Inward rectifying potassium channels in human malignant glioma cells

Human glioma cells obtained from established cell lines (Tp-276MG, Tp-301MG, Tp-378MG, Tp-483MG and U-251MG) were analyzed for the presence of ion channels with the tight-seal voltage clamp technique. The current-voltage relation revealed a marked inward rectification at hyperpolarizing voltages, due to the presence of inward rectifying K-channels in cells from all studied cell lines. These channels were conducting when the membrane potential was more negative than the K-equilibrium potential. The slope conductance for the inward K-currents (g_Ki) was affected both by [K⁺]_i and [K⁺]₀. g_Ki was proportional to [K⁺]₀ raised to 0.35 or 0.50, of which the larger value was measured in the presence of low [K⁺]_i (25mM). The rectification was not significantly different in cells perfused with Mg-free EDTA-buffered internal solution. Tl⁺ was 3.5 times more permaant than K⁺. g_ki was blocked by Cs⁺ (1 mM) in a voltage-dependent way (more effective in the hyperpolarized membrane), and by Na⁺ (154 mM) depending on voltage and time. From measurements of unitary current events in membrane patches (outside out or cell attached) the conductance of the single inward rectifying channel was estimated to be 27 ± 7 pS. This type of ion channel may be important for K-uptake by glial cells and hence for the K-homeostasis in the brain.     

Voltage-dependent ion channels in glial cells

Glial cells, although non-excitable, express a wealth of voltage-activated ion channels that are typically characteristic of excitable cells. Since these channels are also observed in acutely isolated cells and in brain slices, they have to be considered functional in the intact brain. Numerous studies over the past 10 years have yielded detailed characterizations of glial channels permitting comparison of their properties to those of their neuronal counterparts. While for the most part such comparisons have demonstrated a high degree of similarity, they also provide evidence for the expression of some uniquely glial ion channels. An increasing number of studies indicate that the expression of “glial” channels is influenced by the cells' microenvironment. For example, the presence of neurons can induce or inhibit (depending on the preparation and type of channel studied) the expression of glial ion channels. Like ion channels in excitable cells, glial channels can be functionally regulated by activation of second-messenger pathways, allowing for short-term modulation of their membrane properties. Although the extent to which most of the characterized ion channels are involved in glial function is presently unclear, a growing body of data suggests that certain channels play an active role in glial function. Thus inwardly rectifying K⁺ channels in concert with delayed rectifying K⁺ channels are thought to be involved in the removal and redistribution of excess K⁺ in the brain, a process referred to as “spatial buffering.” Glial K⁺ channels may also be crucial in modulating glial proliferation. Cl^? channels and stretch-activated cation channels are believed to be involved in volume regulation. Na⁺ channels appear to be important in fueling the glial Na⁺/K⁺ -pump, and Ca²⁺ channels are likely involved in numerous cellular events in which intracellular Ca²⁺ is a critical second messenger. © 1994 Wiley-Liss, Inc.     

Roles of glial ion transporters in brain diseases

Glial ion transporters are important in regulation of ionic homeostasis, cell volume, and cellular signal transduction under physiological conditions of the central nervous system (CNS). In response to acute or chronic brain injuries, these ion transporters can be activated and differentially regulate glial functions, which has subsequent impact on brain injury or tissue repair and functional recovery. In this review, we summarized the current knowledge about major glial ion transporters, including Na⁺/H⁺ exchangers (NHE), Na⁺/Ca²⁺ exchangers (NCX), Na⁺–K⁺–Cl⁻ cotransporters (NKCC), and Na⁺–HCO₃⁻ cotransporters (NBC). In acute neurological diseases, such as ischemic stroke and traumatic brain injury (TBI), these ion transporters are rapidly activated and play significant roles in regulation of the intra- and extracellular pH, Na⁺, K⁺, and Ca²⁺ homeostasis, synaptic plasticity, and myelin formation. However, overstimulation of these ion transporters can contribute to glial apoptosis, demyelination, inflammation, and excitotoxicity. In chronic brain diseases, such as glioma, Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS), glial ion transporters are involved in the glioma Warburg effect, glial activation, neuroinflammation, and neuronal damages. These findings suggest that glial ion transporters are involved in tissue structural and functional restoration, or brain injury and neurological disease development and progression. A better understanding of these ion transporters in acute and chronic neurological diseases will provide insights for their potential as therapeutic targets.     

