Ca2+ -activated K+ channels regulate cell volume in human glioblastoma cells.

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.

Ion Channels in Glioma Malignancy.

Brain tumors come in many types and differ greatly in outcome. They are classified by the cell of origin (astrocytoma, ependymoma, meningioma, medulloblastoma, glioma), although more recently molecular markers are used in addition to histology. Brain tumors are graded (from I to IV) to measure their malignancy. Glioblastoma, one of the most common adult primary brain tumors, displays the highest malignancy (grade IV), and median survival of about 15 months. Main reasons for poor outcome are incomplete surgical resection, due to the highly invasive potential of glioblastoma cells, and chemoresistance that commonly develops during drug treatment. An important role in brain tumor malignancy is played by ion channels. The Ca2+-activated K+ channels of large and intermediate conductance, KCa3.1 and KCa1.1, and the volume-regulated anion channel, whose combined activity results in the extrusion of KCl and osmotic water, control cell volume, and in turn migration, invasion, and apoptotic cell death. The transient receptor potential (TRP) channels and low threshold-activated Ca (T-type) channels have equally critical role in brain tumor malignancy, as dysregulated Ca2+ signals heavily impact on glioma cell proliferation, migration, invasion. The review provides an overview of the current evidence involving these channels in brain tumor malignancy, and the application of these insights in the light of future prospects for experimental and clinical practice.

The 70-year search for the voltage-sensing mechanism of ion channels.

This retrospective on the voltage-sensing mechanisms and gating models of ion channels begins in 1952 with the charged gating particles postulated by Hodgkin and Huxley, viewed as charges moving across the membrane and controlling its permeability to Na+ and K+ ions. Hodgkin and Huxley postulated that their movement should generate small and fast capacitive currents, which were recorded 20 years later as gating currents. In the early 1980s, several voltage-dependent channels were cloned and found to share a common architecture: four homologous domains or subunits, each displaying six transmembrane α-helical segments, with the fourth segment (S4) displaying four to seven positive charges invariably separated by two non-charged residues. This immediately suggested that this segment was serving as the voltage sensor of the channel (the molecular counterpart of the charged gating particle postulated by Hodgkin and Huxley) and led to the development of the sliding helix model. Twenty years later, the X-ray crystallographic structures of many voltage-dependent channels allowed investigation of their gating by molecular dynamics. Further understanding of how channels gate will benefit greatly from the acquisition of high-resolution structures of each of their relevant functional or structural states. This will allow the application of molecular dynamics and other approaches. It will also be key to investigate the energetics of channel gating, permitting an understanding of the physical and molecular determinants of gating. The use of multiscale hierarchical approaches might finally prove to be a rewarding strategy to overcome the limits of the various single approaches to the study of channel gating.

Piezo1 controls cell volume and migration by modulating swelling-activated chloride current through Ca2+ influx.

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.

Gating current noise produced by Brownian models of a voltage sensor.

The activation of voltage-dependent ion channels is associated with the movement of gating charges, which give rise to gating currents. Although gating currents from a single channel are too small to be detected, analysis of the fluctuations of macroscopic gating currents from a population of channels allows a good guess of their magnitude. The analysis of experimental gating current fluctuations, when interpreted in terms of a rate model of channel activation and assuming sufficiently high bandwidth, is in accordance with the presence of a main step along the activation pathway carrying a charge of 2.3-2.4 e0. To give a physical interpretation to these results and to relate them to the known atomic structure of the voltage sensor domain, we used a Brownian model of voltage-dependent gating based on atomic detail structure, that follows the laws of electrodynamics. The model predicts gating currents and gating current fluctuations essentially similar to those experimentally observed. The detailed study of the model output, also performed by making several simplifications aimed at understanding the basic dependencies of the gating current fluctuations, suggests that in real channels the voltage sensor moves along a sequence of intermediate states separated by relatively low (<5 kT) energy barriers. As a consequence, crossings of successive gating charges through the gating pore become very frequent, and the corresponding current shots are often seen to overlap because of the relatively high filtering. Notably, this limited bandwidth effect is at the origin of the relatively high single-step charge experimentally detected.

Voltage-dependent gating in K channels: experimental results and quantitative models.

