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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

Little is known about how conformation changes of proteins control biological function. How do the locations reveal themselves as measurable physical properties of the protein?One place these questions can be (mostly) answered is in the proteins that form the voltage activated channels of nerve. We calculate conformation currentin Na Vand K V channels in response to a step of voltage. Changing isoleucine of K V channels to threonine of Na V channels speeds responses to voltage, andallows the separation of opening of Na V and K V channels needed to create nerve signals. The polarization mechanism we propose is both logically sufficient and logically necessary to account for the difference in speed and the structural movements of voltage sensors. It is sufficient because we reproduce properties of gating current under a realistic range of conditions.It is necessary because the movement of chargeswe calculate must produce the currents we calculate given the universal and exact nature of the Maxwell equations that link charge and current. Our conclusions arise fromthe universal and exact conservation of total current implied by the Maxwell equations. Of course, evolution is not logical. It often provides redundant mechanismsbeyond the necessary and sufficient. These mechanisms may provide properties not glimpsed here. These mechanisms may also control the speed of gating and give the gating system biologically and evolutionarily useful properties unknown to us.

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.

MLC1 is a membrane protein highly expressed by brain perivascular astrocytes. Mutations in the MLC1 gene account for megalencephalic leukoencephalopathy with subcortical cysts (MLC), an incurable leukodystrophy characterized by macrocephaly, brain edema and cysts, myelin vacuolation and astrocyte swelling, causing cognitive and motor dysfunctions. It has been demonstrated that MLC1 mutations affect the swelling-activated Cl - currents (I Cl,swell ) mediated by volume-regulated anion channel (VRAC) and the consequent regulatory volume decrease (RVD) and lead to abnormal activation of intracellular signaling pathways linked to inflammation/osmotic stress. Despite this knowledge, the MLC1 physiological role and MLC molecular pathogenesis are still elusive. Following the observations that Ca 2+ regulates all the MLC1-modulated processes and that intracellular Ca 2+ homeostasis is altered in MLC1-defective cells, we applied a multidisciplinary approach including biochemistry, molecular biology, video imaging, electrophysiology and proteomic techniques on cultured astrocytes to uncover new Ca 2+ -dependent signaling pathways controlling MLC1 function. Here, we revealed that MLC1 binds the Ca 2+ effector proteins calmodulin (CaM) and Ca 2+ /CaM-dependent protein kinase II (CaMKII) and, as result, changes its assembly, localization and functional properties in response to Ca 2+ changes. Noteworthy, CaM binding to the COOH terminal promotes MLC1 trafficking to the plasma membrane, while CaMKII phosphorylation of the NH 2 -terminal potentiates MLC1 activation of I Cl,swell . Overall, these results revealed that MLC1 is a Ca 2+ -regulated protein linking VRAC function and, possibly, volume regulation to Ca 2+ signaling in astrocytes. These findings open new avenues of investigations aimed at clarifying the abnormal molecular pathways underlying MLC and other diseases characterized by astrocyte swelling and brain edema.

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

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 Ca

The generation of action potentials in excitable cells requires different activation kinetics of voltage gated Na (NaV) and K (KV) channels. NaV channels activate much faster and allow the initial Na+ influx that generates the depolarizing phase and propagates the signal. Recent experimental results suggest that the molecular basis for this kinetic difference is an amino acid side chain located in the gating pore of the voltage sensor domain, which is a highly conserved isoleucine in KV channels, but an equally highly conserved threonine in NaV channels. Mutagenesis suggests that the hydrophobicity of this side chain in Shaker KV channels regulates the energetic barrier that gating charges need to overcome to move through the gating pore, and ultimately the rate of channel opening. We use a multi-scale modeling approach to test this hypothesis. We use high resolution molecular dynamics to study the effect of the mutation on polarization charge within the gating pore. We then incorporate these results in a lower resolution model of voltage gating to predict the effect of the mutation on the movement of gating charges. The predictions of our hierarchical model are fully consistent with the tested hypothesis, thus suggesting that the faster activation kinetics of NaV channels comes from a stronger dielectric polarization by threonine (NaV channel) produced as the first gating charge enters the gating pore, compared to isoleucine (KV channel).eTOC SummaryVoltage-gated Na+ channels activate faster than K+ channels in excitable cells. Catacuzzeno et al. develop a model that shows how the dielectric properties of a divergent side-chain produce this difference in speed.

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.