Effect of kainate on the membrane conductance of hilar glial precursor cells recorded in the perforated-patch configuration

The effects of kainate on membrane current and membrane conductance were investigated in presumed hilar glial precursor cells of juvenile rats. The perforated-patch configuration was used also to reveal possible second-messenger effects. Kainate evoked an inward current that was accompanied by a biphasic change in membrane conductance in 69% of the cells. An initial conductance increase with a time course similar to that of the inward current was followed by a second delayed conductance increase. This second conductance was absent in whole-cell-clamp recordings, suggesting that it was mediated by a second messenger effect. Analysis of the reversal potentials of the membrane current during both phases of the kainate-induced conductance change revealed that the first conductance increase reflected the activation of AMPA receptors. Several lines of evidence suggest that the delayed second conductance increase was due to the indirect activation of Ca²⁺-dependent K⁺ channels via Ca²⁺-influx through AMPA receptors. (1) the delayed second conductance increase was blocked by Ba²⁺ and the reversal of its underlying current was significantly shifted towards E_K+, suggesting that it is due to the activation of K⁺ channels. (2) The delayed second conductance increase disappeared in a Ca²⁺-free saline buffered with BAPTA, indicating that it depended on Ca²⁺-influx. (3) Co²⁺, Cd²⁺ and nimodipine failed to block the delayed second conductance increase excluding a major contribution of voltage-dependent Ca²⁺ channels. (4) The involvement of metabotropic glutamate receptors also appeared unlikely, because the kainate-induced delayed second conductance increase could not be blocked by a depletion of the intracellular Ca²⁺ stores with the Ca²⁺-ATPase inhibitor thapsigargin, and t-ACPD exerted no effect on membrane current and conductance. We conclude that kainate activates directly AMPA receptors in presumed hilar glial precursor cells. This results in a Ca²⁺ influx that could lead indirectly to the activation of Ca²⁺-dependent K⁺ channels. GLIA 23:35–44, 1998. © 1998 Wiley-Liss, Inc.     

Molecular dissection of the myelinated axon

The membrane of the myelinated axon expresses a rich repertoire of physiologically active molecules: (1) Voltagesensitive NA⁺ channels are clustered at high density (～1,000/μm²) in the nodal axon membrane and are present at lower density(<25/μm²) in the internodal axon membrane under the myelin. Na⁺ channels are also present within Schwann cell processes (in peripheral nerve) and perinodal astrocyte processes (in the central nervous system) which contact the Na⁺ channel–rich axon membrane at the node. In some demyelinated fibers, the bared (formerly internodal) axon membrane reorganizes and expresses a higher-than-normal Na⁺ channel density, providing a basis for restoration of conduction. The presence of glial cell processes, adjacent to foci of Na⁺ channels in immature and demyelinated axons, suggests that glial cells participate in the clustering of Na⁺ channels in the axon membrane. (2) “Fast” K⁺ channels, sensitive to 4-aminopyridine, are present in the paranodal of internodal axon membrane under the myelin; these channels may function to prevent reexcitation following action potentials, or participate in the generation of an internodal resting potential. (3) “Slow” K⁺ channels, sensitive to tetraethylammonium, are present in the nodal axon membrane and, in lower densities, in the internodal axon membrane; their activation produces a hyperpolarizing afterpotential which modulates repetitive firing. (4) The “inward rectifier” is activated by hyperpolarization. This channel is permeable to both Na⁺ and K⁺ ions and may modulate axonal excitability or participate in ionic reuptake following activity. (5) Na⁺/K⁺-ATPase and (6) Ca²⁺-ATPase are also present in the axon membrane and function to maintain transmembrane gradients of Na⁺, K⁺, and Ca²⁺. (7) A specialized antiporter molecule, the Na⁺/Ca²⁺ exchanger, is present in myelinated axons within central nervous system white matter. Following anoxia, the Na⁺/Ca²⁺ exchanger mediates an influx of Ca²⁺ which damages the axon. The molecular organization of the myelinated axon has important pathophysiological implications. Blockade of fast K⁺ channels and Na⁺/K⁺-ATPase improves action potential conduction in some demyelinated axons, and block of the Na⁺/Ca²⁺ exchanger protects white matter axons from anoxic injury. Modification of ion channels, pumps, and exchangers in myelinated fibers may thus provide an important therapeutic approach for a number of neurological disorders.     