Voltage-dependent K channels open and close in response to voltage changes across the cell membrane. This voltage dependence was postulated to depend on the presence of charged particles moving through the membrane in response to voltage changes. Recording of gating currents originating from the movement of these particles fully confirmed this hypothesis, and gave substantial experimental clues useful for the detailed understanding of the process. In the absence of structural information, the voltage-dependent gating was initially investigated using discrete Markov models, an approach only capable of providing a kinetic and thermodynamic comprehension of the process. The elucidation of the crystal structure of the first voltage-dependent channel brought in a dramatic change of pace in the understanding of channel gating, and in modeling the underlying processes. It was now possible to construct quantitative models using molecular dynamics, where all the interactions of each individual atom with the surroundings were taken into account, and its motion predicted by Newton's laws. Unfortunately, this modeling is computationally very demanding, and in spite of the advances in simulation procedures and computer technology, it is still limited in its predictive ability. To overcome these limitations, several groups have developed more macroscopic voltage gating models. Their approaches understandably require a number of approximations, which must however be physically well justified. One of these models, based on the description of the voltage sensor as a Brownian particle, that we have recently developed, is able to simultaneously describe the behavior of a single voltage sensor and to predict the macroscopic gating current originating from a population of sensors. The basics of this model are here described, and a typical application using the Kv1.2/2.1 chimera channel structure is also presented.

Simulation of Gating Currents of the Shaker K Channel Using a Brownian Model of the Voltage Sensor.

The physical mechanism underlying the voltage-dependent gating of K channels is usually addressed theoretically using molecular dynamics simulations. However, besides being computationally very expensive, this approach is presently unable to fully predict the behavior of fundamental variables of channel gating such as the macroscopic gating current, and hence, it is presently unable to validate the model. To fill this gap, here we propose a voltage-gating model that treats the S4 segment as a Brownian particle moving through a gating channel pore and adjacent internal and external vestibules. In our model, charges on the S4 segment are screened by charged residues localized on neighboring segments of the channel protein and by ions present in the vestibules, whose dynamics are assessed using a flux conservation equation. The electrostatic voltage spatial profile is consistently assessed by applying the Poisson equation to all the charges present in the system. The treatment of the S4 segment as a Brownian particle allows description of the dynamics of a single S4 segment using the Langevin stochastic differential equation or the behavior of a population of S4 segments-useful for assessing the macroscopic gating current-using the Fokker-Planck equation. The proposed model confirms the gating charge transfer hypothesis with the movement of the S4 segment among five different stable positions where the gating charges interact in succession with the negatively charged residues on the channel protein. This behavior produces macroscopic gating currents quite similar to those experimentally found.

Ca2+ -dependent and Ca2+ -independent somatic release from trigeminal neurons.

Besides the nerve endings, the soma of trigeminal neurons also respond to membrane depolarizations with the release of neurotransmitters and neuromodulators in the extracellular space within the ganglion, a process potentially important for the cross-communication between neighboring sensory neurons. In this study, we addressed the dependence of somatic release on Ca2+ influx in trigeminal neurons and the involvement of the different types of voltage-gated Ca2+ (Cav) channels in the process. Similar to the closely related dorsal root ganglion neurons, we found two kinetically distinct components of somatic release, a faster component stimulated by voltage but independent of the Ca2+ influx, and a slower component triggered by Ca2+ influx. The Ca2+ -dependent component was inhibited 80% by ω-conotoxin-MVIIC, an inhibitor of both N- and P/Q-type Cav channels, and 55% by the P/Q-type selective inhibitor ω-agatoxin-IVA. The selective L-type Ca2+ channel inhibitor nimodipine was instead without effect. These results suggest a major involvement of N- and P/Q-, but not L-type Cav channels in the somatic release of trigeminal neurons. Thus antinociceptive Cav channel antagonists acting on the N- and P/Q-type channels may exert their function by also modulating the somatic release and cross-communication between sensory neurons.

BK channels blockage inhibits hypoxia-induced migration and chemoresistance to cisplatin in human glioblastoma cells.

Glioblastoma (GBM) cells express large-conductance, calcium-activated potassium (BK) channels, whose activity is important for several critical aspects of the tumor, such as migration/invasion and cell death. GBMs are also characterized by a heavy hypoxic microenvironment that exacerbates tumor aggressiveness. Since hypoxia modulates the activity of BK channels in many tissues, we hypothesized that a hypoxia-induced modulation of these channels may contribute to the hypoxia-induced GBM aggressiveness. In U87-MG cells, hypoxia induced a functional upregulation of BK channel activity, without interfering with their plasma membrane expression. Wound healing and transwell migration assays showed that hypoxia increased the migratory ability of U87-MG cells, an effect that could be prevented by BK channel inhibition. Toxicological experiments showed that hypoxia was able to induce chemoresistance to cisplatin in U87-MG cells and that the inhibition of BK channels prevented the hypoxia-induced chemoresistance. Clonogenic assays showed that BK channels are also used to increase the clonogenic ability of U87-MG GBM cells in presence, but not in absence, of cisplatin. BK channels were also found to be essential for the hypoxia-induced de-differentiation of GBM cells. Finally, using immunohistochemical analysis, we highlighted the presence of BK channels in hypoxic areas of human GBM tissues, suggesting that our findings may have physiopathological relevance in vivo. In conclusion, our data show that BK channels promote several aspects of the aggressive potential of GBM cells induced by hypoxia, such as migration and chemoresistance to cisplatin, suggesting it as a potential therapeutic target in the treatment of GBM.

