RESUMEN
We have previously demonstrated the expression of BK channels in human glioma cells. There was a curious feature to the whole-cell currents of glioma cells seen during whole-cell patch-clamp: large, outward current transients accompanied repolarization of the cell membrane following an activating voltage step. This transient current, Itransient, activated and inactivated rapidly (approximately 1 ms). The I-V relationship of Itransient had features that were inconsistent with simple ionic current through open ion channels: (i) Itransient amplitude peaked with a -80 mV voltage change and was invariant over a 200 mV range, and (ii) Itransient remained large and outward at -140 mV. We provide evidence for a direct relationship of Itransient to glioma BK currents. They had an identical time course of activation, identical pharmacology, identical voltage-dependence, and small, random variations in the amplitude of the steady-state BK current and Itransient seen over time were often perfectly in phase. Substituting intracellular K+ with Cs+, Li+, or Na+ ions reversibly reduced Itransient and BK currents. Itransient was not observed in recordings of other BK currents (hbr5 expressed in HEK cells and BK currents in rat neurons), suggesting Itransient is unique to BK currents in human glioma cells. We conclude that Itransient is generated by a mechanism related to the deactivation, and level of prior activation, of glioma BK channels. To account for these findings we propose that K+ ions are "trapped" within glioma BK channels during deactivation and are forced to exit to the extracellular side in a manner independent of membrane potential.
Asunto(s)
Membrana Celular/metabolismo , Glioma/fisiopatología , Activación del Canal Iónico , Riñón/fisiología , Potenciales de la Membrana , Canales de Potasio Calcio-Activados/metabolismo , Potasio/metabolismo , Adaptación Fisiológica , Transporte Biológico Activo , Línea Celular Tumoral , Humanos , Riñón/embriología , Canales de Potasio de Gran Conductancia Activados por el CalcioRESUMEN
We used an in vitro model for glioma cell invasion (transwell migration assay) and patch-clamp techniques to investigate the role of volume-activated Cl(-) currents (I(Cl,Vol)) in glioma cell invasion. Hypotonic solutions ( approximately 230 mOsm) activated outwardly rectifying currents that reversed near the equilibrium potential for Cl(-) ions (E(Cl)). These currents (I(Cl,Vol)) were sensitive to several known Cl(-) channel inhibitors, including DIDS, tamoxifen, and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). The IC(50) for NPPB inhibition of I(Cl,Vol) was 21 microm. Under isotonic conditions, NPPB (165 microm) blocked inward currents (at -40 mV) and increased input resistance in both standard whole-cell recordings and amphotericin perforated-patch recordings. Reducing [Cl(-)](o) under isotonic conditions positively shifted the reversal potential of whole-cell currents. These findings suggest a significant resting Cl(-) conductance in glioma cells. Under isotonic and hypotonic conditions, Cl(-) channels displayed voltage- and time-dependent inactivation and had an I(-) > Cl(-) permeability. To assess the potential role of these channels in cell migration, we studied the chemotactic migration of glioma cells toward laminin or vitronectin in a Boyden chamber containing transwell filters with 8 microm pores. Inhibition of I(Cl,Vol) with NPPB reduced chemotactic migration in a dose-dependent fashion with an IC(50) of 27 microm. Time-lapse video microscopy during patch-clamp recordings revealed visible changes in cell shape and/or movement that accompanied spontaneous activation of I(Cl,Vol), suggesting that I(Cl,Vol) is activated during cell movement, consistent with the effects of NPPB in migration assays. We propose that I(Cl,Vol) contributes to cell shape and volume changes required for glioma cell migration through brain tissue.
Asunto(s)
Movimiento Celular , Canales de Cloruro/metabolismo , Glioma/metabolismo , Glioma/patología , Invasividad Neoplásica/fisiopatología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Canales de Cloruro/antagonistas & inhibidores , Cámaras de Difusión de Cultivos , Relación Dosis-Respuesta a Droga , Electrofisiología , Humanos , Laminina/farmacología , Microscopía por Video/métodos , Nitrobenzoatos/farmacología , Técnicas de Placa-Clamp , Tamoxifeno/farmacología , Células Tumorales Cultivadas , Vitronectina/farmacologíaRESUMEN
Ion channels in inexcitable cells are involved in proliferation and volume regulation. Glioma cells robustly proliferate and undergo shape and volume changes during invasive migration. We investigated ion channel expression in two human glioma cell lines (D54MG and STTG-1). With low [Ca2+]i, both cell types displayed voltage-dependent currents that activated at positive voltages (more than +50 mV). Current density was sensitive to intracellular cation replacement with the following rank order; K+ > Cs+ approximately = Li+ > Na+. Currents were >80% inhibited by iberiotoxin (33 nM), charybdotoxin (50 nM), quinine (1 mM), tetrandrine (30 microM), and tetraethylammonium ion (TEA; 1 mM). Extracellular phloretin (100 microM), an activator of BK(Ca2+) channels, and elevated intracellular Ca2+ negatively shifted the I-V curve of whole cell currents. With 0, 0.1, and 1 microM [Ca2+]i, the half-maximal voltages, V(0.5), for whole cell current activation were +150, +65, and +12 mV, respectively. Elevating [K+]o potentiated whole cell currents in a fashion proportional to the square-root of [K+]o. Recording from cell-attached patches revealed large conductance channels (150-200 pS) with similar voltage dependence and activation kinetics as whole cell currents. These data indicate that human glioma cells express large-conductance, Ca2+ activated K+ (BK) channels. In amphotericin-perforated patches bradykinin (1 microM) activated TEA-sensitive currents that were abolished by preincubation with bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM). The BK channels described here may influence the responses of glioma cells to stimuli that increase [Ca2+]i.