4-aminopyridine causes apoptosis and blocks an outward rectifier K+ channel in malignant astrocytoma cell lines

Among the ion channels and pumps activated by growth factor stimulation, K⁺ channels have been implicated in the growth and proliferation of several cancer cell lines. The role of these channels in central nervous system tumors, however, has not been described. This study used the malignant astrocytoma cell lines U87 and A172. 4-Aminopyridine (4-AP) inhibition of proliferation was dose dependent, and assessment using a TUNEL in situ assay revealed that apoptosis occurred in U87 cells with wild-type p53 but not in A172 cells with mutant p53 (24-hr incubation with 4 mM 4-AP). In patch clamp experiments, we identified two types of K⁺ currents in both cell lines, a charybdotoxin-sensitive Ca²⁺-activated K⁺ channel and a 4-AP-sensitive outward rectifier K⁺ current. The outward rectifier current was blocked by 4-AP in a dose-dependent manner, with half-maximal block occurring at 3.9 mM. The blocking effect of 4 mM 4-AP was noticeable at potentials as low as −65 mV and was statistically significant at −60 mV and above, suggesting that 4-AP-sensitive current is active at physiological potentials. By contrast, charybdotoxin (1 μM) and tetraethylammonium · Cl (2 mM) blocked the Ca²⁺-activated K⁺ channel in both cell lines but had no appreciable effect on cell growth. Our findings reveal that 4-AP inhibits proliferation and the outward rectifier K⁺ channel in both U87 and A172 cells. More studies are needed, however, to describe the mechanism by which K⁺ channels influence proliferation and induce apoptosis. J. Neurosci. Res. 48:122–127, 1997. © 1997 Wiley-Liss, Inc.     

Subcellular heterogeneity of voltage-gated Ca2+ channels in cells of the oligodendrocyte lineage

We studied the distribution of voltage-gated Ca²⁺ channels in cells of the oligodendrocyte lineage from retinal and cortical cultures. Influx Of ca²⁺ via voltagegated channels was activated by membrane depolarization with elevated extracellular K⁺ concentration ([K⁺]_e) and local, subcellular increases in cytosolic free Ca²⁺ concentration ([Ca²⁺]_in) could be monitored with a fluometric system connected to a laser scanning confocal microscope. In glial precursor cells from both retina and cortex, small depolarizations (with 10 or 20 mM K⁺) activated Ca²⁺ transients in processes indicating the presence of low-voltage-activated Ca²⁺ channels. Larger depolarizations (with 50 mM K⁺) additionally activated high-voltage-activated Ca²⁺ channels in the soma. An uneven distribution of Ca²⁺ channels was also observed in the mature oligodendrocytes; Ca²⁺ trasients in processes were considerably larger. Recovery of Ca²⁺ levels after the voltage-induced influx was achieved by the activity of the plasmalemmal Ca²⁺ pump, while mitochondria played a minor role to restore²⁺ levels after an influx through voltageoperated channels. During the development of white matter tracts, cells of the oligodendrocyte lineage contact axons to form myelin. Neuronal activity is accompanied by increases in [K⁺]_e; this may lead to Ca²⁺ changes in the processes and the Ca²⁺ increase might be a signal for the glial precursor cell to start myelin formation. © 1995 Wiley-Liss, Inc.     

Ca2+ influx into leech glial cells and neurones caused by pharmacologically distinct glutamate receptors

The effect of glutamatergic agonists on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) of neuropile glial cells and Retzius neurones in intact segmental ganglia of the medicinal leech Hirudo medicinalis was investigated by using iontophoretically injected fura-2. In physiological Ringer solution the [Ca²⁺]_i levels of both cell types were almost the ssame (glial cells: 58 ± 30 nM, n = 51; Retzius neurones: 61 ± 27 nM, n = 64). In both cell types glutamate, kainate, and quisqualate induced an increase in [Ca²⁺]_i which was inhibited by 6,7-dinitroquinoxaline-2,3-dione (DNQX). This increase was caused by a Ca²⁺ influx from the extracellular space because the response was greatly diminished upon removal of extracellular Ca²⁺. The glutamate receptors of neuropile glial cells and Retzius neurones differed with respect to the relative effectiveness of the agonists used, as well as with regard to the inhibitory strenght of DNQX. In Retzius neurones the agonist-induced [Ca²⁺]_i increase was abolished after replacing extracellular Na⁺ by organic cations or by mM amounts of Ni²⁺, whereas in glial cells the [Ca²⁺]_i increase was largely preserved under both conditions. It is concluded that in Retzius neurones the Ca²⁺ influx is predominantly mediated by voltage-dependent Ca²⁺ channels, whereas in neuropile glial cells the major influx occurs via the ion channels that are associated with the glutamate receptors.     

