Harald Sontheimer

Activity‐dependent extracellular K+ accumulation in rat optic nerve: the role of glial and axonal Na+ pumps

We measured activity‐dependent changes in [K+]o with K+‐selective microelectrodes in adult rat optic nerve, a CNS white matter tract, to investigate the factors responsible for post‐stimulus recovery of [K+]o. Post‐stimulus recovery of [K+]o followed a double‐exponential time course with an initial, fast time constant, τfast, of 0.9 ± 0.2 s (mean ±s.d.) and a later, slow time constant, τslow, of 4.2 ± 1 s following a 1 s, 100 Hz stimulus. τfast, but not τslow, decreased with increasing activity‐dependent rises in [K+]o. τslow, but not τfast, increased with increasing stimulus duration. Post‐stimulus recovery of [K+]o was temperature sensitive. The apparent temperature coefficients (Q10, 27–37°C) for the fast and slow components following a 1 s, 100 Hz stimulus were 1.7 and 2.6, respectively. Post‐stimulus recovery of [K+]o was sensitive to Na+ pump inhibition with 50 μM strophanthidin. Following a 1 s, 100 Hz stimulus, 50 μM strophanthidin increased τfast and τslow by 81 and 464%, respectively. Strophanthidin reduced the temperature sensitivity of post‐stimulus recovery of [K+]o. Post‐stimulus recovery of [K+]o was minimally affected by the K+ channel blocker Ba2+ (0.2 mm). Following a 10 s, 100 Hz stimulus, 0.2 mm Ba2+ increased τfast and τslow by 24 and 18%, respectively. Stimulated increases in [K+]o were followed by undershoots of [K+]o. Post‐stimulus undershoot amplitude increased with stimulus duration but was independent of the peak preceding [K+]o increase. These observations imply that two distinct processes contribute to post‐stimulus recovery of [K+]o in central white matter. The results are compatible with a model of K+ removal that attributes the fast, initial phase of K+ removal to K+ uptake by glial Na+ pumps and the slower, sustained decline to K+ uptake via axonal Na+ pumps.

A role for ion channels in glioma cell invasion

Many cells, including neuronal and glial progenitor cells, stem cells and microglial cells, have the capacity to move through the extracellular spaces of the developing and mature brain. This is particularly pronounced in astrocyte-derived tumors, gliomas, which diffusely infiltrate the normal brain. Although a significant body of literature exists regarding signals that are involved in the guidance of cells and their processes, little attention has been paid to cell-shape and cell-volume changes of migratory cells. However, extracellular spaces in the brain are very narrow and represent a major obstacle that requires cells to dynamically regulate their volume. Recent studies in glioma cells show that this involves the secretion of Cl(-) and K(+) with water. Pharmacological inhibition of Cl(-) channels impairs their ability to migrate and limits tumor progression in experimental tumor models. One Cl(-)-channel inhibitor, chlorotoxin, is currently in Phase II clinical trials to treat malignant glioma. This article reviews our current knowledge of cell-volume changes and the role of ion channels during the migration of glioma cells. It also discusses evidence that supports the importance of channel-mediated cell-volume changes in the migration of immature neurons and progenitor cells during development. New unpublished data is presented, which demonstrates that Cl(-) and K(+) channels involved in cell shrinkage localize to lipid-raft domains on the invadipodia of glioma cells and that their presence might be regulated by trafficking of these proteins in and out of lipid rafts.

Anion channels in astrocytes: Biophysics, pharmacology, and function

The chloride/anion channels that have been so far identified in cultured astrocytes and those that have been confirmed in situ by a combination of mRNA identification, immunocytochemistry, and biophysical studies are reviewed. It is emphasized that we are just beginning to describe such channels and analyze their functions in astrocytes. The best‐studied anion channels studied so far are those known as volume‐regulated anion channels (VRACs). These, as for most channels, have been mainly studied in cultured astrocytes, but some correlative studies have been done in situ, because these channels have been emphasized as release routes for transmitters; namely, excitatory amino acids and ATP. They are activated by cell shape changes and cell swelling, and the release of amino acids and ATP and chloride currents, measured by whole cell clamping, by these processes has been well described, as is also their activation by low concentrations of extracellular ATP. However, the identity of these channels in astrocytes, as in all other cells, remains elusive. The potential involvement of VRACs in pathological states such as stroke, metastasis, and spreading depression is also discussed.

Expression and function of calcium-activated potassium channels in human glioma cells.

