From the * Department of Anesthesiology and Department of Pharmacology, Anesthesiology Research Division, Vanderbilt University
Medical Center, Nashville, Tennessee 37232
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ABSTRACT |
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Swelling-induced activation of the outwardly rectifying anion current, ICl, swell, is modulated by intracellular ATP. The mechanisms by which ATP controls channel activation, however, are unknown. Whole cell patch
clamp was employed to begin addressing this issue. Endogenous ATP production was inhibited by dialyzing N1E115 neuroblastoma cells for 4-5 min with solutions containing (µM): 40 oligomycin, 5 iodoacetate, and 20 rotenone. The effect of ATP on current activation was observed in the absence of intracellular Mg2+, in cells exposed to extracellular metabolic inhibitors for 25-35 min followed by intracellular dialysis with oligomycin, iodoacetate, and rotenone, after substitution of ATP with the nonhydrolyzable analogue AMP-PNP, and in the presence of AMP-PNP and alkaline phosphatase to dephosphorylate intracellular proteins. These results demonstrate
that the ATP dependence of the channel requires ATP binding rather than hydrolysis and/or phosphorylation reactions. When cells were swollen at 15-55%/min in the absence of intracellular ATP, current activation was slow
(0.3-0.8 pA/pF per min). ATP concentration increased the rate of current activation up to maximal values of 4-6
pA/pF per min, but had no effect on the sensitivity of the channel to cell swelling. Rate of current activation was a
saturable, hyperbolic function of ATP concentration. The EC50 for ATP varied inversely with the rate of cell swelling. Activation of current was rapid (4-6 pA/pF per min) in the absence of ATP when cells were swollen at rates
65%/min. Intracellular ATP concentration had no effect on current activation induced by high rates of swelling. Current activation was transient when endogenous ATP was dialyzed out of the cytoplasm of cells swollen at
15%/min. Rundown of the current was reversed by increasing the rate of swelling to 65%/min. These results indicate that the channel and/or associated regulatory proteins are capable of sensing the rate of cell volume increase. We suggest that channel activation occurs via ATP-dependent and -independent mechanisms. Increasing
the rate of cell swelling appears to increase the proportion of channels activating via the ATP-independent pathway. These findings have important physiological implications for understanding ICl, swell regulation, the mechanisms by which cells sense volume changes, and volume homeostasis under conditions where cell metabolism is compromised.
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INTRODUCTION |
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Maintenance and regulation of cell volume are fundamental physiological processes. In response to swelling, cells activate anion and cation channels that allow the passive loss of inorganic ions and organic solutes. Net solute and osmotically obliged water efflux functions to return cell volume towards its original value, a process termed regulatory volume decrease.
Swelling of vertebrate cells activates an outwardly rectifying anion current termed ICl, swell. The general characteristics of ICl, swell include an Eisenman type I anion
permeability sequence (I > Br
> Cl
> F
), modest
outward rectification, voltage-dependent inactivation at
potentials above ECl, inhibition by a wide variety of compounds, including conventional anion transport inhibitors, and block by extracellular nucleotides (Strange et
al., 1996
; Nilius et al., 1997
; Okada, 1997
). The degree
of rectification, voltage sensitivity, and pharmacology of
ICl, swell can vary somewhat between different cell types. It
is not clear whether the differences observed reflect the
existence of distinct channels or whether they are due
to experimental and/or physiological variables.
The mechanism by which swelling activates ICl, swell is
unknown (Strange et al., 1996; Nilius et al., 1997
;
Okada, 1997
). However, two important physiological
parameters have been shown to modulate channel activation. Changes in cytoplasmic ionic strength alter
both the rate of channel activation and channel volume sensitivity (Emma et al., 1997
; Cannon et al., 1998
;
Nilius et al., 1998
), and swelling-induced activation requires the presence of intracellular ATP (reviewed by
Strange et al., 1996
; Nilius et al., 1997
; Okada, 1997
).
Intracellular ATP plays a crucial role in regulating
many ion channels and transporters (e.g., Bryan and
Aguilar-Bryan, 1997; Hilgemann, 1997
). In most cases,
the underlying cellular and molecular mechanisms of
ATP-dependent regulation are incompletely understood. Hilgemann (1997)
recently reviewed the mechanisms by which ATP can regulate channel/transporter
function. These mechanisms include protein and lipid
phosphorylation, ATP binding, ATP hydrolysis, modulation of cytoskeletal function, chelation of polyvalent
cations, and modulation of membrane trafficking.
The mechanisms of ATP-dependent regulation of the
ICl, swell channel are largely unknown. Most studies of ICl,
swell have shown that nonhydrolyzable ATP analogues
readily substitute for intracellular ATP, suggesting that
the requirement for ATP involves binding rather than
phosphorylation or hydrolysis reactions (reviewed by
Strange et al., 1996; Nilius et al., 1997
; Okada, 1997
).
