Separate, Ca2+-activated K+ and Cl− transport pathways in Ehrlich ascites tumor cells (original) (raw)
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Ca2+-dependent K+ transport in the Ehrlich ascites tumor cell
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1982
The possible presence and properties of the Ca2+-dependent K + channel have been investigated in the Ehrlich ascites tumor cell. The treatment with ionophore A23187+Ca 2+, propranolol or the electron donor system ascorbate-pbenazine methosulphate, all of which activate that transport system in the human erythrocyte, produces in the Ehrlich cell a net loss of K + (balanced by the uptake of Na t) and a stimulation of both the influx and the efflux of s6 Rb. These effects were antagonized by quinine, a known inhibitor of the Ca2+-dependent K ÷ channel in other cell systems, and by the addition of EGTA to the incubation medium. Ouabain did not have an inhibitory effect. These results suggests that the Ehrlich cell posseses a Ca2+-dependent K ÷ channel whose characteristics are similar to those described in other cell systems.
The Journal of Membrane Biology, 1984
Ehrlich ascites tumor cells resuspended in hypotonic medium initially swell as nearly perfect osmometers, but subsequently recover their volume within 5 to 10 min with an associated KCI loss. 1. The regulatory volume decrease was unaffected when nitrate was substituted for C1-, and was insensitive to bumetanide and DIDS. 2. Quinine, an inhibitor of the Ca 2 § activated K + pathway, blocked the volume recovery. 3. The hypotonic response was augmented by addition of the Ca 2 + ionophore A23187 in the presence of external Ca z+, and also by a sudden increase in external Ca z+. The volume response was accelerated at alkaline pH. 4. The anti-calmodulin drugs trifluoperazine, pimozide, flupentixol, and chlorpromazine blocked the volume response. 5. Depletion of intracellular Ca z + stores inhibited the regulatory volume decrease. 6. Consistent with the low conductive C1 -permeability of the cell membrane there was no change in cell volume or C1 content when the K + permeability was increased with valinomycin in isotonic medium. In contrast, addition of the Ca z+ ionophore A23187 in isotonic medium promoted CI-loss and cell shrinkage. During regulatory volume decrease valinomycin accelerated the net loss of KC1, indicating that the conductive C1-permeability was increased in parallel with and even more than the K + permeability. It is proposed that separate conductive K + and C1-channels are activated during regulatory volume decrease by reiease of Ca z+ from internal stores, and that the effect is mediated by calmodulin.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1996
Submembrane [Ca2+]i changes were examined in rat chromaffin cells by monitoring the activity of an endogenous Ca(2+)-dependent protein: the large conductance Ca(2+)-and voltage-activated K+ channel (also known as the BK channel). The Ca2+ and voltage dependence of BK current inactivation and conductance were calibrated first by using defined [Ca2+]i salines. This information was used to examine submembrane [Ca2+]i elevations arising out of Ca2+ influx and muscarine-mediated release of Ca2+ from intracellular stores. During Ca2+ influx, some BK channels are exposed to [Ca2+]i of at least 60 microM. However, the distribution of this [Ca2+]i elevation is highly nonuniform so that the average [Ca2+]i detected when all BK channels are activated is only approximately 10 microM. Intracellular dialysis with 1 mM or higher EGTA spares only the BK channels activated by the highest [Ca2+]i during influx, whereas dialysis with 1 mM or higher BAPTA blocks activation of all BK channels. Submembra...
The Journal of Physiology, 1993
1. To study Ca2"-activated K' currents in dimethyl sulphoxide (DMSO)differentiated HL-60 cells (HL-60 granulocytes), we have combined the patch clamp technique with microfluorimetric measurements of the cytosolic free Ca2+ concentration ([Ca2+]i). 2. Elevations of [Ca2+]i induced by the receptor agonist N-formyl-L-methionyl-Lphenylalanine (f-MLP), by cellular spreading or by the Ca2+ ionophore ionomycin, activated whole-cell currents. The kinetics of the current elevations closely paralleled the kinetics of the elevations in [Ca2+]i. Cellular spreading induced oscillations in [Ca2 ]i and parallel oscillatory changes in the amplitude of the recorded currents. 3. The reversal potential of the Ca2+-activated current was a function of the extracellular K+ concentration (56-1 mV per log [K+]), demonstrating that the underlying conductance was selective for K+. 4. The current was blocked by charybdotoxin, but insensitive to apamin. 5. The whole-cell current was inwardly rectifying. No time-dependent activation or inactivation of the current could be observed within the range of voltages tested (-100 to + 100 mV). 6. The dependence of the current amplitude on the measured [Ca2+]i revealed a half-maximal activation at approximately 350 nm [Ca2+]i, and a highly cooperative activation by [Ca2+]i with an apparent Hill coefficient of approximately 8. Neither the half-maximal activation by [Ca2+]i nor the apparent Hill coefficient depended on the voltage, and they were identical for Ca2+ elevations caused by the ionophore and the receptor agonist. 7. Analysis of Ca2+-activated single-channel events in cell-attached recordings revealed an inwardly rectifying K+ channel with a slope conductance of 35 pS. Fluctuation analysis of the Ca2+-activated whole-cell current suggested an underlying single-channel conductance of a similar size (28 pS).