Ca2+‐activated K+ (KCa) channels are a unique family of ion channels because they are capable of directly communicating calcium signals to changes in cell membrane potential required for cellular processes including but not limited to cellular proliferation and migration. It is now possible to distinguish three families of KCa channels based on differences in their biophysical and pharmacological properties as well as genomic sequence. Using a combination of biochemical, molecular, and biophysical approaches, we show that human tumor cells of astrocytic origin, i.e. glioma cells, express transcripts for all three family members of KCa channels including BK, IK, and all three SK channel types (SK1, SK2, and SK3). The use of selective pharmacological inhibitors shows prominent expression of currents that are inhibited by the BK channel specific inhibitors iberiotoxin and paxilline. However, despite the presence of transcripts for IK and SK, neither clotrimazole, an inhibitor of IK channels, nor apamin, known to block most SK channels inhibited any current. The exclusive expression of functional BK channels was further substantiated by shRNA knockdown experiments, which selectively reduced iberiotoxin sensitive currents. Western blotting of patient biopsies with antibodies specific for all three KCa channel types further substantiated the exclusive expression of BK type KCa channels in vivo. This finding is in sharp contrast to other cancers that express primarily IK channels.

Chloride accumulation drives volume dynamics underlying cell proliferation and migration.

During brain development, progenitor cells migrate over long distances through narrow and tortuous extracellular spaces posing significant demands on the cells ability to alter cell volume. This phenotype is recapitulated in primary brain tumors. We demonstrate here that volume changes occurring spontaneously in these cells are mediated by the flux of Cl- along with obligated water across the cell membrane. To do so, glioma cells accumulate Cl- to approximately 100 mM, a concentration threefold greater than predicted by the Nernst equation. Shunting this gradient through the sustained opening of exogenously expressed GABA-gated Cl- channels caused a 33% decrease in cell volume and impaired the ability of cells to migrate in a spatially constrained environment. Further, dividing cells condense their cytoplasm prior to mitosis, a phenomenon which is associated with the release of intracellular Cl- as indicated by a 40-mM decrease in [Cl-]i. These findings provide a new framework for considering the role of intracellular Cl- in glioma cells. Here, Cl- serves as an important osmotically active regulator of cell volume being the energetic driving force for volume changes required by immature cells in cell migration and proliferation. This mechanism that was studied in CNS malignancies may be shared with other immature cells in the brain as well.

Mislocalization of Kir channels in malignant glia

Inwardly rectifying potassium (Kir) channels are a prominent feature of mature, postmitotic astrocytes. These channels are believed to set the resting membrane potential near the potassium equilibrium potential (EK) and are implicated in potassium buffering. A number of previous studies suggest that Kir channel expression is indicative of cell differentiation. We therefore set out to examine Kir channel expression in malignant glia, which are incapable of differentiation. We used two established and widely used glioma cell lines, D54MG (a WHO grade 4 glioma) and STTG‐1 (a WHO grade 3 glioma), and compared them to immature and differentiated astrocytes. Both glioma cell lines were characterized by large outward K+ currents, depolarized resting membrane potentials (Vm) (−38.5 ± 4.2 mV, D54 and −28.1 ± 3.5 mV, STTG1), and relatively high input resistances (Rm) (260.6 ± 64.7 MΩ, D54 and 687.2 ± 160.3 MΩ, STTG1). These features were reminiscent of immature astrocytes, which also displayed large outward K+ currents, had a mean Vm of −51.1 ± 3.7 and a mean Rm value of 627.5 ± 164 MΩ. In contrast, mature astrocytes had a significantly more negative resting membrane potential (−75.2 ± 0.56 mV), and a mean Rm of 25.4 ± 7.4 MΩ. Barium (Ba2+) sensitive Kir currents were >20‐fold larger in mature astrocytes (4.06 ± 1.1 nS/pF) than in glioma cells (0.169 ± 0.033 nS/pF D54, 0.244 ± 0.04 nS/pF STTG1), which had current densities closer to those of dividing, immature astrocytes (0.474 ± 0.12 nS/pF). Surprisingly, Western blot analysis shows expression of several Kir channel subunits in glioma cells (Kir2.3, 3.1, and 4.1). However, while in astrocytes these channels localize diffusely throughout the cell, in glioma cells they are found almost exclusively in either the cell nucleus (Kir2.3 and 4.1) or ER/Golgi (3.1). These data suggest that mislocalization of Kir channel proteins to intracellular compartments is responsible for a lack of appreciable Kir currents in glioma cells.

BK channels in human glioma cells have enhanced calcium sensitivity

We have previously demonstrated the expression of large‐conductance, calcium‐activated potassium (BK) channels in human glioma cells. In the present study, we characterized the calcium sensitivity of glioma BK channels in excised membrane patches. Channels in inside‐out patches were activated at −60 mV by 2.1 × 10−6 M cytosolic Ca2+, were highly K+‐selective, and had a slope conductance of ≈210 pS. We characterized the Ca2+ sensitivity of these channels in detail by isolating BK currents in outside‐out patches with different free [Ca2+]i. The half‐maximal voltage for channel activation, V0.5, of glioma BK currents in outside‐out patches was +138 mV with 0 Ca2+/10 EGTA. V0.5 was shifted to +81 mV and −14 mV with free [Ca2+]i of 1.5 × 10−7 M and 2.1 × 10−6 M, respectively. These results suggest that glioma BK channels have a higher Ca2+ sensitivity than that described in many other human preparations. Data obtained from a cloned BK channel (hbr5) expressed in HEK cells support the conclusion that glioma BK channels have an unusually high sensitivity to calcium. In addition, the sensitivity of glioma BK channels to the BK inhibitor tetrandrine suggests the expression of BK channel auxiliary β‐subunits by glioma cells. Expression of the auxiliary β‐subunit of BK channels by glioma cells may relate to the high Ca2+ sensitivity of glioma BK channels. GLIA 38:281–291, 2002.