However, studies in cultured mouse cortical collecting
duct (Meyer and Korbmacher, 1996
) and carotid body
type I cells (Carpenter and Peers, 1997
) indicate that
phosphorylation of the ICl, swell channel and/or accessory
proteins is required for swelling-induced activation.
In the present investigation, we demonstrate that ICl, swell activation in N1E115 neuroblastoma cells does not involve ATP hydrolysis or phosphorylation reactions. Furthermore, we demonstrate that the ATP dependence of the ICl, swell channel is modulated by the rate of cell volume change. At high rates of swelling, ICl, swell activates rapidly and activation is insensitive to intracellular ATP concentration. Our results indicate that there are ATP-dependent and -independent mechanisms by which ICl, swell can be activated. In addition, they demonstrate that cells sense not only the magnitude of volume change, but also the rate at which volume is perturbed. These findings have important physiological implications for control of both cell volume and metabolic homeostasis, and they provide new insights into regulation of the ICl, swell channel.
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MATERIALS AND METHODS |
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Cell Culture
N1E115 mouse neuroblastoma cells were cultured in high glucose Dulbecco's modified eagle's medium (DMEM; GIBCO BRL) with 10% fetal bovine serum, 50 U/ml penicillin, 50 µg/ml streptomycin, and 25 mM HEPES in the presence of 5% CO2/ 95% air. Cells were used between passages 30 and 45. The osmolality of the growth medium was measured by vapor pressure osmometry (5520; Wescor Inc.) and was 295-305 mOsm.
Patch Clamp Recordings
N1E115 cells were grown in 35-mm culture dishes and dissociated by brief treatment with Ca2+- and Mg2+-free modified Hank's solution. Dissociated cells were allowed to reattach to the poly-L-lysine-coated cover slip bottom of a bath chamber (R-26G; Warner Instrument Corp.) that was mounted onto the stage of an inverted microscope (TE 300; Nikon Inc.). Patch electrodes were pulled from 1.5-mm outer diameter borosilicate glass microhematocrit tubes (Fisher Scientific Co.) that had been salinized with dimethyl-dichloro silane (Sigma Chemical Co.). Electrodes were not fire polished before use.
The standard bath solution contained (mM): 70 N-methyl- D-glucamine (NMDG)1-Cl, 5 MgSO4, 1.3 CaCl2, 12 HEPES, 8 Tris, 5 glucose, 2 glutamine, 120 sucrose (pH 7.4; 300 mOsm).
Bath was reduced to 150-250 mOsm by reducing sucrose concentration. In one set of experiments, bath was reduced to 100 mOsm. For these studies, the NMDG-Cl concentration in the control (i.e., 300 mOsm) bath solution was reduced to 40 mM and sucrose concentration was increased to 200 mM.
Patch clamping was carried out using a pH 7.2 pipette solution
that contained 125 mM CsCl, 20 mM HEPES, 6 mM CsOH, 1 mM
EDTA, 40 µM oligomycin, 5 µM iodoacetate, and 20 µM rotenone.
Sodium and K+-free bath and pipette solutions were used to minimize the contribution of cation currents to the whole cell conductance. To prevent spontaneous cell swelling, the osmolality of
the pipette solution was hypotonic (280 mOsm) with respect to
the bath. Metabolic inhibitors were added from concentrated
stock solutions dissolved in DMSO. Final DMSO concentration in
the pipette solutions was 0.15%. ATP and ATP analogues were
added as sodium and lithium salts, respectively. Electrodes had DC
resistances of 3-5 m. Cells were used only if the series resistance
was no greater than ~150% of the pipette resistance and the reversal potential (Erev) was within ±3 mV of the calculated value.
An Axopatch 200A (Axon Instruments) patch clamp amplifier
was used to voltage clamp N1E115 cells after gigaseal formation and attainment of whole cell access. Command voltage generation, data digitization, and data analysis were carried out on a
200 mHz Pentium computer (Dimension XPS M200s; Dell Computer Corp.) using a DigiData 1200 AD/DA interface with
pClamp 6 software (Axon Instruments). Data were digitized at
5 kHz and filtered at 0.5 kHz using an eight pole Bessel filter
(902; Frequency Devices). Electrical connections to the amplifier
were made using Ag/AgCl pellets and 3 M KCl/agar bridges.
Whole cell currents were measured by varying membrane potential from 80 to +80 mV at 80 mV/s every 5-15 s.
Cells were swollen by reduction of bath osmolality. At the end of a patch clamp recording, bath osmolality was returned to 300 mOsm and another cell was selected for study. Cells were exposed to hypotonicity approximately every 15-25 min and to at most four to five hypotonic shocks before they were removed and replaced with fresh cells. This number of repetitive hypotonic shocks had no effect on the rate or extent of current activation (data not shown).