Molecular targets for antiepileptic drug development

This review considers how recent advances in the physiology of ion channels and other potential molecular targets, in conjunction with new information on the genetics of idiopathic epilepsies, can be applied to the search for improved antiepileptic drugs (AEDs). Marketed AEDs predominantly target voltage-gated cation channels (the α subunits of voltage-gated Na⁺ channels and also T-type voltage-gated Ca²⁺ channels) or influence GABA-mediated inhibition. Recently, α2-δ voltage-gated Ca²⁺ channel subunits and the SV2A synaptic vesicle protein have been recognized as likely targets. Genetic studies of familial idiopathic epilepsies have identified numerous genes associated with diverse epilepsy syndromes, including genes encoding Na⁺ channels and GABA_A receptors, which are known AED targets. A strategy based on genes associated with epilepsy in animal models and humans suggests other potential AED targets, including various voltage-gated Ca²⁺ channel subunits and auxiliary proteins, A- or M-type voltage-gated K⁺ channels, and ionotropic glutamate receptors. Recent progress in ion channel research brought about by molecular cloning of the channel subunit proteins and studies in epilepsy models suggest additional targets, including G-protein-coupled receptors, such as GABA_B and metabotropic glutamate receptors; hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel subunits, responsible for hyperpolarization-activated currentI _h; connexins, which make up gap junctions; and neurotransmitter transporters, particularly plasma membrane and vesicular transporters for GABA and glutamate. New information from the structural characterization of ion channels, along with better understanding of ion channel function, may allow for more selective targeting. For example, Na⁺ channels underlying persistent Na⁺ currents or GABA_A receptor isoforms responsible for tonic (extrasynaptic) currents represent attractive targets. The growing understanding of the pathophysiology of epilepsy and the structural and functional characterization of the molecular targets provide many opportunities to create improved epilepsy therapies.     

Electrophysiological studies of NMDA receptors

Recent electrophysiological studies of NMDA receptors and of the associated ion channels (NMDA channels) have revealed five properties of this system: (1) the NMDA channels are blocked by Mg²⁺ in a voltage-dependent way; (2) the NMDA channels are permeable to Ca²⁺ as well as to Na⁺ and K⁺; (3) the NMDA channels may adopt multiple conductance states; some of the minor states (small conductances) resemble the major conductance states opened by non-NMDA agonists; (4) continued exposure to NMDA agonists produces short-term and long-term decreases in the sensitivity of the NMDA system; and (5) glycine potentiates the response to NMDA.     

Single ion channel currents in neuropile glial cells of the leech central nervous system

The patch-clamp technique was used to investigate the activity of single ion channels in neuropile glial (NG) cells in the central nervous system (CNS) of the medicinal leech, Hirudo medicinalis. We found evidence for two distinct Cl^? channels that could be distinguished by their basic electrical properties and their responses to different inhibitors on single ion channels currents. In the Inside-out configuration in symmetrical Cl solutions, these channels showed current-voltage relationships with slight outward rectification, mean conductances of 70 and 80 pS, and reversal potentials near 0 mV. Significant permeability to Na⁺, K⁺, or SO₄^2? could not be detected. The open-state probability of the 70 pS Cl^? channel increased with membrane depolarization, whereas the open-state probability of the 80 pS Cl^? channel was voltage-independent. The application of the stilbene derivative DIDS (100 μM) to the cytoplasmic side of the glial cell membrane blocked both Cl^? channels. The activity of the 70 pS channel was blocked irreversibly by DIDS, whereas the activity of the 80 pS channel reappeared after wash-out of DIDS. Both channels were blocked reversibly by 1 mM Zn²⁺. K⁺ channels could only be observed occasionally in the soma membrane of the NG cells. We have characterized a 60 pS K⁺ channel with a high selectivity for K⁺ over Na⁺. The low density of K⁺ channels in the soma membrane may indicate a non-uniform distribution of this channel type in NG cells. © 1993 Wiley-Liss, Inc.     