Asunto(s)
Glioma/fisiopatología , Canales de Potasio Calcio-Activados , Canales de Potasio/fisiología , Bradiquinina/farmacología , Calcio/metabolismo , Electrofisiología , Glioma/patología , Humanos , Membranas Intracelulares/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio , Concentración Osmolar , Potasio/metabolismo , Canales de Potasio/efectos de los fármacos , Estimulación Química , Células Tumorales CultivadasRESUMEN
1. 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. 2. Post-stimulus recovery of [K+]o followed a double-exponential time course with an initial, fast time constant, tau fast, of 0.9 +/- 0.2 s (mean +/- S.D.) and a later, slow time constant, tau slow, of 4.2 +/- 1 s following a 1 s, 100 Hz stimulus. tau fast, but not tau slow, decreased with increasing activity-dependent rises in [K+]o. tau slow, but not tau fast, increased with increasing stimulus duration. 3. Post-stimulus recovery of [K+]o was temperature sensitive. The apparent temperature coefficients (Q10, 27-37 degrees C) for the fast and slow components following a 1 s, 100 Hz stimulus were 1.7 and 2.6, respectively. 4. Post-stimulus recovery of [K+]o was sensitive to Na+ pump inhibition with 50 microM strophanthidin. Following a 1 s, 100 Hz stimulus, 50 microM strophanthidin increased tau fast and tau slow by 81 and 464%, respectively. Strophanthidin reduced the temperature sensitivity of post-stimulus recovery of [K+]o. 5. 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 tau fast and tau slow by 24 and 18%, respectively. 6. 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. 7. 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.
Asunto(s)
Axones/metabolismo , Espacio Extracelular/metabolismo , Neuroglía/metabolismo , Nervio Óptico/metabolismo , Potasio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Bario/farmacología , Técnicas de Cultivo , Estimulación Eléctrica , Masculino , Microelectrodos , Neuroglía/citología , Nervio Óptico/citología , Nervio Óptico/efectos de los fármacos , Bloqueadores de los Canales de Potasio , Canales de Potasio/metabolismo , Ratas , Ratas Long-Evans , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Estrofantidina/farmacología , Temperatura , Factores de TiempoRESUMEN
1. Two subtypes of astrocytes that expressed distinctly different ion channel complements were identified in primary cultures from rat spinal cord and hippocampus using whole cell patch-clamp techniques. One population of cells expressed voltage-activated Na+ currents and displayed outwardly rectifying I-V relationships; the other group of cells had no detectable Na+ currents and pronounced inwardly rectifying I-V curves. 2. Astrocytes expressing Na+ currents were hyperpolarized (by approximately 7 mV) upon removal of external sodium, suggesting a resting Na+ conductance in these cells. In contrast, cells expressing primarily inwardly rectifying K+ currents, Kir, depolarized (by approximately 4-6 mV) in low-sodium solutions. 3. Removal of external Na+ ions increased the input resistance (189% of control) and reduced the whole cell current amplitude (60% of control at -120 mV) of cells with Kir. The reduction in current amplitude was dose-dependent and became apparent after a 10% reduction of [Na+]0 in 7/7 cells tested. At -120 mV, the effect was near maximal in response to a 50% reduction of [Na+]0. 4. The outward potassium currents of cells expressing Na(+)-currents were unaffected by removal of bath Na+. 5. We conclude that the conductance of glial inwardly rectifying K+ channels is dependent on external sodium ions via a mechanism that does not involve sodium ion permeation or blockade of these channels.
Asunto(s)
Astrocitos/fisiología , Hipocampo/fisiología , Canales de Potasio/fisiología , Sodio/fisiología , Médula Espinal/fisiología , Animales , Conductividad Eléctrica , Hipocampo/citología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Médula Espinal/citologíaRESUMEN
1. Whole cell and cell-attached patch-clamp recordings were obtained from rat spinal cord astrocytes maintained in culture for 6-14 days. It was found that the resting conductance in these astrocytes is primarily due to inwardly rectifying K+ (Kir) channels. 2. Two types of astrocytic Kir channels were identified with single-channel conductances of approximately 28 and approximately 80 pS, respectively. Channels displayed some voltage dependence in their open probability, which was largest (0.8-0.9) near the K+ equilibrium potential (Ek) and decreased at more negative potentials. The resting potential closely followed Ek, so it can be assumed that Kir channels have a high open probability at the resting potential. 3. The conductance of inwardly rectifying K+ currents (Kir) depended strongly on [K+]o and was approximately proportional to the square-root of [K+]o. 4. Kir currents inactivated in a time- and voltage-dependent manner. The Na+ dependence of inactivation was studied with ion substitution experiments. Replacement of [Na+]o with choline or Li+ removed inactivation. This dependence of current inactivation on [Na+]o resembles the previously described block of Kir channels in other systems by [Na+]o. 5. Kir currents were also blocked in a dose-dependent manner by Cs+ (Kd = 189 microM at -140 mV), Ba2+ (Kd = 3.5 microM), and tetraethylammonium (TEA; 90% block at 10 mM) but were insensitive to 4-aminopyridine (4-AP; 5 mM). In the current-clamp mode, Ba2+ and TEA inhibition of Kir currents was associated with a marked depolarization, suggesting that Kir channel activity played a role in the establishment of the negative resting potential typical of astrocytes. 6. These biophysical features of astrocyte inwardly rectifying K+ channels are consistent with those properties required for their proposed involvement in [K+]o clearance: 1) high open probability at the resting potential, 2) increasing conductance with increasing [K+]o, and 3) rectification, e.g., channel closure, at positive potentials. It is proposed, therefore, that the dissipation of [K+]o following neuronal activity is mediated primarily by the activity of astrocytic Kir channels.