Megalencephalic leukoencephalopathy with subcortical cysts protein-1 regulates epidermal growth factor receptor signaling in astrocytes.

Mutations in the MLC1 gene, which encodes a protein expressed in brain astrocytes, are the leading cause of MLC, a rare leukodystrophy characterized by macrocephaly, brain edema, subcortical cysts, myelin and astrocyte vacuolation. Although recent studies indicate that MLC1 protein is implicated in the regulation of cell volume changes, the exact role of MLC1 in brain physiology and in the pathogenesis of MLC disease remains to be clarified. In preliminary experiments, we observed that MLC1 was poorly expressed in highly proliferating astrocytoma cells when compared with primary astrocytes, and that modulation of MLC1 expression influenced astrocyte growth. Because volume changes are key events in cell proliferation and during brain development MLC1 expression is inversely correlated to astrocyte progenitor proliferation levels, we investigated the possible role for MLC1 in the control of astrocyte proliferation. We found that overexpression of wild type but not mutant MLC1 in human astrocytoma cells hampered cell growth by favoring epidermal growth factor receptor (EGFR) degradation and by inhibiting EGF-induced Ca(+) entry, ERK1/2 and PLCγ1 activation, and calcium-activated KCa3.1 potassium channel function, all molecular pathways involved in astrocyte proliferation stimulation. Interestingly, MLC1 did not influence AKT, an EGFR-stimulated kinase involved in cell survival. Moreover, EGFR expression was higher in macrophages derived from MLC patients than from healthy individuals. Since reactive astrocytes proliferate and re-express EGFR in response to different pathological stimuli, the present findings provide new information on MLC pathogenesis and unravel an important role for MLC1 in other brain pathological conditions where astrocyte activation occurs.

Role of KCa3.1 Channels in Modulating Ca2+ Oscillations during Glioblastoma Cell Migration and Invasion.

Cell migration and invasion in glioblastoma (GBM), the most lethal form of primary brain tumors, are critically dependent on Ca2+ signaling. Increases of [Ca2+]i in GBM cells often result from Ca2+ release from the endoplasmic reticulum (ER), promoted by a variety of agents present in the tumor microenvironment and able to activate the phospholipase C/inositol 1,4,5-trisphosphate PLC/IP3 pathway. The Ca2+ signaling is further strengthened by the Ca2+ influx from the extracellular space through Ca2+ release-activated Ca2+ (CRAC) currents sustained by Orai/STIM channels, meant to replenish the partially depleted ER. Notably, the elevated cytosolic [Ca2+]i activates the intermediate conductance Ca2+-activated K (KCa3.1) channels highly expressed in the plasma membrane of GBM cells, and the resulting K⁺ efflux hyperpolarizes the cell membrane. This translates to an enhancement of Ca2+ entry through Orai/STIM channels as a result of the increased electromotive (driving) force on Ca2+ influx, ending with the establishment of a recurrent cycle reinforcing the Ca2+ signal. Ca2+ signaling in migrating GBM cells often emerges in the form of intracellular Ca2+ oscillations, instrumental to promote key processes in the migratory cycle. This has suggested that KCa3.1 channels may promote GBM cell migration by inducing or modulating the shape of Ca2+ oscillations. In accordance, we recently built a theoretical model of Ca2+ oscillations incorporating the KCa3.1 channel-dependent dynamics of the membrane potential, and found that the KCa3.1 channel activity could significantly affect the IP3 driven Ca2+ oscillations. Here we review our new theoretical model of Ca2+ oscillations in GBM, upgraded in the light of better knowledge of the KCa3.1 channel kinetics and Ca2+ sensitivity, the dynamics of the Orai/STIM channel modulation, the migration and invasion mechanisms of GBM cells, and their regulation by Ca2+ signals.