Physiology of microglial cells

Microglial cells in culture and in situ express a defined pattern of K⁺ channels, which is distinct from that of other glial cells and neurons. This pattern undergoes defined changes with microglial activation. As expected for a cell with immunological properties, microglia express a variety of cytokine and chemokine receptors, which are linked to the mobilization of Ca²⁺ (cytosolic free calcium) from internal stores. Microglial cells also have the capacity to respond to neuronal activity: they express receptors for the major excitatory receptor glutamate and the main inhibitory receptor GABA (γ-amino butyric acid). By expressing purinergic receptors, microglia can sense astrocyte activity in the form of Ca²⁺ waves. Activation of transmitter receptors can affect cytokine release which is a potential means as to how brain activity can affect immune function.     

Types and activities of voltage-operated calcium channels change during development of rat pituitary neurointermediate lobe

Cultures of pituitary neurointermediate lobe cells were established from rats aged 1, 12, and 42 days to identify the types and assess the activities of Ca²⁺ channels present in melanotropes, glial-like cells, and fibroblasts during development. Day 12 represents the time at which dopaminergic axons have become distributed throughout the lobe, glial cells begin to lose their radial orientation, and melanotropes robustly express the short isoform of the dopamine D₂ receptor. Thus, we studied Ca²⁺ channels in relation to the event of innervation of melanotropes. Real-time fluorescence video microscopy, in the presence of pharmacological agents, which block L-, N-, P-, and T-type channels, was used as an indirect measurement of channel activity. Assessment of cell type was verified by triple-label fluorescence immunohistochemistry. In melanotropes, extracellular Ca²⁺ addition caused Ca²⁺ influx through ω-conotoxin GVIA-sensitive, N-type channels on days 1 and 12 but not on day 42. The K⁺ depolarization induced an increase in intracellular Ca²⁺ concentration in all age-groups. This effect was decreased by nifedipinc, an L-type channel blocker, at all ages, and by ω-agatoxin IVa, a P-type blocker, only on day 42. These results demonstrate that the predominance of N- or P-type channels on melanotropes is age-dependent and can be correlated with other developmental changes. The T-type blocker, NiSO₄, had no effect. In glial-like cells of all ages, extracellular Ca²⁺ addition resulted in an increase in intracellular Ca²⁺ concentration, which was inhibited only by NiSO₄. The percentage of responsive glial-like cells was equally high in days 1 and 12 cultures, then declined by day 42. The K⁺ depolarization had no effect on glial-like cells. Fibroblasts did not respond significantly to extracellular Ca²⁺ or K⁺ depolarization, indicating little detectable activity by this methodology from functional voltage-operated Ca²⁺ channels.     

Inhibition of cell growth and intracellular Ca2+ mobilization in human brain tumor cells by Ca2+ channel antagonists

The effects of various Ca²⁺ channel agonists and antagonists on tumor cell growth were investigated using U-373 MG human astrocytoma and SK-N-MC human neuroblastoma cell lines. Classical Ca²⁺ channel antagonists, verapamil, nifedipine, and diltiazem, and inorganic Ca²⁺ channel antagonists, Ni²⁺ and Co²⁺, inhibited growth of these tumor cells in a dose-dependent manner. Except Ni²⁺, these Ca²⁺ channel antagonists did not induce a significant cytotoxicity, suggesting that the growth-inhibitory effects of these drugs may be the result of the influence on the proliferative signaling mechanisms of these tumor cells. In contrast, Bay K-8644, a Ca²⁺ channel agonist, neither enhanced the growth of tumor cells nor increased intracellular Ca²⁺ concentration, indicating that voltage-sensitive Ca²⁺ channels may not be involved in tumor cell proliferation. Moreover, growth-inhibitory concentrations of Ca²⁺ channel antagonists significantly blocked agonist (carbachol or serum)-induced intracellular Ca²⁺ mobilization, which was monitored using Fura-2 fluorescence technique. These results suggest that the inhibition of the growth of human brain tumor cells induced by Ca²⁺ channel antagonists may not be the result of interaction with Ca²⁺ channels, but may be the result of the interference with agonist-induced intracellular Ca²⁺ mobilization, which is an important proliferative signaling mechanism.     