Whole cell current measured before induction of cell swelling is defined as "baseline current." This current was recorded every 5 s for 60 s before the bath osmolality was reduced. These 13 data points were averaged to yield a mean baseline current. This current was subsequently subtracted from all data points within a given current record to correct for variability in resting current levels between different cells.
Measurement of Relative Cell Volume Changes
Whole cell currents and volume changes were measured simultaneously in single, patch clamped cells. Cells attached to the cover
slip bottom of the patch clamp bath chamber were visualized by
video-enhanced differential interference contrast microscopy (Strange and Spring, 1986) using a 63× oil-immersion objective lens (1.25 numerical aperture; Carl Zeiss, Inc.) and a 32× condenser lens (0.4 numerical aperture; Leitz). Optical sectioning
(Strange and Spring, 1986
) demonstrated that the cells maintained a spherical morphology for at least 60 min after attachment to the cover slip. Cells were routinely removed from the
bath chamber and replaced with fresh ones every 30-45 min.
Given that the cells have a spherical morphology, relative cell volume change can be determined as:
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where CSA is the cell cross-sectional area (CSA) measured at a single focal plane. In all CSA measurements described in this paper, we used focal planes located at the point of maximum cell diameter.
Cell images were recorded continuously throughout a patch
clamp experiment using a super VHS video cassette recorder
(SVO-2000; Sony Electronics, Inc.) and a CCD camera (C2400;
Hamamatsu Photonics K.K.). Cross-sectional areas of single cells
were quantified by digitizing recorded video images with an image processing computer board (MV-1000; MuTech) with 512 × 480 × 8 bit resolution and a 300 mHz Pentium II computer (Dimension XPS D300; Dell Computer Corp.). Digitized images
were displayed on the computer monitor and cell borders were
traced using a mouse and a computer-generated cursor. The
CSA of a traced region was determined by image analysis software (Optimas; Bioscan, Inc.). This image acquisition and analysis system allows detection of changes in CSA with an accuracy of
±2-3%. Optical sectioning methods (Strange and Spring, 1986) have demonstrated that this approach reliably tracks relative cell
volume increases up to 250% of control values (our unpublished observations).
During swelling, a small percentage of cells exhibited bleb formation. These cells were excluded from the analysis of both volume changes and current activation.
Cell volume typically increased in a linear fashion when bath osmolality was reduced. The rate of cell swelling was therefore determined by performing linear regression analysis on volume changes measured for 30-60 s after swelling was initiated.
Data Analysis
The effect of intracellular ATP concentration on ICl, swell was quantified using two parameters, the rate of current activation initiated by cell swelling and the cell volume set-point of the channel. Cell volume set-point is defined as the volume of the cell at which current activation is initiated.
Rate of current activation and cell volume set-point were measured simultaneously in single, patch clamped cells. Fig. 1 shows volume and current measurements in a single cell and illustrates how the set-point and rate of current activation were quantified. Current activation is defined as the point at which there is a significant and continuous increase in current amplitude above the baseline current. Rate of current activation was quantified by performing linear regression analysis on whole cell currents measured for 30-60 s after activation was detected. The threshold for current activation is the intercept between the line defining rate of current activation and the current immediately before activation begins (see Fig. 1). The relative volume of the cell at the current activation threshold is defined as the cell volume set-point of the channel.
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Statistical Analysis
Data are presented as means ± SEM. Statistical significance was
determined using Student's two-tailed t test for unpaired, independent means. When comparing three or more groups, statistical significance was determined by one-way analysis of variance.
P 0.05 indicated statistical significance.
Enzymes
Grade VI apyrase was purchased from Sigma Chemical Co. Alkaline phosphatase and creatine kinase were purchased from Boehringer Mannheim Biochemicals.
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RESULTS |
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The ATP Requirement of ICl, swell Activation Does Not Involve Hydrolysis and/or Phosphorylation Reactions
In most cell types, ICl, swell activation is supported normally by nonhydrolyzable ATP analogues (reviewed by
Strange et al., 1996; Nilius et al., 1997
; Okada, 1997
).
However, in carotid body type 1 and mouse cortical collecting duct cells, phosphorylation reactions appear to
be needed for channel activation (Meyer and Korbmacher, 1996
; Carpenter and Peers, 1997
). The reason for
these contradictory findings is unclear and we therefore carried out a series of experiments to look more
closely at the role of phosphorylation.