Background The KCa3.1 channel is the intermediate-conductance member of the Ca2+- activated K channel superfamily. It is widely expressed in excitable and non-excitable cells, where it plays a major role in a number of cell functions. This paper aims at illustrating the main structural, biophysical and modulatory properties of the KCa3.1 channel, and providing an account of experimental data on its role in volume regulation and Ca2+ signals. Methods Research and online content related to the structure, structure/function relationship, and physiological role of the KCa3.1 channel are reviewed. Results Expressed in excitable and non-excitable cells, the KCa3.1 channel is voltage independent, its opening being exclusively gated by the binding of intracellular Ca2+ to calmodulin, a Ca2+- binding protein constitutively associated with the C-terminus of each KCa3.1 channel α subunit. The KCa3.1 channel activates upon high affinity Ca2+ binding, and in highly coordinated fashion giving steep Hill functions and relatively low EC50 values (100-350 nM). This high Ca2+ sensitivity is physiologically modulated by closely associated kinases and phosphatases. The KCa3.1 channel is normally activated by global Ca2+ signals as resulting from Ca2+ released from intracellular stores, or by the refilling influx through store operated Ca2+ channels, but cases of strict functional coupling with Ca2+-selective channels are also found. KCa3.1 channels are highly expressed in many types of cells, where they play major roles in cell migration and death. The control of these complex cellular processes is achieved by KCa3.1 channel regulation of the driving force for Ca2+ entry from the extracellular medium, and by mediating the K+ efflux required for cell volume control. Conclusion Much work remains to be done to fully understand the structure/function relationship of the KCa3.1 channels. Hopefully, this effort will provide the basis for a beneficial modulation of channel activity under pathological conditions.

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.

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.

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.

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.

Malignancy of glioblastoma multiforme (GBM), the most common and aggressive form of human brain tumor, strongly depends on its enhanced cell invasion and death evasion which make surgery and accompanying therapies highly ineffective. Several ion channels that regulate membrane potential, cytosolic Ca2+ concentration and cell volume in GBM cells play significant roles in sustaining these processes. Among them, the volume-regulated anion channel (VRAC), which mediates the swelling-activated chloride current (IClswell) and is highly expressed in GBM cells, arguably plays a major role. VRAC is primarily involved in reestablishing the original cell volume that may be lost under several physiopathological conditions, but also in sustaining the shape and cell volume changes needed for cell migration and proliferation. While experimentally VRAC is activated by exposing cells to hypotonic solutions that cause the increase of cell volume, in vivo it is thought to be controlled by several different stimuli and modulators. In this review we focus on our recent work showing that two conditions normally occurring in pathological GBM tissues, namely high serum levels and severe hypoxia, were both able to activate VRAC, and their activation was found to promote cell migration and resistance to cell death, both features enhancing GBM malignancy. Also, the fact that the signal transduction pathway leading to VRAC activation appears to involve GBM specific intracellular components, such as diacylglicerol kinase and phosphatidic acid, reportedly not involved in the activation of VRAC in healthy tissues, is a relevant finding. Based on these observations and the impact of VRAC in the physiopathology of GBM, targeting this channel or its intracellular regulators may represent an effective strategy to contrast this lethal tumor.

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.

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.

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 Ca

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 IDRKfound 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 IDRKwas 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 IDRKcontrols the membrane resting potential, the firing frequency, and the AHP duration. A biophysical study of the CP-sensitive IDRKindicated 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 V1/2for steady-state inactivation ( IDRKs).

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.

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.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 the other segments of the channel protein, and by ions present in the vestibules, whose dynamics is 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 to alternatively describe the dynamics of a single S4 segment, using the Langevin stochastic differential equation, or the behavior of a population of S4 segments, useful to assess 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 5 different stable positions where the gating charges differently interact with the negatively charged residues on the channel protein. This behavior results in macroscopic gating currents displaying properties quite similar to those experimentally found, including a very fast component followed by a slower one with an evident rising phase at high depolarizing voltages.

This work shows experimentally how electrical energy can be harvested directly from cell membrane potential and used to power a wireless communication. The experiment is performed by exploiting the membrane potential of Xenopus oocytes taken from female frogs. Electrical potential energy of the membrane is transferred to a capacitor connected to the cell via a proper electrical circuit. Once the capacitor has reached the planned amount of energy, the circuit is disconnected from the cell and the stored energy is used to power a radio frequency communication that carries bio-sensed information to a distanced receiving circuit. Our result shows that electrical energy can be harvested directly from biological cells and used for a number of purposes, including wireless communication of sensed biological quantities to a remote receiving hub.

