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Chloride channels in mast cells: block by DIDS and role in exocytosis.

In rat peritoneal mast cells, we have investigated the influence of the chloride transport blocker 4,4'-diisothiocyanato-stilbene-2,2'- disulfonic acid (DIDS) and the extracellular chloride concentration on the chloride current induced by intracellular application of cyclic AMP (cAMP) and on hexosaminidase secretion from intact cells stimulated with compound 48/80. The inhibition of the Cl-current by extracellular DIDS is voltage and time dependent. Upon depolarization from -10 to +70 mV, the outward current diminishes with millisecond kinetics. The size of the steady state current and the time constant of the decrease both decrease with increasing DIDS concentrations. The steady state current at +70 mV is blocked by DIDS with an IC50 of 2.3 microM. The number of open channels at -10 mV is reduced with an IC50 of 22 microM. The electrophysiological and pharmacological properties of this current are most similar to those of the Cl- current in T lymphocytes activated by osmotic stress (Lewis, R. S., P. E. Ross, and M. D. Cahalan. 1993. Journal of General Physiology. 101:801-826). Extracellular DIDS also inhibits exocytosis. At optimal stimulation with 10 micrograms/ml compound 48/80 secretion is inhibited with an IC50 = 50 microM and a Hill coefficient n = 10. At half optimal stimulation with 1 microgram/ml inhibition occurs with an IC50 = 10 microM and n = 1. Substitution of extracellular chloride by glutamate has only very small effects on secretion stimulated with 10 micrograms/ml compound 48/80. We conclude that activation of the chloride current in mast cells is not essential for stimulation of exocytosis but may enhance secretion at suboptimal stimulation. Alternatively, the channel may play a role in volume regulation following degranulation.


INTRODUCTION
In mast cells, exocytosis is stimulated via activation of GTP-binding proteins (for review see Lindau and Gomperts, 1991). Although large calcium transients occur in response to stimulation by extracellular application of compound 48/80 (Almers and Neher, 1985) or intracellular application of GTP~/S (Neher, 1988), the significance of the calcium change for activation of secretion has been questioned because granule fusion occurs at resting and even lower intraceUular calcium concentrations (Neher and Almers, 1986;Lindau and Nfige, 1987;Neher, 1988). Negative membrane potentials drive a calcium influx across the plasma membrane (Penner, Matthews, and Neher, 1988;Matthews, Neher, and Penner, 1989a). The resting potential rat peritoneal mast cells, however, is unstable as measured in the permeabilized patch configuration using ATP in the pipette and fluctuates between 0 and -30 mV (Lindau and Fernandez, 1986). An inward Ca 2+ current would thus easily depolarize the cell. Activation of the C1-channels would clamp the membrane potential at Ecv Although the cytoplamic [C1-] in mast cells is not exactly known, Ecl may be expected to be ~-40 mV, thus, supporting Ca 2+ influx driven by hyperpolarized membrane potentials (Penner et al., 1988;Matthews, Neher, and Penner, 1989b). However, when mast cells are stimulated with compound 48/80 or substance P, the development of the C1-current (Penner et al., 1988;Matthews et al., 1989b) is much slower than the exocytotic response (Fernandez, Lindau, and Eckstein, 1987) suggesting that the CI-permeability may not be essential for exocytosis.
Stilbene disulfonic acid anion channel blockers have been reported to inhibit exocytosis of platelets (Pollard, Tack-Goldman, Pazoles, Creutz, and Shulman, 1977) and neutrophils (Korchak, Eisenstat, Hoffstein, Dunham, and Weissmann, 1980). However, it was suggested that these compounds may interfere with the agonist rather than the exocytotic response in the case of thrombin stimulation of platelets (Vostal, Reid, Jones, and Shulman, 1989). In neutrophils, the effects of DIDS and 4-acetamido-4'isothiocyano-2,2'-disulfonic acid (SITS) are not limited to the anion channel (Korchak, Eisenstat, Smolen, Rutherford, Dunham, and Weissmann, 1982) and they may inhibit the activity of the secreted product [3-glucuronidase, rather than inhibiting exocytosis by an effect on CI-channels (Vostal et al., 1989). We have compared the influence of the CI-channel blocker DIDS and the extracellular CIconcentration on the C1-current and exocytosis in intact mast cells. The results indicate that, as in platelets, the inhibitory effect of DIDS on secretion is mostly not due to CI-channel block.