In their studies on cortical collecting duct cells,
Meyer and Korbmacher (1996) exposed cells to extracellular metabolic inhibitors for 20-45 min before they
were patch clamped. Such prolonged metabolic inhibition may result in dephosphorylation of the channel
and/or other proteins. If the putative phosphorylation
of these proteins is crucial to channel function, then intracellular ATP rather than nonhydrolyzable analogues
would be required for swelling-induced activation. We
therefore pretreated N1E115 neuroblastoma cells with
5 mM 2-deoxyglucose and 100 nM rotenone for 25-35
min, and then patch clamped them with the standard
pipette solution containing (µM) 40 oligomycin, 5 iodoacetate, and 20 rotenone. Both 2-deoxyglucose and
rotenone were present in the bath throughout the entire experiment. Cells were dialyzed for 4-5 min with
the metabolic inhibitors before swelling was induced.
Preliminary studies demonstrated that a dialysis period
of 2-3 min was sufficient to maximally inhibit swelling-induced current activation, presumably by maximally reducing intracellular ATP levels.
In the presence of intracellular metabolic inhibitors
and ATP or ATP analogues, an outwardly rectifying (data
not shown) current was activated when cells were swollen by reduction of bath osmolality. With 73 and 125 mM
Cl in the bath and pipette solutions, respectively, the
mean ± SEM Erev observed was 13.1 ± 0.3 mV (n = 109). This value is close to the value of 12.8 mV predicted from the Goldman-Hodgkin-Katz equation and
the previously measured relative cation conductance (Pcation/PCl) of 0.03 (Jackson and Strange, 1993
). These
results indicate that the current activated by swelling is
a Cl
current (i.e., ICl, swell).
Results of the metabolic inhibition studies are shown in Fig. 2 A. When exposed to intracellular metabolic inhibitors alone, and a Mg2+-free pipette solution containing 2 mM ATP, the mean ± SEM rate of chloride current activation measured at +60 mV was 3.95 ± 0.26 pA/pF per min (n = 28). This value was not significantly (P > 0.1) different from those obtained in cells pretreated for 25-35 min with 2-deoxyglucose and rotenone, and then dialyzed with metabolic inhibitors in the presence of 2 mM ATP and 0 mM Mg2+ or 2 mM ATP and 2 mM Mg2+ (Fig. 2 A).
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As a further test for the involvement of phosphorylation in regulating ICl, swell, we patch clamped cells with a pipette solution containing alkaline phosphatase. As shown in Fig. 2 B, there was no significant (P > 0.8) difference in the rate of swelling-induced current activation in the presence of 2 mM ATP and 0 mM Mg2+, 2 mM AMP-PNP, and 0 mM Mg2+, or 2 mM AMP-PNP, 0 mM Mg2+, and 30 U/ml of alkaline phosphatase. We conclude from the results shown in Fig. 2 that swelling- induced activation of ICl, swell in N1E115 neuroblastoma cells requires only ATP binding, and not phosphorylation events and/or ATP hydrolysis.
The Rate of ICl, swell Activation Is a Saturable Function of Intracellular ATP Concentration
To further characterize the dependence of ICl, swell activation on ATP binding, we quantified the effect of ATP concentration on the rate of current activation, the rate of cell swelling, and the cell volume set-point of the channel simultaneously in single patch-clamped cells. We define cell volume set-point as the relative cell volume at which current activation is initiated (see Fig. 1). In initial experiments, cells were swollen at a rate of 35-40%/min via reduction of bath osmolality by 100 mOsm (see Fig. 3 and Table I). As shown in Fig. 3 (right), intracellular ATP concentration had no effect on the rate of cell swelling. The rate of current activation, however, was increased by increasing ATP levels in the patch pipette solution (Fig. 3, left). The rate of current activation was a saturable function of intracellular ATP concentration (Fig. 4). The maximal rate of current activation (Rmax) was 4.11 pA/pF per min and the EC50 for ATP was 0.32 mM. The cell volume set-point of the channel was 1.09-1.15 and was unaffected by ATP concentration (Fig. 4, inset, and Table II).
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With prolonged exposure to hypotonic medium,
N1E115 cells underwent dramatic volume increases
(see Figs. 3 and 9). The ability to withstand such large
degrees of swelling most likely reflects the presence of
extensive membrane microvilli (Walum and Marchner, 1983) and possibly membrane infolding. Similar large
volume increases have been observed in patch-clamped
lymphocytes (Ross et al., 1994
) and have been attributed to the presence of numerous microvilli that amplify the total membrane surface area.
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It is likely that ATP-consuming reactions are active in
patch-clamped cells. These reactions may occur at a
rate that approaches the rate of ATP diffusion from the
patch pipette solution. Thus, it was conceivable that
ATP-consuming reactions close to the channel and/or
its activation machinery may have reduced local ATP
concentrations, resulting in an overestimation of the
concentration of the nucleotide required for any given
rate of current activation. To address this important issue, we dialyzed cells with an ATP regeneration system
consisting of 10 mM creatine phosphate and 80 U/ml
of creatine kinase. The solution also contained 0.25 mM
ATP. This concentration was close to the EC50, and was
chosen so that small increases in ATP concentration
potentially brought about by the regeneration system
would be detected as relatively large increases in the rate of current activation. The rate of current activation
in the presence of the ATP regeneration system (Fig. 4,
) was not significantly (P > 0.15) different from that
observed with 0.25 mM ATP alone. We conclude that
the ATP concentration sensed by the channel and/or
its regulatory machinery is similar to that in the bulk
pipette solution.
