induced glioblastoma migration requires both ERK1/2 and PI3K activity. All together these findings suggest that the efficacy of glioblastoma invasiveness might be related to an array of nonoverlapping mechanisms activated by different chemotactic agents. chemokines; transwell; tumor; TRAM-34; short hairpin RNA doi: 10.1152/ajpcell.00344.2009.

The effects of histamine on the membrane potential and currents of human glioblastoma (GL-15) cells were investigated. In perforated whole cell configuration, short (3 s) applications of histamine (100 [micro]M) hyperpolarized the membrane by activating a [K.sup.+]-selective current. The response involved the activation of the pyrilamine-sensitive [H.sub.1] receptor and [Ca.sup.2+] release from thapsigargin-sensitive intracellular stores. The histamine-activated current was insensitive to tetraethylammonium (3 mM), iberiotoxin (100 nM), and d-tubocurarine (100 [micro]M) but was markedly inhibited by charybdotoxin (100 nM), clotrimazole (1 [micro]M), and 1-[(2-chlorophenyl)diphenyl-methyl]-1H-pyrazole (TRAM-34, 1 [micro]M), a pharmacological profile congruent with the intermediate conductance [Ca.sup.2+]-activated [K.sup.+] ([IK.sub.Ca]) channel. Cell-attached recordings confirmed that histamine activated a [K.sup.+] channel with properties congruent with the [IK.sub.Ca] channel (voltage independence, 22 pS unitary conductance and slight inward rectification in symmetrical 140 mM [K.sup.+]). More prolonged histamine applications (2-3 min) often evoked a sustained [IK.sub.Ca] channel activity, which depended on a [La.sup.2+] (10 [micro]M)-sensitive [Ca.sup.2+] influx. Intracellular [Ca.sup.2+] measurements revealed that the sustained [IK.sub.Ca] channel activity enhanced the histamine-induced [Ca.sup.2+] signal, most likely by a hyperpolarization-induced increase in the driving force for [Ca.sup.2+] influx. In virtually all cells examined we also observed the expression of the large conductance [Ca.sup.2+]-activated [K.sup.+] ([BK.sub.Ca]) channel, with a unitary conductance of ca. 230 pS in symmetrical 140 mM [K.sup.+], and a [Ca.sup.2+] dissociation constant [[K.sub.D(Ca)]] of ca. 3 [micro]M, at -40 mV. Notably in no instance was the [BK.sub.Ca] channel activated by histamine under physiological conditions. The most parsimonious explanation based on the different [K.sub.D(Ca)] for the two [K.sub.Ca] channels is provided. histamine; intermediate conductance [Ca.sup.2+]-activated [K.sup.+] channels; large conductance [Ca.sup.2+]-activated [K.sup.+] channels; human glioblastoma GL-15 cells

We report here the expression in [C.sub.2][C.sub.12] myoblasts of the intermediate-conductance [Ca.sup.2+]-activated [K.sup.+] ([IK.sub.Ca]) channel. The [IK.sub.Ca] current, recorded under perforated-patch configuration, had a transient time course when activated by ionomycin (0.5 [micro]M; peak current density 26.2 [+ or -] 3.7 pA/pF; n = 10), but ionomycin (0.5 [micro]M) + 5,6-dichloro1-ethyl-l,3-dihydro-2H- benzimidazol-2-one (100 [micro]M) evoked a stable outward current (28.4 [+ or -] 8.2 pA/pF; n = 11). The current was fully inhibited by charybdotoxin (200 nM), clotrimazole (2 [micro]M), and 5-nitro-2- (3-phenylpropylamino)benzoic acid (300 [micro]M), but not by tetraethylammonium (1 mM) or D-tubocurarine (300 [micro]M). Congruent with the [IK.sub.Ca] channel, elevation of intracellular [Ca.sup.2+] in inside-out patches resulted in the activation of a voltage-insensitive [K.sup.+] channel with weak inward rectification, a unitary conductance of 38 [+ or -] 6 pS (at negative voltages), and an [IC.sub.50] for [Ca.sup.2+] of 530 nM. The [IK.sub.Ca] channel was activated metabotropically by external application of ATP (100 [micro]M), an intracellular [Ca.sup.2+] mobilizer. Under current-clamp conditions, ATP application resulted in a membrane hyperpolarization of ~35 mV. The [IK.sub.Ca] current downregulated during myogenesis, ceasing to be detectable 4 days after the myoblasts were placed in differentiating medium. Downregulation was prevented by the myogenic suppressor agent basic FGF (bFGF). We also found that block of the [IK.sub.Ca] channel by charybdotoxin did not inhibit bFGF-sustained myoblast proliferation. These observations show that in [C.sub.2][C.sub.12] myoblasts the [IK.sub.Ca] channel expression correlates inversely with differentiation, yet it does not appear to have a role in myoblast proliferation. ATP; cell proliferation

Regulatory volume decrease (RVD), a homeostatic process responsible for the re‐establishment of the original cell volume upon swelling, is critical in controlling several functions, including migration. RVD is mainly sustained by the swelling‐activated Cl− current (ICl,swell), which can be modulated by cytoplasmic Ca2+. Cell swelling also activates mechanosensitive channels, including the ubiquitously expressed Ca2+‐permeable channel Piezo1. We hypothesized that, by controlling cytoplasmic Ca2+ and in turn ICl,swell, Piezo1 is involved in the fine regulation of RVD and cell migration. We compared RVD and ICl,swell in wild‐type (WT) HEK293T cells, which express endogenous levels of Piezo1, and in cells overexpressing (OVER) or knockout (KO) for Piezo1. Compared to WT, RVD was markedly increased in OVER, while virtually absent in KO cells. Consistently, ICl,swell amplitude was highest in OVER and lowest in KO cells, with WT cells displaying an intermediate level, suggesting a Ca2+‐dependent modulation of the current by Piezo1 channels. Indeed, in the absence of external Ca2+, ICl,swell in both WT and OVER cells, as well as the RVD probed in OVER cells, were significantly lower than in the presence of Ca2+ and no longer different compared to KO cells. However, the Piezo‐mediated Ca2+ influx was ineffective in enhancing ICl,swell in the absence of releasable Ca2+ from intracellular stores. The different expression levels of Piezo1 affected also cell migration which was strongly enhanced in OVER, while reduced in KO cells, as compared to WT. Taken together, our data indicate that Piezo1 controls RVD and migration in HEK293T cells by modulating ICl,swell through Ca2+ influx. [ABSTRACT FROM AUTHOR]