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

The malignancy of glioblastoma multiforme (GBM), the most common human brain tumor, correlates with the presence of hypoxic areas, but the underlying mechanisms are unclear. GBM cells express abundant Cl channels whose activity supports cell volume and membrane potential changes, ultimately leading to cell proliferation, migration, and escaping death. In non-tumor tissues Cl channels are modulated by hypoxia, which prompted us to verify whether hypoxia would also modulate Cl channels in GBM cells. Our results show that in GBM cell lines, acute application of a hypoxic solution activates a Cl current displaying the biophysical and pharmacological features of the swelling-activated Cl current (ICl,swell ). We also found that acute hypoxia increased the cell volume by about 20%, and a 30% hypertonic solution partially inhibited the hypoxia-activated Cl current, suggesting that cell swelling and the activation of the Cl current are sequential events. Notably, the hypoxia-induced cell swelling was followed by a regulatory volume decrease (RVD) mediated mainly by ICl,swell . Since, a hypoxia-induced prolonged cell swelling is usually regarded as a death insult, we hypothesized that the hypoxia-activated Cl current could limit cell swelling and prevent necrotic death of GBM cells under hypoxic conditions. In accordance, we found that the ICl,swell inhibitor DCPIB hampered the RVD process, and more importantly it sensibly increased the hypoxia-induced necrotic death in these cells. Taken together, these results suggest that Cl channels are strongly involved in the survival of GBM cells in a hypoxic environment, and may thus represent a new therapeutic target for this malignant tumor. J. Cell. Physiol. 232: 91-100, 2017. © 2016 Wiley Periodicals, Inc.

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. © 2016 Wiley Periodicals, Inc.

Genetically induced dysfunctions of Kir2.1 channels: implications for short QT3 syndrome and autism-epilepsy phenotype.

Short QT3 syndrome (SQT3S) is a cardiac disorder characterized by a high risk of mortality and associated with mutations in Kir2.1 (KCNJ2) channels. The molecular mechanisms leading to channel dysfunction, cardiac rhythm disturbances and neurodevelopmental disorders, potentially associated with SQT3S, remain incompletely understood. Here, we report on monozygotic twins displaying a short QT interval on electrocardiogram recordings and autism-epilepsy phenotype. Genetic screening identified a novel KCNJ2 variant in Kir2.1 that (i) enhanced the channel's surface expression and stability at the plasma membrane, (ii) reduced protein ubiquitylation and degradation, (iii) altered protein compartmentalization in lipid rafts by targeting more channels to cholesterol-poor domains and (iv) reduced interactions with caveolin 2. Importantly, our study reveals novel physiological mechanisms concerning wild-type Kir2.1 channel processing by the cell, such as binding to both caveolin 1 and 2, protein degradation through the ubiquitin-proteasome pathway; in addition, it uncovers a potential multifunctional site that controls Kir2.1 surface expression, protein half-life and partitioning to lipid rafts. The reported mechanisms emerge as crucial also for proper astrocyte function, suggesting the need for a neuropsychiatric evaluation in patients with SQT3S and offering new opportunities for disease management.

Expression and function of a CP339,818-sensitive K⁺ current in a subpopulation of putative nociceptive neurons from adult mouse trigeminal ganglia.

Trigeminal ganglion (TG) neurons are functionally and morphologically heterogeneous, and the molecular basis of this heterogeneity is still not fully understood. Here we describe experiments showing that a subpopulation of neurons expresses a delayed-rectifying K(+) current (IDRK) with a characteristically high (nanomolar) sensitivity to the dihydroquinoline CP339,818 (CP). Although submicromolar CP has previously been shown to selectively block Kv1.3 and Kv1.4 channels, the CP-sensitive IDRK found in TG neurons could not be associated with either of these two K(+) channels. It could neither be associated with Kv2.1 channels homomeric or heteromerically associated with the Kv9.2, Kv9.3, or Kv6.4 subunits, whose block by CP, tested using two-electrode voltage-clamp recordings from Xenopus oocytes, resulted in the low micromolar range, nor to the Kv7 subfamily, given the lack of blocking efficacy of 3 µM XE991. Within the group of multiple-firing neurons considered in this study, the CP-sensitive IDRK was preferentially expressed in a subpopulation showing several nociceptive markers, such as small membrane capacitance, sensitivity to capsaicin, and slow afterhyperpolarization (AHP); in these neurons the CP-sensitive IDRK controls the membrane resting potential, the firing frequency, and the AHP duration. A biophysical study of the CP-sensitive IDRK indicated the presence of two kinetically distinct components: a fast deactivating component having a relatively depolarized steady-state inactivation (IDRKf) and a slow deactivating component with a more hyperpolarized V1/2 for steady-state inactivation (IDRKs).