Solutions
The compositions of the solutions used for cell preparation and for the experiments are listed in Table I. The variable concentration of glucose in the external saline (ES) for patch clamp experiments was used to adjust the osmolality to 290 mosm. The pipette solution was supplemented with variable amounts of NaCI, MgCI2, EGTA, CaCI2, GTP, ATP, cAMP, or GTP~,S as described in the text. In most experiments we used 20 mM NaCI, 300 ~M GTP, 200 p.M EGTA and 50 p~M cAMP.

Cell Preparation
Peritoneal mast cells were obtained from Sprague Dawley rats by peritoneal lavage with isotonic NaCI containing 1 mg/ml BSA. For purification, cells were centrifuged at 220 g, resuspended in HEPES buffer, layered on top of an isotonic percoll layer and centrifuged for 15 rain at 400 g. The pellet was washed twice with HEPES buffer and the cells were stored at room temperature. The resulting cell suspension had a purity > 85% and a yield of 0.4-1.1 x 106 cells. Patch Clamp Experiments 100-200 I~1 of a suspension of nonpurified peritoneal cells or of purified mast cells were placed in a recording chamber. After a few minutes, allowing the cells to settle on the glass bottom, the dish was perfused with ES buffer to remove floating cells. Recordings were made at room temperature in the patch clamp whole-cell configuration using an EPC-7 patch clamp amplifier (List Electronics, Darmstadt, Germany) in the voltage clamp mode without capacitance compensation. The gain was usually set to 2 mV/pA. The command input was controlled by a PDP 11/73 via a D/A converter. The current monitor output was filtered at 29 kHz using an 8pole Bessel filter (LPF 902B, Frequency Devices, Haverhill, MA). The internal filters of the EPC-7 were switched off. In a typical experiment, the holding potential was -10 mV and a sequence of 10 pulses was repeatedly given. All potentials are corrected for the liquid junction potential which develops at the pipette tip when the pipette is immersed into the bath (Fenwick, Marty, and Neher, 1982) as previously described for the solutions used here (Lindau and Fernandez, 1986). First, a 1 A-ms pulse with -20 mV amplitude was given. The current was sampled (12-bit A/D converter, DT2782A, Data Translation, Marlboro, MA) with a dwell time of 7 p.s before, during, and after the pulse. 256 data points were stored on disk for off-line determination of the cell capacitance (Lindau and Neher, 1988). Subsequently, 200-ms pulses were given to test potentials between -90 and +70 mV in 20-mV intervals. For each pulse, the current was sampled with a dwell time of 25 Vs. A total of 10,240 data points was sampled with 1,280 points before the pulse, 8,000 points during the pulse and the remaining 960 points after repolarization. 40 subsequent current values were added and divided by 4 reducing the amount of data to 256 points per pulse (l-ms averages) with reduced noise and improved resolution. These data were also stored on disk for later analysis. A complete sequence required 9.1 s and was continuously repeated. The interval between individual pulses was ~ 1 s. To illustrate the time course of the currents during individual 200 ms pulses the data point containing the capacitive transient was set equal to the preceding point. External solutions were exchanged by bath perfusion.

Secretion Measurements
All experiments were done in HEPES or ES buffer. After incubating the cells at 37~ for 5 min, 25 Izl of the cell suspension was added to 25 Izl of buffer containing DIDS at twice the final concentration. After 2 min, 50 o,l of buffer containing the final DIDS concentration and the appropriate amount of the stimulator compound 48/80, was added. After 10 min at 37~ the samples were cooled on ice and cells were pelleted at 3,000 rpm at 4~ 50 ~1 of the supernatant were transferred to 5-ml tubes and 50 Ixl of buffer containing a 4-methylumbelliferyl [3-glucosamindase substrate were added and the mixture was kept at 37~ for 30-120 rain. The hexosaminidase reaction was quenched by adding 1 ml of ice-cold carbonate buffer (pH 10). To determine a possible direct effect of DIDS on the [~-glucosaminidase activity DIDS was added 2 min after, rather than before stimulation. At this time, the secretory response to compound 48/80 is complete. A similar protocol was used to determine the effects of anion substitution in the buffer. Fluorescence of the reaction product 4-methyl-umbelliferone (4-MU) was measured in a fluorescence spectrometer (Shimadzu RF 540) (excitation, 360 nm; emission, 448 nm). DIDS exhibits strong fluorescence in the same spectral range. After 30 min of the enzyme reaction the DIDS fluorescence was comparable to the 4-MU fluorescence. Recording of the fluorescence time course after opening of the excitation light beam revealed time-dependent bleaching of DIDS in the fluorimeter which had to be accounted for. When the time for the enzyme reaction was prolonged to 2 h, an irreversible decay of the DIDS fluorescence was observed which, together with the increased amount of the reaction product, reduced the DIDS fluorescence to less than 7% of the 4-MU fluorescence.