Surprisingly, swelling-induced activation of an outwardly rectifying anion current was also detected in
metabolically poisoned cells dialyzed with an ATP-free
pipette solution, albeit at a greatly reduced rate (Figs.
3-5). The mean ± SEM rate of current activation observed was 0.44 ± 0.09 pA/pF per min (n = 10). This suggested that the cells may contain trace amounts of
ATP. To test for this possibility, we dialyzed cells for
5 min with an ATP-free pipette solution containing
metabolic inhibitors and the ATP-consuming enzyme
apyrase. Apyrase was present at a concentration of 2 U/
ml. Concentrations of apyrase of 0.5-1 U/ml have been shown previously to be effective in depleting both intracellular (Wang et al., 1996) and extracellular (Schwiebert et al., 1995
) ATP. The presence of apyrase had no
significant (P > 0.25) effect on the rate of current activation (Figs. 4,
, and 5, bottom two traces plotted with
and
).
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The observation that a small amount of current is activated in the absence of intracellular ATP is consistent
with findings of Volk et al. (1996). These investigators
noted that swelling-induced anion current activation
required intracellular ATP when inner medullary collecting duct cells were swollen by a 50-mOsm reduction in bath. In contrast, when swelling was induced by 100-mOsm reduction in bath osmolality, an ATP-independent anion current was activated.
Given these results, we examined the effect of increasing the rate of swelling on the ATP dependence of
ICl, swell. N1E115 cells were dialyzed for 4-5 min with an
ATP-free pipette solution containing metabolic inhibitors, and then exposed to a 150-mOsm reduction in
bath. Under these conditions, the cells swelled at a rate
of 65-70%/min (Fig. 3, Table I). As shown in Figs. 3
and 5, cell swelling induced a rapid increase in whole
cell current. The mean ± SEM Erev for the current was
12.8 ± 1.2 mV (n = 17), indicating that it is carried by
Cl. The mean ± SEM rate of current activation was
5.0 ± 0.8 pA/pF per min (n = 17). Addition of 2 U/ml
of apyrase to the patch pipette solution had no significant (P > 0.4) effect on the rate of current activation
(Fig. 5, top two traces plotted with
and
). Activation of the ATP-independent current was reversed
by cell shrinkage. Exposure of swollen cells to a 300-mOsm bath solution induced cell shrinkage and a
rapid reduction in whole cell current (Fig. 6; see also
Fig. 9 B).
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To determine whether the ICl, swell channel was responsible for this ATP-independent current, we compared its biophysical and pharmacological characteristics to the current activated by a 100-mOsm reduction in bath in metabolically poisoned cells dialyzed with 2 mM ATP. As shown in Table III, both currents had similar reversal potentials, rectification and voltage sensitivity, and were inhibited in a voltage-dependent manner and to the same extent by 100 µM DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate). In addition, the channels responsible for the currents had similar relative anion permeabilities. We conclude from these results that the ICl, swell channel gives rise to both currents.
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The effect of ATP concentration on ICl, swell activation in cells swollen by a 150-mOsm hypotonic shock is shown in Fig. 7. There was no significant difference (P > 0.2) between rates of current activation measured in the presence of different intracellular concentrations of ATP. Therefore, at high rates of cell swelling, current activation is insensitive to cytoplasmic ATP levels. The cell volume set-point of the channel was also unaffected by ATP concentration and was similar to that observed in cells swollen more slowly with smaller hypotonic shocks (Table II).3
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The effect of high rates of swelling on the ATP sensitivity of current activation suggested that it would also be altered by slower rates of cell swelling. Fig. 3 shows the effects of intracellular ATP concentrations and different osmotic perturbations on whole cell current activation and the rate of cell swelling. When cells were swollen at a rate of 13-17%/min by a 50-mOsm hypotonic shock, current activation was again a saturable function of ATP concentration (Fig. 7), but both the EC50 (0.8 mM) and Rmax (1.37 pA/pF per min) were dramatically altered compared with cells swollen by a 100-mOsm reduction in bath (Fig. 7). The cell volume set-point of the channel was unaffected by ATP concentration and was similar to that observed at higher rates of swelling (Table II).