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.

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. J. Cell. Physiol. 229: 783–790, 2014. © 2013 Wiley Periodicals, Inc.

The hypotheses is forwarded that the introduction in the environment of high concentrations of phenols and other aromatic compounds (AC) is one, perhaps the main cause of the continuously rising trend of malignant melanoma (MM) incidence. Two, non-mutually exclusive, possibilities could explain how AC may induce MM: (1) AC may act as inhibitors or alternative substrates of tyrosinase, the enzyme synthesizing melanin, thus impairing the melanocyte photoprotection. (2) AC may impair, directly or indirectly, the activity or synthesis of the melanocorticotropin receptor (MC1R), which photoprotects melanocytes from the UV rays (UVR) by stimulating the DNA repair system. Particularly suspected are sunscreens, as they contain high concentrations of a large variety of AC, three of which are known to be tyrosinase inhibitors. AC that may interfere with tyrosinase are also present in a large variety of medicines used orally or as creams, and in many industrial products with which man is frequently in contact.

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 Ca2+-activated K (IKCa) 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 IKCachannel with its specific inhibitor 1-[(2-chlorophenyl) diphenylmethyl]-1 H-pyrazole (TRAM-34) or IKCachannel 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 IKCachannels in the glioblastoma-derived cell line GL15, stimulate cell chemotaxis even if the IKCachannels have been blocked or silenced. Furthermore, we demonstrate that both CXCL12 and EGF induce Ca2+mobilization and IKCachannel activation but only CXCL12 induces a long-term upregulation of the IKCachannel activity. Furthermore, the Ca2+-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.

In adult mouse trigeminal ganglion (TG) neurones we identified three neuronal subpopulations, defined in terms of their firing response to protracted depolarizations, namely MF neurones, characterized by a multiple tonic firing; DMF neurones, characterized by a delay before the beginning of repetitive firing; and SS neurones, characterized by a strongly adapting response. The three subpopulations also differed in several other properties important for defining their functional role in vivo, namely soma size, action potential (AP) shape and capsaicin sensitivity. MF neurones had small soma, markedly long AP and mostly responded to capsaicin, properties typical of a subgroup of C-type nociceptors. SS neurones had large soma, short AP duration and were mostly capsaicin insensitive, suggesting that most of them have functions other than nociception. DMF neurones were all capsaicin insensitive, had a small soma size and intermediate AP duration, making them functionally distinct from both MF and SS neurones. We investigated the ionic basis underlying the delay to the generation of the first AP of DMF neurones, and the strong adaptation of SS neurones. We found that the expression of a fast-inactivating, 4-AP- and CP-339,818-sensitive K+ current (IA) in DMF neurones plays a critical role in the generation of the delay, whereas a DTX-sensitive K+ current (IDTX) selectively expressed in SS neurones appeared to be determinant for their strong firing adaptation. A minimal theoretical model of TG neuronal excitability confirmed that IA and IDTX have properties congruent with their suggested role.

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.

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. Supplementary information accompanies this paper in the files section., peer-reviewed

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"., peer-reviewed

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., peer-reviewed

In this study we investigated the role of extracellular 5′-guanosine-triphosphate (GTP) on early phases of skeletal muscle differentiation using the widely used C2C12 mouse cells as a myogenic model. We show that extracellular GTP binding to specific sites activates a metabotropic cascade that leads to a transient intracellular Ca2+ mobilization, consequent activation of the intermediate Ca2+-activated K+ channels (IKCa), and hyperpolarization of the plasma membrane. We further show that in differentiating C2C12 myoblasts GTP induces a proliferative boost, and increases the number of cells positive for the myosin heavy chain (MyHC) proteins. These effects were shown to be mediated by the IKCa channel-dependent hyperpolarization, as evidenced by their disappearance when myoblasts were incubated with the IKCa channel inhibitor charybdotoxin. These data give new insights into nucleotide purinergic signalling pathways, and address the role of the GTP-dependent IKCa channel activation and hyperpolarization in myogenesis.