Curve Fitting
The concentration dependence of the DIDS inhibition was analyzed using the Hill equation . For the secretion experiments the ICs0, the Hill coefficient n and the maximal inhibition /max were determined by a nonlinear least squares fit. For the inhibition of the chloride current only ICs0 and n were used as free parameters. Small leak currents were neglected assuming that the maximal block at high DIDS concentrations was complete (Im~x = 1).

cAMP-activated Cl-Current
Intracellular dialysis of a mast cell with a pipette solution containing 50 o~M cAMP induced the slow development of an outward rectifying current as previously described (Penner et al., 1988;Matthews et al., 1989a, b) (Fig. 1). Fig. 1A shows the currents during 200 ms pulses given from a holding potential of -10 mV to test potentials between -90 and +70 mV in 20-mV intervals. To follow the time course of membrane conductance, the pulse sequence was continuously repeated. A complete cycle was acquired every 9.1 s. In Fig. 1 B, the mean currents during each pulse are plotted as a function of time and the points measured at a particular potential were connected by straight lines. When external [CI-] was reduced from 151 to 11 mM by exchange for L-glutamate, the outward currents were markedly reduced. The anion substitution effect is reversible. The outward rectification is illustrated in the I/V relationship for normal extracellular [CI-] ( Fig. 1 C, open circles). At low extraceUular [CI-], the I/V relationship became almost linear and the reversal potential was shifted to ~ 0 mV ( Fig. 1 C,   The observed reversal potential shift indicates CI-selectivity and for a CI-channel a reduction of the outward currents would be expected as a result of the reduced [C1-]. Alternatively, the channel could be completely blocked by extraceUular glutamate and the remaining current could be due to a nonspecific leak at the pipette-membrane seal which would shift the reversal potential to 0 mV independent of channel selectivity. A leak at the seal persists when an outside-out patch is pulled. However, when this was done at the end of the experiment (Fig. 1 B, horizontal  bar), the remaining conductance markedly decreased (to 20 pS). The reversal potentials measured here are, thus, not distorted by leak conductances, confirming CI-selectivity. The shift is, however, less than the 65 mV expected for perfect a C1channel, indicating poor CI-selectivity and a significant glutamate permeability.
The amplitude of the CI-current induced by 50 p,M cAMP was not affected by including GTP (0.3 mM) or MgATP (1 mM) in the pipette. However, in the presence of 23-25 mM internal Na § the current amplitude was 230 -50 pA (SEM, n = 22), which is ~5 times larger than with 5-15 mM Na + (48 -16 pA, n = 18). Variation of intracellular CI-between 11 and 34 mM had no detectable effect on current activation. With 0.2 or 1 mM intracellular ATP the development of the current was much slower (0.44 -+ 0.26 pA/s) than without ATP (3.5 -+ 1.5 pA/s). These results indicate that current activation is not mediated by activation of protein kinase A.

