Luigi Sforna

Serum-activated K and Cl currents underlay U87-MG glioblastoma cell migration

Glioblastoma cells in vivo are exposed to a variety of promigratory signals, including undefined serum components that infiltrate into high grade gliomas as result of blood–brain barrier breakdown. Glioblastoma cell migration has been further shown to depend heavily on ion channels activity. We have then investigated the modulatory effects of fetal calf serum (FCS) on ion channels, and their involvement in U87‐MG cells migration. Using the perforated patch‐clamp technique we have found that, in a subpopulation of cells (42%), FCS induced: (1) an oscillatory activity of TRAM‐34 sensitive, intermediate‐conductance calcium‐activated K (IKCa) channels, mediated by calcium oscillations previously shown to be induced by FCS in this cell line; (2) a stable activation of a DIDS‐ and NPPB‐sensitive Cl current displaying an outward rectifying instantaneous current‐voltage relationship and a slow, voltage‐dependent inactivation. By contrast, in another subpopulation of cells (32%) FCS induced a single, transient IKCa current activation, always accompanied by a stable activation of the Cl current. The remaining cells did not respond to FCS. In order to understand whether the FCS‐induced ion channel activities are instrumental to promoting cell migration, we tested the effects of TRAM‐34 and DIDS on the FCS‐induced U87‐MG cell migration using transwell migration assays. We found that these inhibitors were able to markedly reduce U87‐MG cell migration in the presence of FCS, and that their co‐application resulted in an almost complete arrest of migration. It is concluded that the modulation of K and Cl ion fluxes is essential for the FCS‐induced glioblastoma cell migration. J. Cell. Physiol. 226: 1926–1933, 2011.

Histamine hyperpolarizes human glioblastoma cells by activating the intermediate-conductance Ca2+-activated K+ channel

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

Thymosin α1 represents a potential potent single-molecule-based therapy for cystic fibrosis

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that compromise its chloride channel activity. The most common mutation, p.Phe508del, results in the production of a misfolded CFTR protein, which has residual channel activity but is prematurely degraded. Because of the inherent complexity of the pathogenetic mechanisms involved in CF, which include impaired chloride permeability and persistent lung inflammation, a multidrug approach is required for efficacious CF therapy. To date, no individual drug with pleiotropic beneficial effects is available for CF. Here we report on the ability of thymosin alpha 1 (Tα1)—a naturally occurring polypeptide with an excellent safety profile in the clinic when used as an adjuvant or an immunotherapeutic agent—to rectify the multiple tissue defects in mice with CF as well as in cells from subjects with the p.Phe508del mutation. Tα1 displayed two combined properties that favorably opposed CF symptomatology: it reduced inflammation and increased CFTR maturation, stability and activity. By virtue of this two-pronged action, Tα1 has strong potential to be an efficacious single-molecule-based therapeutic agent for CF.

Gain-of-function defects of astrocytic Kir4.1 channels in children with autism spectrum disorders and epilepsy

Dysfunction of the inwardly-rectifying potassium channels Kir4.1 (KCNJ10) represents a pathogenic mechanism contributing to Autism-Epilepsy comorbidity. To define the role of Kir4.1 variants in the disorder, we sequenced KCNJ10 in a sample of affected individuals, and performed genotype-phenotype correlations. The effects of mutations on channel activity, protein trafficking, and astrocyte function were investigated in Xenopus laevis oocytes, and in human astrocytoma cell lines. An in vivo model of the disorder was also explored through generation of kcnj10a morphant zebrafish overexpressing the mutated human KCNJ10. We detected germline heterozygous KCNJ10 variants in 19/175 affected children. Epileptic spasms with dysregulated sensory processing represented the main disease phenotype. When investigated on astrocyte-like cells, the p.R18Q mutation exerted a gain-of-function effect by enhancing Kir4.1 membrane expression and current density. Similarly, the p.R348H variant led to gain of channel function through hindrance of pH-dependent current inhibition. The frequent polymorphism p.R271C seemed, instead, to have no obvious functional effects. Our results confirm that variants in KCNJ10 deserve attention in autism-epilepsy, and provide insight into the molecular mechanisms of autism and seizures. Similar to neurons, astrocyte dysfunction may result in abnormal synaptic transmission and electrical discharge, and should be regarded as a possible pharmacological target in autism-epilepsy.