We report here the expression and properties of the intermediate-conductance Ca2+-activated K+ (IKCa) channel in the GL-15 human glioblastoma cell line. Macroscopic

Intracellular Ca2+ oscillations are observed in a large number of non-excitable cells. While most appear to reflect an intermittent Ca2+ release from intracellular stores, in some instances intracellular Ca2+ oscillations strongly depend on Ca2+ influx, and are coupled to oscillations of the membrane potential, suggesting that a plasma membrane-based mechanism may be involved. We have developed a theoretical model for the latter type of intracellular Ca2+ oscillations based on the Ca2+-dependent modulation of the intermediate-conductance, Ca2+-activated K+ (IKCa) channel. The functioning of this model relies on the Ca2+-dependent activation, and the much slower Ca2+-dependent rundown of this channel. We have shown that Ca2+-dependent activation of the IKCa channels, the consequent membrane hyperpolarization and the resulting increase in Ca2+ influx may confer the positive feedback mechanism required for the ascending phase of the oscillation. The much slower Ca2+-dependent rundown process will conversely halt this positive loop, and establish the descending phase of the intracellular Ca2+ oscillation. We found that this simple model gives rise to intracellular Ca2+ oscillations when using physiologically reasonable parameters, suggesting that IKCa channels could participate in the generation of intracellular Ca2+ oscillations.

Spontaneous membrane voltage oscillations were found in 27 of 130 isolated frog saccular hair cells. Voltage oscillations had a mean peak-to-peak amplitude of 23 mV and a mean oscillatory frequency of 4.6 Hz. When compared with non-oscillatory cells, oscillatory cells had significantly greater hyperpolarization-activated and lower depolarization-activated current densities. Two components, the hyperpolarization-activated cation current, Ih, and the K+-selective inward-rectifier current, IK1, contributed to the hyperpolarization-activated current, as assessed by the use of the IK1-selective inhibitor Ba2+ and the Ih-selective inhibitor ZD-7288. Five depolarization-activated currents were present in these cells (transient IBK, sustained IBK, IDRK, IA, and ICa), and all were found to have significantly lower densities in oscillatory cells than in non-oscillatory cells (revealed by using TEA to block IBK, 4-AP to block IDRK, and prepulses at different voltages to isolate IA). Bath application of either Ba2+ or ZD-7288 suppressed spontaneous voltage oscillations, indicating that Ih and IK1 are required for generating this activity. On the contrary, TEA or Cd2+ did not inhibit this activity, suggesting that IBK and ICa do not contribute. A mathematical model has been developed to test the interpretation derived from the pharmacological and biophysical data. This model indicates that spontaneous voltage oscillations can be generated when the electrophysiological features of oscillatory cells are used. The oscillatory behaviour is principally driven by the activity of IK1 and Ih, with IA playing a modulatory role. In addition, the model indicates that the high densities of depolarization-activated currents expressed by non-oscillatory cells help to stabilize the resting membrane potential, thus preventing the spontaneous oscillations.

Outward K currents and electrical resonance of frog (Rana esculenta) saccular hair cells isolated enzymatically with bacterial protease VIII were investigated using the perforated patch-clamp method. Under voltage-clamp conditions we identified two K currents, a voltage-dependent K (K(V)) current, and a partially inactivating iberiotoxin-sensitive K (BK) current. The K(V) current activated at a membrane potential of approximately -50 mV (from a holding potential of -70 mV). Its activation rate was rather slow, having a time constant in the range 5-8 ms at 0 mV. The K(V) current was resistant to tetraethylammonium (10 mM), but was inhibited by 4-aminopyridine (1 mM). A striking feature of the BK current was its inactivation; this was monoexponential and had fast kinetics (tau(inact)=2.7 ms +/-1.2, at -10 mV; n=8). Inactivation of the current was incomplete, a residual sustained component remaining. This varied considerably among hair cells (mean ratio between peak transient and sustained component was 1.22+/-0.18, range 0.53-1.8; n=8). In current-clamp mode steady depolarizing current pulses evoked membrane potential oscillatory responses, with mean frequencies varying between 30 and 100 Hz for membrane potentials from -60 to -40 mV (n=18). Most hair cells (14/18) exhibited damped oscillations, and in the remainder a few initial damped oscillations were succeeded by smaller, undamped voltage oscillations. The peak quality factor and the characteristic frequency assessed on 14 cells displaying only damped oscillatory responses were 2.4+/-1.3 and 59+/-39 Hz, respectively. In contrast, papain-dissociated frog saccular hair cells possess solely a sustained BK current, and exhibited significantly higher resonant frequencies and quality factors. In conclusion, the K currents and the electrical resonance of hair cells dissociated in protease VIII differ markedly from those dissociated with papain, but are similar to those reported for in situ preparations, suggesting that our dissociation procedure preserves the electrophysiological profile of in situ frog saccular hair cells.