Voltage-dependent Block by DIDS
It was previously reported that the Cl-current in mast cells is blocked by DIDS (Matthews et al., 1989b). After step depolarization from -10 mV holding potential in the presence of DIDS in the extracellular saline, the outward currents declined with time at positive potentials ( Fig. 2 A). At +70 mV, the half time for the decrease was 8 ms with 30 ~M DIDS and 40 ms with 3 ~M DIDS. In the presence of DIDS, small tail currents are observed upon repolarization. These tail currents depend on the pulse potential and apparently reflect the kinetics of binding and dissociation of DIDS at the holding potential.
The DIDS block of the outward currents is further illustrated in Fig. 2 (B and C). The cell was first perfused with 11 mM C1-to identify the current as the C1-current. The cell was then exposed to increasing DIDS concentrations. The final and initial currents at the end (IF) and onset (If) of the 200 ms pulses are plotted as a function of time where the pulse was given. The final currents are reduced by about half in the presence of 3 ~M DIDS and are virtually abolished at 30 p,M DIDS. After a 2-rain exposure to high concentrations of DIDS the inhibition became partly irreversible (Fig. 2 B, 30 p,M). Fig. 2 indicates that the DIDS block is voltage dependent. For comparison with the effect of DIDS on secretion from intact cells, the block at physiological membrane potentials must be determined. The membrane potential of mast cells shows slow fluctuations between 0 and -30 mV (Lindau and Fernandez, 1986) and activation of the CI-current sets the membrane potential to the reversal potential of this current (Matthews et al., 1989b). However, the Cl-currents are extremely small between 0 and -30 mV and the DIDS sensitivity cannot be accurately determined from the steady state currents at these potentials. As shown in Fig. 2 A the currents in the presence of DIDS show time-dependent inactivation. The instantaneous current Ii after depolarization from -10 to +70 mV is thus determined by the number of unblocked channels at the holding potential of -10 mV. As shown in Fig. 2 C, a 50% block Ofll is observed at ~ 30 IxM DIDS which is 10 times higher then the ICs0 of IF. Hill coefficient n = 0.7 +-0.1. For Ii the ICs0 is shifted by a factor of 10-22 -+ 3 p~M with n = 0.7 -0.1. DIDS also blocks the CI-current at -10 mV, but ~ 10-fold higher concentrations are required as compared to the block at +70 mV. The Hill coefficients may be underestimated because small leak currents would artificially increase the currents at high DIDS concentrations and would flatten the doseresponse curve. For comparison, the irreversible block after 50-150 s exposure to different DIDS concentrations is also shown (x).
Because the Hill coefficient for the DIDS block is close to 1 for the steady state current at +70 mV as well as for the number of open channels at -10 mV, the simplest model for the DIDS block would be  With tl/2 = 40 ms, we obtain k + = 10 s -1 and k-= 8 s -l at 3 IxM DIDS. At 30 ~M DIDS with tl/2 = 8 ms the corresponding values are k + = 75 s -l and k-= 13 s -l. The off rate constants are thus similar at the two concentrations, whereas the on rate constant is nearly 10-fold higher at 30 p.M. The simple reaction scheme used here thus agrees quite well with the measured properties of the block. For the single channels, the above rate constants correspond to a mean blocked time of ~ 100 ms and a mean open time of 100 ms at 3 IxM DIDS and ~ 13 ms at 30 ~M.
When DIDS is added, the decrease of the current is accompanied by an increase of the variance (Fig. 2A). This indicates that without DIDS, the channels are indeed open most of the time and the channels may close only in the presence of the blocker. For high [DIDS], the open probability is small and from the variance measured by ensemble averaging, we estimate the single-channel current (Anderson and Stevens, 1973) to be i0 = 0.2 _+ 0.05 pA. The voltage dependence of the block correlates with the negative charge of DIDS and suggests that the DIDS binding site is inside the channel. However, we cannot exclude the possibility that the blocking site becomes more accessible at positive membrane potentials.

DIDS Blocks Secretion Stimulated with Compound 48/80
If the cAMP activated CI-current plays a role in mediating stimulus secretion coupling in mast cells, then the anion channel blocker DIDS should inhibit exocytosis. GTP~/S stimulates both, exocytosis (Fernandez, Neher, and Gomperts, 1984) and activation of the CI-current (Penner et al., 1988;Matthews et al., 1989b). By capacitance measurements, we found that 200 ~M DIDS has no detectable inhibitory effect on GTP~/S-stimulated degranulation as expected for a voltage clamp experiment with an intracellular CI-concentration determined by the pipette solution. Furthermore, GTP~S may activate exocytosis at a very late stage bypassing other second messenger-mediated events (Lindau and Gomperts, 1991). We have thus measured release of glucosaminidase from intact cells stimulated with compound 48/80. Fig. 3 shows the dose response curves obtained with two different concentrations of compound 48/80 inducing maximal (10 p.g/ml) or half maximal (1 p.g/ml) release. At 1 p~g/ml, the fit of the Hill equation yields an ICs0 of 9.7 p~M and a Hill coefficient of 1.1 -0.3. At 10 p.g/ml inhibition requires much higher concentrations (IC50 = 51 pLM, Hill coefficient = 10). It is possible that DIDS reacts with compound 48/80 or the target of the stimulus since in the presence of 50 p~M DIDS, the 48/80 dose response curve was shifted to ~ 10-fold higher concentrations (data not shown). A direct inhibitory effect of DIDS on the glucosaminidase activity was observed only above 200 ~M DIDS (data not shown).