A proinvasive role for the Ca2+‐activated K+ channel KCa3.1 in malignant glioma

Glioblastoma multiforme are highly motile primary brain tumors. Diffuse tissue invasion hampers surgical resection leading to poor patient prognosis. Recent studies suggest that intracellular Ca²⁺ acts as a master regulator for cell motility and engages a number of downstream signals including Ca²⁺‐activated ion channels. Querying the REepository of Molecular BRAin Neoplasia DaTa (REMBRANDT), an annotated patient gene database maintained by the National Cancer Institute, we identified the intermediate conductance Ca²⁺‐activated K⁺ channels, KCa3.1, being overexpressed in 32% of glioma patients where protein expression significantly correlated with poor patient survival. To mechanistically link KCa3.1 expression to glioma invasion, we selected patient gliomas that, when propagated as xenolines in vivo, present with either high or low KCa3.1 expression. In addition, we generated U251 glioma cells that stably express an inducible knockdown shRNA to experimentally eliminate KCa3.1 expression. Subjecting these cells to a combination of in vitro and in situ invasion assays, we demonstrate that KCa3.1 expression significantly enhances glioma invasion and that either specific pharmacological inhibition with TRAM‐34 or elimination of the channel impairs invasion. Importantly, after intracranial implantation into SCID mice, ablation of KCa3.1 with inducible shRNA resulted in a significant reduction in tumor invasion into surrounding brain in vivo. These results show that KCa3.1 confers an invasive phenotype that significantly worsens a patient's outlook, and suggests that KCa3.1 represents a viable therapeutic target to reduce glioma invasion. GLIA 2014;62:971–981     

Two types of potassium channels in the PC12 cell line

Activation of K⁺ channels in the PC12 cell line was studied by comparing⁸⁶Rb⁺ efflux under depolarizing and non-depolarizing conditions. Evidence for both Ca²⁺-dependent and voltage-dependent K⁺ channels was obtained by studying depolarization-induced⁸⁶Rb⁺ efflux in solutions of varying Ca²⁺ concentration and in the presence of K⁺ and Ca²⁺ channel blocking agents.     

Independent changes of intracellular calcium and pH in identified leech glial cells

The intracellular Ca²⁺ (Ca²⁺_i) and the intracellular pH (pH_i) were measured in identified neuropile glial cells in the central nervous system of the leech Hirudo medicinalis, using the fluorescent dye fura-2, and double-barrelled, neutral carrier, pH-sensitive microelectrodes. Different stimuli were used to elicit Ca²⁺_i and/or pH_i changes, such as application of ammonium, high external K⁺-concentration, and low external pH. Ammonium (20 mM) and high external K⁺ (20 mM), which depolarized the glial membrane by 20–30 mV, evoked rapid and large rises of Ca²⁺_i. In contrast to the Ca²_i changes, amplitude and direction of the pH_i changes were dependent on the presence of CO₂/HCO₃^? in the saline. The addition of CO₂/HCO₃^?, and the subsequent reduction of external pH from 7.4 to 7.0, had no effect on Ca²⁺_i, but caused significant changes of pH_i. The results suggest that the ammonium- and K⁺-induced Ca²⁺_i rises were due to the membrane depolarization leading to a Ca²⁺ influx through voltage-gated Ca²⁺ channels in the glial membrane, while the pH_i changes resulted from movements of ammonia and from the activation or inhibition of the Na⁺-HCO₃^? cotransporter. This indicates that changes of intracellular Ca²⁺ and pH can occur independently of each other, suggesting that the homeostasis of these ions is not necessarily interrelated in these glial cells.     

Adult rat optic nerve oligodendrocyte progenitor cells express a distinct repertoire of voltage- and ligand-gated ion channels