The effects of varying the rate of cell swelling on ATP dependence are shown in Figs. 3 and 7 and summarized in Fig. 8. Fig. 8 A shows the rate of current activation in metabolically poisoned cells dialyzed with an ATP-free pipette solution (i.e., ATP-independent current activation). ICl, swell activation was relatively slow (0.26-0.44 pA/pF per min) when the rate of cell swelling was 15-40%/min (50-100-mOsm reductions in bath). The rates of ATP-independent current activation observed under these conditions were not significantly different (P > 0.3), indicating that they were relatively insensitive to the rate of cell volume increase. In only 1 (swollen by a 50-mOsm reduction in bath) of the 20 cells used in these experiments, were we unable to detect swelling-induced, ATP-independent current activation.
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When cells were swollen at 55%/min (120-mOsm reduction in bath), the rate of ATP-independent current activation increased significantly (P < 0.05) to 0.85 pA/pF per min (Fig. 8 A). Importantly, current activation under these conditions was still a saturable function of intracellular ATP concentration (Figs. 3 and 7).
ATP-independent current activation increased dramatically to ~5 pA/pF per min when the rate of cell swelling was increased to 65-70%/min (150-mOsm reduction in bath). Increasing the rate of cell swelling to 100%/min (200-mOsm reduction in bath)4 had no further effect on the rate of ATP-independent current activation (Fig. 8 A).
The EC50 for ATP varied inversely with the rate of cell swelling (Fig. 8 B). At low rates of swelling, half maximal rates of current activation required higher concentrations of ATP compared with that observed in cells swollen at higher rates. Rmax was a direct function of the rate of cell swelling (Fig. 8 C).
Current Rundown
Transient activation of ICl, swell has been observed in several cell types when they are patch clamped with ATP-free solutions and activated shortly after obtaining the
whole cell configuration (Lewis et al., 1993; Jackson et al.,
1994
; Oike et al., 1994
). This "rundown" of the current
is prevented when the pipette solution contains ATP or
ATP analogues. The simplest interpretation of these
findings is that current activation is initially supported by the presence of endogenous ATP. As this ATP is dialyzed away, the ICl, swell channels spontaneously inactivate (e.g., Lewis et al., 1993
; Jackson et al., 1994
; Oike
et al., 1994
).
To assess whether N1E115 neuroblastoma cells also exhibited such rundown behavior, we patch clamped cells with an ATP-free pipette solution containing metabolic inhibitors. Cells were swollen by exposure to a 50-mOsm reduction in bath within 60 s after obtaining the whole cell configuration. As shown in Fig. 9 A, ICl, swell activated in response to cell swelling. The activation was transient, however, and the current slowly inactivated despite continued volume increase.
Fig. 9 B shows the effect of increasing the rate of swelling on current rundown. The cell shown in the figure was initially swollen at a rate of 12%/min by exposure to a 50-mOsm reduction in bath. Current activation was initiated at a relative cell volume of 1.04 and spontaneous rundown was observed when the relative cell volume was 1.38. Approximately 5 min after rundown began, relative cell volume was 1.8, and the cell was then swollen at a rate of 63%/min by exposure to a 150-mOsm reduction in bath. Rapid current reactivation occurred immediately upon switching the solution. Similar experiments were carried out in five additional cells. In all cases, exposure to a 150-mOsm reduction in bath induced a rapid reactivation of current (data not shown). The amount of additional swelling that occurred before current reactivation was initiated was between 6 and 12% in three cells. In three other cells, current reactivation was initiated immediately after the solution switch and it was not possible to accurately measure the cell volume change at which it occurred.
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DISCUSSION |
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Role of Phosphorylation in ICl, swell Activation
Most studies of ICl, swell activation that have examined
channel ATP dependence have concluded that nonhydrolyzable ATP analogues substitute normally for ATP
(reviewed by Strange et al., 1996; Nilius et al., 1997
;
Okada, 1997
). However, studies in cultured mouse cortical collecting duct (Meyer and Korbmacher, 1996
)
and carotid body type I cells (Carpenter and Peers,
1997
) have shown that activation only occurs with ATP
(Meyer and Korbmacher, 1996
; Carpenter and Peers,
1997
) or ATP
S (Meyer and Korbmacher, 1996
), that it
requires the presence of intracellular Mg2+ (Meyer
and Korbmacher, 1996
; Carpenter and Peers, 1997
),
which is needed for kinase activity, and that it can be
inhibited by the protein kinase inhibitor staurosporine
(Meyer and Korbmacher, 1996
). These results have
been interpreted as indicating that phosphorylation of
the ICl, swell channel and/or accessory proteins is required for swelling-induced activation.
The reasons for the contradictory findings on the
role of intracellular ATP are unclear. One possibility is
that methodological differences may unmask constitutive phosphorylation of the channel and/or accessory
proteins. Meyer and Korbmacher (1996) exposed collecting duct cells to metabolic inhibitors for 20-45 min
before they were patch clamped. Such prolonged metabolic inhibition is likely to lead to dephosphorylation
of the channel and/or important regulatory proteins.