Cl-Dependence of 48~80-induced Secretion
To investigate the effect of the external CI-concentration we have replaced CI-by L-glutamate, or acetate (Fig. 4). The effect on glucosaminidase activity in the supernatant after stimulation with 10 I~g/ml 48/80 is shown in Fig. 4A. Since the anion substitution may affect the exocytotic response as well as the enzyme activity, we also measured the effect of anion substitution on enzyme activity alone by changing the anion concentration after release was complete. The strong inhibition in the presence of acetate is due to a direct effect on the glucosaminidase activity as shown in Fig. 4 B. The inhibitory effect of L-glutamate substitution on secretion, evaluated as the difference between the corresponding results of Fig. 4 A and B, is shown in Fig. 4 C. There is a gradual decrease of secretion reaching ~ 60% of the control level at 16 mM external Cl-. Similar results were obtained using D-glutamate indicating that the observed effects are due to a reduction in Cl-rather than a specific effect of L-glutamate.

Activation of the Cl-Current
Intracellular application of cAMP induces an outward rectifying CI-current in the plasma membrane (Penner et al., 1988;Matthews et al., 1989b). The slow activation indicates that cAMP does not directly activate the channel. This current has several similarities with the cAMP-dependent CI-conductance in cardiac myocytes (Ackerman and Clapham, 1993). The cardiac CI-current has a similar voltage dependence when similar solutions are used and is also inhibited by low concentrations of stilbene Dose-response curves for DIDS inhibition of secretion stimulated by compound 48/80 ( 9 10 p.g/ ml; O, 1 txg/ml) together with the fits of the Hill equation (continuous lines).
blockers (Bahinsky, Nairn, Greengard, and Gadsby, 1989). Furthermore, the cardiac current is sensitive to intracellular Na + (Matsuoka, Ehara, and Nomaet, 1990;Harvey, Jurevicius, and Hume, 1991) which fits with our observation that the mast cell CI-currents are much larger in the presence of ~25 mM Na + than with 5-15 mM Na +. It remains to be determined if this is an effect on the activation process or on the channel itself. There is, however, one major difference: the cAMP activation of the heart channel is apparently mediated by activation of protein kinase A (PKA) (Bahinsky et al., 1989)whereas in mast cells, we found that the current amplitude is independent of ATP and the activation is even slower in the presence of ATP, indicating that current activation is not mediated by a phosphorylation through PKA.
By fluctuation analysis we estimate an apparent single-channel current of ~ 0.2 pA at +70 mV, which agrees with the previous estimate of 0.13 -0.06 pA at +40 mV (Matthews et al., 1989b). These values are certainly smaller, but still comparable to those of the cardiac cAMP-activated CI-channel. A low conductance cAMP-activated CI-channel has been described in epithelial cells, which is defective in cystic fibrosis, but this channel is insensitive to DIDS (Tabcharani, Low, Elie, and Hanrahan, 1990;Egan, Flotte, Afione, Solow, Zeitlin, Carter, and Guggino, 1992;Cunningham, Worrell, Benos, and Frizzell, 1992) and only weakly affected by the extracellular CIconcentration (Cunningham et al., 1992). It also requires the presence of PKA and ATP to be active. The outwardly rectifying C1-channel which is also defective in cystic fibrosis is DIDS sensitive, but it has a much larger conductance. Furthermore, the block by 4,4'-dinitrostilbene-2-2'-disulphonic acid (DNDS) is not voltage dependent and the DIDS block is irreversible (Bridges, Worrell, Frizzell, and Benos, 1989). A low conductance, C1-channel similar to that in mast cells has recently been described in chromaffin cells (Doroshenko, Penner, and Neher, 1991). This channel is not activated by cAMP, but via GTP-binding protein-mediated activation of phospholipase A2 and generation of arachidonic acid metabolites (Doroshenko, 1991). It is also blocked by micromolar concentrations of DIDS. A channel which has a similar unitary conductance and which is blocked by DIDS in a similar voltage and time dependent manner was recently observed in T lymphocytes (Lewis, Ross, and Cahalan, 1993). The electrophysiological properties of this channels appear to be virtually identical to those of the mast cell channel described here. It also has a substantial permeability for organic anions like aspartate or gluconate and activates slowly on a time scale of minutes (Lewis et al., 1993). However, the CI-conductance in lymphocytes is activated by osmotic stress and requires intracellular ATP. It is possible that in this case ATP is required for the generation of cAMP in response to the osmotic change.
It appears that the low conductance CI-channel is expressed in a wide variety of cell types, but the mechanisms for its activation may be different depending on the cell type.