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

AbstractThe swelling-activated chloride current (ICl,Vol) is abundantly expressed in glioblastoma (GBM) cells, where it controls cell volume and invasive migration. The transduction pathway mediating ICl,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 ICl,Vol activation by hypotonic swelling required the activity of a U73122-sensitive phospholipase C (PLC). ICl,Vol activation could also be induced by the membrane-permeable diacylglycerol (DAG) analog OAG. In contrast, neither calcium (Ca2+) chelation by BAPTA-AM nor changes in PKC activity were able to affect ICl,Vol activation by hypotonic swelling. We further found that R59022, an inhibitor of diacylglycerol kinase (DGK), reverted ICl,Vol activation, suggesting the involvement of phosphatidic acid. In addition, ICl,Vol activation required the activity of a EHT1864-sensitive Rac1 small GTPase and the resulting actin polymerization, as ICl,Vol activation was prevented by cytochalasin B. We finally show that ICl,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 ICl,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 ICl,Vol may represent potential therapeutic targets against this lethal cancer.

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

Glioblastomas (GBMs) are brain tumors characterized by diffuse invasion of cancer cells into the healthy brain parenchyma, and establishment of secondary foci. GBM cells abundantly express large‐conductance, calcium‐activated potassium (BK) channels that are thought to promote cell invasion. Recent evidence suggests that the GBM high invasive potential mainly originates from a pool of stem‐like cells, but the expression and function of BK channels in this cell subpopulation have not been studied. We investigated the expression of BK channels in GBM stem‐like cells using electrophysiological and immunochemical techniques, and assessed their involvement in the migratory process of this important cell subpopulation. In U87‐MG cells, BK channel expression and function were markedly upregulated by growth conditions that enriched the culture in GBM stem‐like cells (U87‐NS). Cytofluorimetric analysis further confirmed the appearance of a cell subpopulation that co‐expressed high levels of BK channels and CD133, as well as other stem cell markers. A similar association was also found in cells derived from freshly resected GBM biopsies. Finally, transwell migration tests showed that U87‐NS cells migration was much more sensitive to BK channel block than U87‐MG cells. Our data show that BK channels are highly expressed in GBM stem‐like cells, and participate to their high migratory activity. J. Cell. Physiol. 232: 2478–2488, 2017.

The role of ion channels in the hypoxia-induced aggressiveness of glioblastoma

The malignancy of glioblastoma multiform (GBM), the most common and aggressive form of human brain tumors, strongly correlates with the presence of hypoxic areas, but the mechanisms controlling the hypoxia-induced aggressiveness are still unclear. GBM cells express a number of ion channels whose activity supports cell volume changes and increases in the cytosolic Ca2+ concentration, ultimately leading to cell proliferation, migration or death. In several cell types it has previously been shown that low oxygen levels regulate the expression and activity of these channels, and more recent data indicate that this also occurs in GBM cells. Based on these findings, it may be hypothesized that the modulation of ion channel activity or expression by the hypoxic environment may participate in the acquisition of the aggressive phenotype observed in GBM cells residing in a hypoxic environment. If this hypothesis will be confirmed, the use of available ion channels modulators may be considered for implementing novel therapeutic strategies against these tumors.