If phosphorylation were crucial to the function of these
proteins, rephosphorylation by ATP would have to occur before the channel could be activated by swelling
and ATP binding. Our findings do not support this hypothesis, however. Channel activation is normal in
cells exposed to prolonged metabolic inhibition after
they were dialyzed with Mg-free pipette solutions containing 2 mM ATP (Fig. 1 A). Furthermore, channel activation is unaffected by dialyzing metabolically poisoned cells with Mg-free solutions containing 2 mM
AMP-PNP and 30 U/ml of alkaline phosphatase (Fig. 2
B) to dephosphorylate proteins. We conclude therefore that in N1E115 neuroblastoma cells, activation of
ICl, swell requires ATP binding rather than hydrolysis
and/or phosphorylation reactions. The requirement
for phosphorylation observed in other cell types (Meyer and Korbmacher, 1996
; Carpenter and Peers,
1997
) may reflect the existence of distinct channel
types. Alternatively, it may reflect the existence of multiple signaling/regulatory pathways involved in swelling-induced channel activation. These pathways could
be cell-specific, they may reflect the physiological status of the cell, and/or they may be sensitive to experimental
parameters such as solution composition3 and the mechanism (e.g., Strange, 1994
) or rate of cell swelling.
Characteristics of ICl, swell Activation
There are several aspects of ICl, swell channel activation
that bear on the discussion of the modulatory role of
intracellular ATP. First, it is important to note that
graded increases in cell volume do not activate ICl, swell
by inducing graded changes in the Po of a single, homogeneous population of channels. Instead, channels exist in either an inactive state where Po = 0, or an active
state where Po ~ 1 (Jackson and Strange, 1995; Boese
et al., 1996
; Meyer and Korbmacher, 1996
). Swelling increases the number, n, of active channels in the cell
membrane (Jackson and Strange, 1995
; Meyer and
Korbmacher, 1996
), and n is therefore a direct function of the magnitude of the volume increase.
ICl, swell can be activated by forcing fluid into a cell via
the patch pipette (Cannon et al., 1998). This indicates
that activation does not require transmembrane water
flow or the presence of an osmotic gradient. Spontaneous channel activation does not occur with prolonged dialysis of the cell, suggesting that dilution of a freely diffusible factor is not involved. Activation only requires expansion of the cell volume. Thus, activation must
ultimately involve mechanical events. These mechanical
events may be detected directly by the channel and/or
associated regulatory proteins. Putative regulatory proteins may include components of the cytoskeleton that
are altered by cell swelling. Alternatively, cell swelling may alter the activity of signal transduction pathways that
ultimately are responsible for activating the channel.
When using whole cell patch clamp to study ICl, swell,
current activation can be characterized in terms of a
cell volume set-point of the channel and a rate of current activation. Cell volume set-point is defined as the
relative cell volume at which current activation is detected and is a measure of the sensitivity of the channels to swelling. The set-point is regulated by intracellular ionic strength (Emma et al., 1997; Cannon et al.,
1998
) and G-proteins (Voets et al., 1998
). The fact that
n increases as a direct function of the amount of cell
swelling suggests that individual channels or groups of
channels have different cell volume set-points. Alternatively, macroscopic cell swelling may have different effects
on the mechanical properties of microdomains within
the plasma membrane or in submembrane structures.
Modulation of ICl, swell Activation by Intracellular ATP
It has been concluded previously that ATP is an essential cofactor for swelling-induced activation of ICl, swell
(reviewed by Strange et al., 1996; Nilius et al., 1997
;
Okada, 1997
). However, as shown in Figs. 3-9, Table
III, and by Volk et al. (1996)
, current activation can be
observed under ATP-free conditions. With rates of
swelling between 15 and 55%/min, increases in intracellular ATP increase the rate of ICl, swell activation without changing the cell volume set-point of the channel
(Fig. 7, Table II). The rate of ICl, swell activation is a saturable function of ATP concentration, which can be defined as an EC50 for ATP and a maximal rate of current
activation (Rmax) at saturating ATP levels (Figs. 4, 7, and 8). Two conclusions can be drawn from these observations. First, ATP interacts with a saturable binding
site(s) that selectively recognizes the nucleotide and
certain analogues. Second, ATP binding induces a conformational change(s) in the channel and/or regulatory proteins that is required for channel activation.
The rate at which this conformational change, and
hence current activation, occurs is a saturable function
of ATP concentration.
Rundown of ICl, swell occurs when endogenous ATP is dialyzed out of the cytoplasm (Fig. 9). This indicates that ATP is required to maintain the channel and/or regulatory proteins in the activated conformation. Thus, ATP must be bound to the activated channel and/or regulatory proteins.