DIDS Block of Secretion
An inhibitory effect of DIDS on exocytosis has been described for lysosomal enzyme secretion from neutrophils (Korchak et al., 1980;Korchak et al., 1982), but it has also been suggested that the apparent inhibition may not be restricted to an effect on anion channels (Korchak et al., 1982) and may simply be due to inhibition of ~-glucuronidase activity by DIDS (Vostal et al., 1989). We found that in mast cells an inhibitory effect on the activity of the measured product fl-glucosaminidase is observed above 200 ~M DIDS. At lower concentrations, DIDS also has an inhibitory effect on secretion. With 1 o.g/ml 48/80 which induced half maximal release, ~ 10 ~M DIDS were required for 50% inhibition with a Hill coefficient of n = 1.1. These values are close to those obtained for the CI-channel block at -10 mV (ICs0 --22 ~M, n = 0.7). In contrast, with 10 ~g/ml 48/80 inducing maximal release, ~ 50 ~M DIDS is required for 50% inhibition and the concentration dependence is very steep. The large Hill coefficient (n = 10) indicates that the inhibitory effect of DIDS under these conditions may be due to a completely different mechanism. The fact that the inhibition is relieved by elevating the agonist concentrations would be in agreement with an interference of the anionic DIDS with the cationic stimulator. The similarity of the concentration dependence for inhibition of exocytosis and inhibition of the CI-current at -10 mV, however, suggests that at low agonist concentration the activation of the CI-current may be required for a full response, whereas at high agonist concentrations this current is not required to stimulate complete degranulation.

Cl-Dependence of Secretion
To overcome the difficulty of nonspecific effects of DIDS we did anion substitution experiments. Replacing most of the extraceUular C1-by glutamate reduces the outward current and shifts the reversal potential close to zero. Under these conditions, activation of the CI-conductance will induce neither a significant C1-influx nor a hyperpolarization of the membrane potential. CI-substitution by L-glutamate has a relatively small inhibitory effect on 48/80 induced secretion demonstrating that exocytosis occurs in the absence of C1-influx and membrane hyperpolarization. When exocytosis is stimulated in permeabilized mast cells with GTP,tS and Ca 2+, replacement of most of the CI-by glutamate results in ~ 30% inhibition of secretion (Churcher and Gomperts, 1990), comparable to the effect which we observe on intact cells. The CI-replacement in the external solution will probably induce also a reduction of cytoplasmic [CI-]. The inhibitory effect of CI-substitution described here could thus also be explained by a reduction of intracellular [CI-] and does not necessarily indicate an enhancement of secretion by CI-channel activation.
We conclude that the cAMP-activated CI-current in mast cells is not a necessary event in stimulus-secretion coupling but its activation could possibly enhance secretion at suboptimal stimulation. Since a CI-channel with similar electrophysiological and pharmacological properties is activated by osmotic stress in lymphocytes, it is tempting to speculate that the CI-channel in mast cells may alternatively play a role in volume regulation. After exocytosis and the associated increase of plasma membrane area, mechanisms for volume regulation may indeed be required. The activation of a current involved in volume regulation may thus be needed during the degranulation process without the development of significant membrane stress. Accordingly different activation mechanisms might be required for the Cl-channel in different cell types depending on the physiological conditions where volume regulation is necessary.
We are grateful to Wolf Almers for critically reading our manuscript.
Original version received 21 June 1993 and accepted version received 31 August 1994.