Cultured oligodendrocyte progenitor cells derived from the developing central nervous system (CNS) express a pattern of ion channels that is distinct from mature oligodendrocytes and other cell types of the CNS. In the present study, we used the whole-cell patch-clamp technique and the fura-2-based Ca⁺⁺ imaging system to study the ion channel expression of oligodendrocyte progenitor cells derived from the optic nerves of adult rats. We found that the adult oligodendrocyte progenitor cell membrane is dominated by K⁺ currents, both delayed outward and inward rectifying. The inwardly rectifying K⁺ currents were often as large as the outward delayed rectifying K⁺ currents. The delayed rectifying outward currents were partially blocked by 50 mM tetraethylammonium or 1 mM 4-aminopyridine, but not by 2 or 5 mM BaCl₂. This suggests that the delayed rectifier channels expressed by adult progenitor cells are different from those expressed by perinatal cells. Most adult oligodendrocyte progenitor cells showed no or only small A-type K⁺ currents. Both Ca⁺⁺ and Na⁺ channels were also detected in these cells. Furthermore, adult progenitor cells responded to the neurotransmitters GABA and kainate and the pharmacology of these responses indicated that these cells express GABA_A receptors and kainate receptors that are Ca⁺⁺ -permeable. Our study suggests that adult oligodendrocyte progenitor cells are electrophysiologically distinct and that these cells share electrophysiological characteristics with both perinatal progenitor cells and immature oligodendrocytes. © 1995 Wiley-Liss, Inc.     

Potassium depolarization of mammalian vestibular sensory cells increases [Ca2+]i through voltage‐sensitive calcium channels

The existence of voltage-sensitive Ca²⁺ channels in type I vestibular hair cells of mammals has not been conclusively proven. Furthermore, Ca²⁺ channels present in type II vestibular hair cells of mammals have not been pharmacologically identified. Fura-2 fluorescence was used to estimate, in both cell types, intracellular Ca²⁺ concentration ([Ca²⁺]_i) variations induced by K⁺ depolarization and modified by specific Ca²⁺ channel agonists and antagonists. At rest, [Ca²⁺]_i was 90 ± 20 nm in both cell types. Microperifusion of high-K⁺ solution (50 mm ) for 1 s increased [Ca²⁺]_i to 290 ± 50 nm in type I (n = 20) and to 440 ± 50 nm in type II cells (n = 10). In Ca²⁺-free medium, K⁺ did not alter [Ca²⁺]_i. The specific L-type Ca²⁺ channel agonist, Bay K, and antagonist, nitrendipine, modified in a dose-dependent manner the K⁺-induced [Ca²⁺]_i increase in both cell types with maximum effect at 2 μm and 400 nm , respectively. Ni²⁺, a T-type Ca²⁺ channel blocker, reduced K⁺-evoked Ca²⁺ responses in a dose-dependent manner. For elevated Ni²⁺ concentrations, the response was differently affected by Ni²⁺ alone, or combined to nitrendipine (500 nm ). In optimal conditions, nitrendipine and Ni²⁺ strongly depressed by 95% the [Ca²⁺]_i increases. By contrast, neither ω-agatoxin IVA (1 μm ), a specific P- and Q-type blocker, nor ω-conotoxin GVIA (1 μm ), a specific N-type blocker, affected K⁺-evoked Ca²⁺_i responses. These results provide the first direct evidence that L- and probably T-type channels control the K⁺-induced Ca²⁺ influx in both types of sensory cells.     

Trophic effect of cholera toxin B subunit in cultured cerebellar granule neurons: Modulation of intracellular calcium by GM1 ganglioside

Survival of cerebellar granule cells (CGC) in culture was significantly improved in the presence of cholera toxin B subunit (Ctx B), a ligand which binds to GM1 with specificity and high affinity. This trophic effect was linked to elevation of intracellular calcium ([Ca²⁺]_i), and was additive to that of high K⁺. Survival was optimized when Ctx B was present for several days during the early culture period. ⁴⁵Ca²⁺ and cell survival studies indicated the mechanism to involve enhanced influx of Ca²⁺ through L-type voltage-sensitive channels, since the trophic effect was blocked by antagonists specific for that channel type. Inhibitors of N-methyl-D-aspartate receptor/channels were without effect. During the early stage of culture Ctx B, together with 25 mM K⁺, caused [Ca²⁺]_i to rise to 0.2–0.7 μM in a higher proportion of cells than 25 mM K⁺ alone. A significant change in the nature of GM1 modulation of Ca²⁺ flux occurred after 7 days in culture, at which time Ctx B ceased to elevate and instead reduced [Ca²⁺]_i below the level attained with 25 mM K⁺. GM1 thus appears to serve as intrinsic inhibitor of one or more L-type Ca²⁺ channels during the first 7 days in vitro, and then as intrinsic activator of (possibly other) L-type channels after that period. This is the first demonstration of a modulatory role for GM1 ganglioside affecting Ca²⁺ homeostasis in cultured neurons of the CNS. © 1996 Wiley-Liss, Inc.