Episodic ataxia type 1 (EA1) is a K+ channelopathy characterized by a broad spectrum of symptoms. Generally, patients may experience constant myokymia and dramatic episodes of spastic contractions of the skeletal muscles of the head, arms, and legs with loss of both motor coordination and balance. During attacks additional symptoms may be reported such as vertigo, blurred vision, diplopia, nausea, headache, diaphoresis, clumsiness, stiffening of the body, dysarthric speech, and difficulty in breathing. These episodes may be precipitated by anxiety, emotional stress, fatigue, startle response or sudden postural changes. Epilepsy is overrepresented in EA1. The disease is inherited in an autosomal dominant manner, and genetic analysis of several families has led to the discovery of a number of point mutations in the voltage-dependent K+ channel gene KCNA1 (Kv1.1), on chromosome 12p13. To date KCNA1 is the only gene known to be associated with EA1. Functional studies have shown that these mutations impair Kv1.1 channel function with variable effects on channel assembly, trafficking and biophysics. Despite the solid evidence obtained on the molecular mechanisms underlying EA1, how these cause dysfunctions within the central and peripheral nervous systems circuitries remains elusive. This review summarizes the main breakthrough findings in EA1, discusses the neurophysiological mechanisms underlying the disease, current therapies, future challenges and opens a window onto the role of Kv1.1 channels in central nervous system (CNS) and peripheral nervous system (PNS) functions., peer-reviewed

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., peer-reviewed

Transient outward currents in rat saphenous arterial myocytes were studied using the perforated configuration of the patch-clamp method. When myocytes were bathed in a Na-gluconate solution containing TEA to block large-conductance Ca2+-activated K+ (BK) currents, depolarizing pulses positive to +20 mV from a holding potential of -100 mV induced fast transient outward currents. The activation and inactivation time constants of the current were voltage dependent, and at +40 mV were 3.6 +/- 0.8 ms and 23.9 +/- 6.4 ms (n = 4), respectively. The steady-state inactivation of the transient outward current was steeply voltage dependent (z = 1.7), with 50% of the current inactivated at -55 mV. The current was insensitive to the A-type K+ channel blocker 4-AP (1-5 mM), and was modulated by external Ca, decreasing to approximately 0.85 of control values upon raising Ca2+ from 1 to 10 mM, and increasing approximately 3-fold upon lowering it to 0.1 mM. Transient outward currents were also recorded following replacement of internal K+ with either Na+ or Cs+, raising the possibility that the current was carried by monovalent ions passing through voltage-gated Ca2+ channels. This hypothesis was supported by the finding that the transient outward current had the same inactivation rate as the inward Ba2+ current, and that both currents were effectively blocked by the L-type Ca2+ channel blocker, nifedipine and enhanced by the agonist BAYK8644.

Autism spectrum disorders (ASDs) are characterized by impaired ability to properly implement environmental stimuli that are essential to achieve a state of social and cultural exchange. Indeed, the main features of ASD are impairments of interpersonal relationships, verbal and non-verbal communication and restricted and repetitive behaviors. These aspects are often accompanied by several comorbidities such as motor delay, praxis impairment, gait abnormalities, insomnia, and above all epilepsy. Genetic analyses of autistic individuals uncovered deleterious mutations in several K+ channel types strengthening the notion that their intrinsic dysfunction may play a central etiologic role in ASD. However, indirect implication of K+ channels in ASD has been also reported. For instance, loss offragile X mental retardation protein (FMRP) results in K+ channels deregulation, network dysfunction and ASD-like cognitive and behavioral symptoms. This review provides an update on direct and indirect implications of K+ channels in ASDs. Owing to a mounting body of evidence associating a channelopathy pathogenesis to autism and showing that nearly 500 ion channel proteins are encoded by the human genome, we propose to classify ASDs - whose susceptibility is significantly enhanced by ion channels defects, either in a monogenic or multigenic condition - in a new category named “channelAutismSpectrumDisorder”(channelASD; cASD) and introduce a new taxonomy (e.g., Kvx.y-channelASD and likewise Navx.y-channelASD, Cavx.y-channelASD; etc.). This review also highlights some degree of clinical and genetic overlap between K+ channelASDs and K+ channelepsies, whereby such correlation suggests that a subcategory characterized by achannelASD-channelepsy phenotypemay be distinguished. Ultimately, this overview aims to further understand the different clinical subgroups and help parse out the distinct biological basis of autism that are essential to establish patient-tailored treatments., peer-reviewed