Reconciling the discrepancies on the involvement of large-conductance Ca2+-activated K channels in glioblastoma cell migration

Glioblastoma (GBM) is the most common and aggressive primary brain tumor, and is notable for spreading so effectively through the brain parenchyma to make complete surgical resection virtually impossible, and prospect of life dismal. Several ion channels have been involved in GBM migration and invasion, due to their critical role in supporting volume changes and Ca2+ influx occuring during the process. The large-conductance, Ca2+-activated K (BK) channels, markedly overexpressed in biopsies of patients with GBMs and in GBM cell lines, have attracted much interest and have been suggested to play a central role in cell migration and invasion as candidate channels for providing the ion efflux and consequent water extrusion that allow cell shrinkage during migration. Available experimental data on the role of BK channel in migration and invasion are not consistent though. While BK channels block typically resulted in inhibition of cell migration or in no effect, their activation would either enhance or inhibit the process. This short review reexamines the relevant available data on the topic, and presents a unifying paradigm capable of reconciling present discrepancies. According to this paradigm, BK channels would not contribute to migration under conditions where the [Ca2+]i is too low for their activation. They will instead positively contribute to migration for intermediate [Ca2+]i, insufficient as such to activate BK channels, but capable of predisposing them to cyclic activation following oscillatory [Ca2+]i increases. Finally, steadily active BK channels because of prolonged high [Ca2+]i would inhibit migration as their steady activity would be unsuitable to match the cyclic cell volume changes needed for proper cell migration.

A Calsequestrin-1 mutation associated with a skeletal muscle disease alters sarcoplasmic Ca2+ release

An autosomal dominant protein aggregate myopathy, characterized by high plasma creatine kinase and calsequestrin-1 (CASQ1) accumulation in skeletal muscle, has been recently associated with a missense mutation in CASQ1 gene. The mutation replaces an evolutionarily-conserved aspartic acid with glycine at position 244 (p.D244G) of CASQ1, the main sarcoplasmic reticulum (SR) Ca2+ binding and storage protein localized at the terminal cisternae of skeletal muscle cells. Here, immunocytochemical analysis of myotubes, differentiated from muscle-derived primary myoblasts, shows that sarcoplasmic vacuolar aggregations positive for CASQ1 are significantly larger in CASQ1-mutated cells than control cells. A strong co-immuno staining of both RyR1 and CASQ1 was also noted in the vacuoles of myotubes and muscle biopsies derived from patients. Electrophysiological recordings and sarcoplasmic Ca2+ measurements provide evidence for less Ca2+ release from the SR of mutated myotubes when compared to that of controls. These findings further clarify the pathogenic nature of the p.D244G variant and point out defects in sarcoplasmic Ca2+ homeostasis as a mechanism underlying this human disease, which could be distinctly classified as “CASQ1-couplonopathy”.

Cromakalim activates the KATP and enhances spontaneous transient outward potassium currents in rat saphenous arterial myocytes

Potassium channel openers, e.g. cromakalim are held to relax smooth muscle by hyperpolarizing the cell membrane via activation of ATP-sensitive K(+) (K(ATP)) channels. A recent report indicates that members of this group dilate cerebral arteries also by enhancing the K(Ca)-based spontaneous transient outward currents (STOCs) through the activation of mitochondrial K(ATP) channels. We extended the study to rat saphenous arterial myocytes, a model for peripheral resistance vessels, to investigate the effects of cromakalim on K(ATP) and STOCs, and the underlaying mechanisms. Smooth muscle myocytes were enzymatically dissociated from the saphenous branch of the femoral artery. Macroscopic currents were recorded from acutely isolated cells using the perforated-patch and whole-cell variants of the patch-clamp technique. Predictably metabolic inhibitors and cromakalim activated a background K(+) current blocked by glibenclamide, identified as the K(ATP) channel. However, in addition, cromakalim markedly increased the amplitude and frequency of STOCs. The latter action was not sensitive to the specific K(ATP) channel blocker glibenclamide, excluding the participation of mitocondrial K(ATP) channels in this action. In conclusion, this study suggests that, in addition to the opening of K(ATP) channels, the increased STOC activity may have an important role in the vasorelaxing action of cromakalim, but through a mechanism different from that reported on cerebral artery.