AJP: Cell Physiology, 2004
Volume changes and whole cell ionic currents activated by gradual osmolarity reductions (GOR) of 1.8 mosM/min were characterized in C6 glioma cells. Cells swell less in GOR than after sudden osmolarity reductions (SOR), the extent of swelling being partly Ca(2+) dependent. In nominally Ca(2+)-free conditions, GOR activated predominantly whole cell outward currents. Cells depolarized from the initial -79 mV to a steady state of -54 mV reached at 18% osmolarity reduction [hyposmolarity of -18% (H-18%)]. Recordings of Cl(-) and K(+) currents showed activation at H-3% of an outwardly rectifying Cl(-) current, with conductance of 1.6 nS, sensitive to niflumic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid, followed at H-18% by an outwardly rectifying K(+) current with conductance of 4.1 nS, inhibited by clofilium but insensitive to the typical K(+) channel blockers. With 200 nM Ca(2+) in the patch pipette, whole cell currents activated at H-3% and at H-13% cells depolarized from -77 to -63 mV. A K(+) current activated at H-1%, showing a rapid increase in conductance, suppressed by charybdotoxin and insensitive to clofilium. These results show the operation of two different K(+) channels in response to GOR in the same cell type, activated by Ca(2+) and osmolarity and with different osmolarity activation thresholds. Taurine and glutamate efflux, monitored by labeled tracers, showed delayed osmolarity thresholds of H-39 and H-33%, respectively. This observation clearly separates the Cl(-) and amino acid osmosensitive pathways. The delayed amino acid efflux may contribute to counteract swelling at more stringent osmolarity reductions.
Separate Swelling- and Ca 2+ -activated Anion Currents in Ehrlich Ascites Tumor Cells
Journal of Membrane Biology, 1998
A Ca 2+ -activated (I Cl,Ca ) and a swellingactivated anion current (I Cl,vol ) were investigated in Ehrlich ascites tumor cells using the whole cell patch clamp technique. Large, outwardly rectifying currents were activated by an increase in the free intracellular calcium concentration ([Ca 2+ ] i ), or by hypotonic exposure of the cells, respectively. The reversal potential of both currents was dependent on the extracellular Cl − concentration. I Cl,Ca current density increased with increasing [Ca 2+ ] i , and this current was abolished by lowering [Ca 2+ ] i to <1 nM using 1,2-bis-(o-aminophenoxy)ethane-N,N,NЈ,NЈ-tetra-acetic acid (BAPTA). In contrast, activation of I Cl,vol did not require an increase in [Ca 2+ ] i . The kinetics of I Cl,Ca and I Cl,vol were different: at depolarized potentials, I Cl,Ca as activated in a [Ca 2+ ] i -and voltage-dependent manner, while at hyperpolarized potentials, the current was deactivated. In contrast, I Cl,vol exhibited time-and voltage-dependent deactivation at depolarized potentials and reactivation at hyperpolarized potentials. The deactivation of I Cl,vol was dependent on the extracellular Mg 2+ concentration. The anion permeability sequence for both currents was I − > Cl − > gluconate. I Cl,Ca was inhibited by niflumic acid (100 M), 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 M) and 4,4Ј-diisothiocyano-2,2Ј-stilbenedisulfonic acid (DIDS, 100 M), niflumic acid being the most potent inhibitor. In contrast, I Cl,vol was unaffected by niflumic acid (100 M), but abolished by tamoxifen (10 M). Thus, in Ehrlich cells, separate chloride currents, I Cl,Ca and I Cl,vol , are activated by an increase in [Ca 2+ ] i and by cell swelling, respectively.
Interactions among calcium compartments in C6 rat glioma cells: involvement of potassium channels
The Journal of Physiology, 1994
1. Variations in intracellular free Ca2+ concentration ([Ca2+]1) induced by alteration of the extracellular concentrations of Ca2+ ([Ca2+]0) and K+ ([K+]O) were imaged in single fluo-3-loaded C6 glioma cells. In addition, the effect of membrane potential on [Ca2+] was investigated in fura-2-loaded, voltage-clamped cells. 2. Step alterations of [Ca2+]o from 0 to 10 mm were followed by proportional variations in [Ca2+]i, with a maximum 7-fold increase and an apparent half-maximum at [Ca2+]o of 1P5 mM. 3. The time to half-maximum change (t%) of [Ca2+]0-associated [Ca2"I variations ranged between 10 and 50 s, and was inversely related to the amplitude of [Ca2]O steps. 4. Transient, serotonin-induced [Ca2+]i elevations, used as a measure of Ca2' availability in inositol 1,4,5-trisphosphate-sensitive stores, were diminished within 10 min in 0 mM [Ca2+]O, but were unaffected by [Ca2+]o changes in the 1-5 mm range. 5. Restoration of normal [Ca21]i following its elevation by serotonin was delayed by removal of external Na+ or Cland was enhanced by warming the medium to 37°C. These conditions did not affect [Ca2+]0-associated [Ca2+]i variations. 6. [Ca2+]O-associated [Ca2+]j variations were depressed by La3+ and Ba2+, while blockers of voltage-activated Ca2+ channels were ineffective. 7. Elevated [K+]O depressed the basal level of [Ca21]1, and in high concentrations (70-140 mM) also diminished the response to serotonin. 8. Depolarizing the membrane potential of voltage-clamped cells reversibly reduced [Ca2+]j. These membrane-potential associated [Ca2+]i variations were blocked by La3+, Ba2`and TEA, all of which also depolarized membrane resting potential. 9. Apamin (at 1-10 /bM), a blocker of [Ca2+]1-activated K+ channel, totally and reversibly prevented [Ca2+]O-associated [Ca2+]i variations. 10. These studies indicate that C6 cells are responsive to variations in [Ca2i]0, and that a K+ channel is a possible path through which Ca2`penetrates into the cell. Free cytosolic Ca21 concentration ([Ca21]i) can increase transiently following binding of ligands to receptors, which activates a cascade of events leading to release of Ca2+ from internal stores (reviewed by Berridge & Irvine,