M2 receptors exert analgesic action on DRG sensory neurons by negatively modulating VR1 activity.

The peripheral application of the M2 cholinergic agonist arecaidine on sensory nerve endings shows anti-nociceptive properties. In this work, we analyze in vitro, the mechanisms downstream M2 receptor activation causing the analgesic effects, and in vivo the effects produced by M2 agonist arecaidine administration on nociceptive responses in a murine model of nerve growth factor (NGF)-induced pain. Cultured DRG neurons treated with arecaidine showed a decreased level of VR1 and SP transcripts. Conversely, we found an increased expression of VR1 and SP transcripts in DRG from M2/M4(-/-) mice compared to WT and M1(-/-) mice, confirming the inhibitory effect in particular of M2 receptors on SP and VR1 expression. Patch-clamp experiments in the whole-cell configuration showed that arecaidine treatment caused a reduction of the fraction of capsaicin-responsive cells, without altering the mean capsaicin-activated current in responsive cells. We also demonstrated that arecaidine prevents PKCϵ translocation to the plasma membrane after inflammatory agent stimulation, mainly in medium-small sensory neurons. Finally, in mice, we have observed that intraperitoneal injection of arecaidine reduces VR1 expression blocking hyperalgesia and allodynia caused by NGF intraplantar administration. In conclusion, our data demonstrate that in vivo M2 receptor activation induces desensitization to mechanical and heat stimuli by a down-regulation of VR1 expression and by the inhibition of PKCϵ activity hindering its translocation to the plasma membrane, as suggested by in vitro experiments.

Identification of key signaling molecules involved in the activation of the swelling-activated chloride current in human glioblastoma cells.

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.

The Long Journey from Animal Electricity to the Discovery of Ion Channels and the Modelling of the Human Brain.

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.

Fifty years of gating currents and channel gating.

We celebrate this year the 50th anniversary of the first electrophysiological recordings of the gating currents from voltage-dependent ion channels done in 1973. This retrospective tries to illustrate the context knowledge on channel gating and the impact gating-current recording had then, and how it continued to clarify concepts, elaborate new ideas, and steer the scientific debate in these 50 years. The notion of gating particles and gating currents was first put forward by Hodgkin and Huxley in 1952 as a necessary assumption for interpreting the voltage dependence of the Na and K conductances of the action potential. 20 years later, gating currents were actually recorded, and over the following decades have represented the most direct means of tracing the movement of the gating charges and gaining insights into the mechanisms of channel gating. Most work in the early years was focused on the gating currents from the Na and K channels as found in the squid giant axon. With channel cloning and expression on heterologous systems, other channels as well as voltage-dependent enzymes were investigated. Other approaches were also introduced (cysteine mutagenesis and labeling, site-directed fluorometry, cryo-EM crystallography, and molecular dynamics [MD] modeling) to provide an integrated and coherent view of voltage-dependent gating in biological macromolecules. The layout of this retrospective reflects the past 50 years of investigations on gating currents, first addressing studies done on Na and K channels and then on other voltage-gated channels and non-channel structures. The review closes with a brief overview of how the gating-charge/voltage-sensor movements are translated into pore opening and the pathologies associated with mutations targeting the structures involved with the gating currents.

Hypoxia, Ion Channels and Glioblastoma Malignancy.

The malignancy of glioblastoma (GBM), the most aggressive type of human brain tumor, strongly correlates with the presence of hypoxic areas within the tumor mass. Oxygen levels have been shown to control several critical aspects of tumor aggressiveness, such as migration/invasion and cell death resistance, but the underlying mechanisms are still unclear. GBM cells express abundant K+ and Cl- channels, whose activity supports cell volume and membrane potential changes, critical for cell proliferation, migration and death. Volume-regulated anion channels (VRAC), which mediate the swelling-activated Cl- current, and the large-conductance Ca2+-activated K+ channels (BK) are both functionally upregulated in GBM cells, where they control different aspects underlying GBM malignancy/aggressiveness. The functional expression/activity of both VRAC and BK channels are under the control of the oxygen levels, and these regulations are involved in the hypoxia-induced GBM cell aggressiveness. The present review will provide a comprehensive overview of the literature supporting the role of these two channels in the hypoxia-mediated GBM malignancy, suggesting them as potential therapeutic targets in the treatment of GBM.