Muscarinic activation of BK channels induces membrane oscillations in glioma cells and leads to inhibition of cell migration.

Abstract. Patients with cerebral tumors often present with elevated levels of acetylcholine (ACh) in their cerebrospinal fluid. This motivated us to investigate physiological effects of ACh on cultured human astrocytoma cells (U373) using a combination of videomicroscopy, calcium microspectrofluorimetry and perforated patch-clamp recording. Astrocytoma cells exhibited the typical morphological changes associated with cell migration; polarized cells displayed prominent lamellipodia and associated membrane ruffling at the anterior of the cell, and a long tail region that periodically contracted into the cell body as the cell moved forward. Bath application of the ACh receptor agonist, muscarine, reversibly inhibited cell migration. In conjunction with this inhibition, ACh induced a dose-dependent, biphasic increase in resting intracellular free calcium concentration ([Ca2+]i) associated with periodic Ca2+ oscillations during prolonged ACh applications. The early transient rise in [Ca2+]i was abolished by ionomycin and thapsigargin but was insensitive to caffeine and ryanodine while the plateau phase was strictly dependent on external calcium. The Ca2+ response to ACh was mimicked by muscarine and abolished by the muscarinic antagonists, atropine or 4-DAMP, but not by pirenzepine. Using perforated patch-clamp recordings combined with fluorescent imaging, we demonstrated that ACh-induced [Ca2+]i oscillations triggered membrane voltage oscillations that were due to the activation of voltage-dependent, Ca2+-sensitive K+ currents. These K+ currents were blocked by intracellular injection of EGTA, or by extracellular application of TEA, quinine, or charybdotoxin, but not by apamin. These studies suggest that activation of muscarinic receptors on glioma cells induce the release of Ca2+ from intracellular stores which in turn activate Ca2+-dependent (BK-type) K+ channels. Furthermore, this effect was associated with inhibition of cell migration, suggesting an interaction of this pathway with glioma cell migration.

Ion channels and amino acid transporters support the growth and invasion of primary brain tumors

The malignant growth of glial support cells causes gliomas, highly invasive, primary brain tumors that are largely resistant to therapy. Individual tumor cells spread by active cell migration, invading diffusely into the normal brain. This process is facilitated by Cl− channels that endow glioma cells with an enhanced ability to quickly adjust their shape and cell volume to fit the narrow and tortuous extracellular brain spaces. Once satellite tumors enlarge, their growth is limited by the spatial constraints imposed by the bony cavity of the skull and spinal column. Glioma cells circumvent this limitation by active destruction of peritumoral neural tissue through the release of glutamate, inducing peritumoral seizures and ultimately excitotoxic neuronal cell death. Hence, primary brain tumors support their unusual biology by taking advantage of ion channels and transporters that are designed to support ion homeostatic functions in normal brain.

Chemotaxis of MDCK-F cells toward fibroblast growth factor-2 depends on transient receptor potential canonical channel 1

Movement toward the source of a chemoattractant gradient is a basic cellular property in health and disease. Enhanced migration during metastasis involves deregulated growth factor signaling. Growth factor stimulation and cell migration converge both on the important second messenger Ca2+. To date, the molecular identification of Ca2+ entry pathways activated by growth factors during chemotaxis is still an open issue. We investigated the involvement of the nonselective Ca2+ channel TRPC1 (transient receptor potential canonical 1) in FGF-2 guided chemotaxis by means of time-lapse video microscopy and by functional Ca2+ measurements. To specifically address TRPC1 function in transformed MDCK cells we altered the expression levels by siRNA or overexpression. We report that TRPC1 channels are required for the orientation of transformed MDCK cells in FGF-2 gradients because TRPC1 knockdown or pharmacological blockade prevented chemotaxis. Stimulation with FGF-2 triggered an immediate Ca2+ influx via TRPC1 channels that depended on phospholipase C and phosphatidylinositol 3-kinase signaling. Impeding this Ca2+ influx abolished chemotaxis toward FGF-2. This functional connection correlated with clustering of FGF receptors and TRPC1 channels as was observed by immunolabeling. These findings show the important interplay between growth factor signaling and Ca2+ influx in chemotaxis.