One of the more novel findings of our studies is that the rate of cell swelling alters channel ATP sensitivity (Figs. 3, 7, and 8). As the rate of cell swelling is increased, the EC50 for ATP falls (Figs. 7 and 8). At high rates of volume increase, ICl, swell activation is insensitive to intracellular ATP concentration (Figs. 3, 5, and 7-9).
How does the rate of cell swelling modulate the ATP dependence of ICl, swell activation? There are two obvious models to consider. First, the rate of cell swelling could somehow change the affinity of the ATP binding site(s). Alternatively, channel activation may occur via distinct ATP-dependent and -independent pathways.
Changes in ATP affinity cannot explain all of the experimental observations adequately. As shown in Figs.
4, 5, 7, and 8, ATP-independent current activation occurs, albeit slowly, at rates of cell swelling (15-55%/
min) where there is a strong dependence of current activation on intracellular ATP concentration. Furthermore, if ATP affinity were a function of the rate of volume expansion, the Kd of the binding site will fall as the
rate of swelling is increased. However, this effect would
have to be reversed at high rates of swelling where current activation is independent of intracellular ATP concentration (Figs. 7 and 8). Indeed, it would be necessary to postulate that the Kd for ATP rises to infinity
(i.e., the site is no longer capable of binding ATP)
when cells are swollen at 65%/min.
The alternative and, in our opinion, simpler explanation for the observed changes in ATP sensitivity is that the ICl, swell channel can be activated via distinct ATP- dependent and -independent mechanisms. Swelling- induced changes in the EC50 for ATP can be explained if increases in the rate of cell swelling increase the proportion of channels activating in the ATP-independent mode. As the rate of swelling is increased, less ATP would be needed (i.e., the EC50 for ATP would fall) to drive a given rate of current activation.
The results of our studies have important implications for understanding the regulation of ICl, swell. It is entirely possible that channel activation is modulated by multiple factors and signal transduction pathways. The physiological status of the cell, as well as experimental variables such as the rate and mechanism of swelling, and the magnitude of the volume increase may determine which factors and signaling pathways control channel activation. This possibility needs to be taken into account in the design and interpretation of future studies of ICl, swell regulation.
Clearly, extensive additional biophysical and cellular studies are required to test the models described above. A complete understanding of the ATP dependence of the channel ultimately requires molecular identification of the pore-forming and associated regulatory proteins.
How Is the Rate of Cell Swelling Sensed?
An important finding of these studies is the demonstration that the rate of cell swelling alters the ATP requirement of the ICl, swell channel (Figs. 3, 5, 7, and 8). How does a cell sense the rate at which its volume is changing? One possibility is that rate per se is not sensed, but instead there is some critical volume that is reached more rapidly at higher rates of swelling. This critical volume could in turn determine whether the channel activates via the ATP-independent pathway. The data in Table II argue strongly against this possibility. When cells were swollen at 65-70%/min (150-mOsm reduction in bath) in the absence of intracellular ATP, the cell volume set-point of the channel was 1.14. This value is not significantly different from those observed under all other experimental conditions (Table II). Furthermore, in cells that are exhibiting current rundown, current can be reactivated rapidly at various times after rundown begins simply by increasing the rate of swelling to 65-70%/min (Fig. 9 B). Taken together, these results indicate that the cells are sensing the rate of volume increase and not some absolute volume change.
The molecular mechanisms responsible for sensing
the rate of cell swelling are completely unknown. However, it is interesting to speculate that the cytoskeleton
may play an important role. Numerous studies have
demonstrated that the cytoskeleton undergoes structural reorganization in response to mechanical forces.
These changes can occur on a time scale of seconds to
minutes and appear to play important roles in fundamental processes such as maintenance of cell shape,
cell locomotion, mechanosensitivity, and signal transduction (Choquet et al., 1997; Hamill and McBride,
1997
; Ingber, 1997
; Chicurel et al., 1998
).
It is reasonable to postulate that the mechanical stress associated with cell swelling may also induce changes in cytoskeletal organization. These putative changes could be important for maintaining plasma membrane-cytoskeleton interactions, the spatial organization of membrane and submembrane proteins, and the overall mechanical properties of the cell. If the rate of volume perturbation was more rapid than the rate at which cytoskeletal reorganization could take place, it is easy to envision that protein-protein and protein-membrane interactions might be altered. Changes in protein-protein interactions could in turn modulate the ATP dependence, as well as other aspects of ICl, swell activation. Assessment of the role of the cytoskeleton in regulation of ICl, swell will require extensive additional investigation using electrophysiological, pharmacological, molecular, and biophysical approaches.
Physiological Significance of ATP-dependent Channel Regulation
Volume regulatory efflux of structurally diverse organic
osmolytes appears to occur primarily via the ICl, swell
channel (Kirk and Strange, 1998). In addition, the
channel is highly permeable to important metabolic intermediates such as pyruvate, the short-chain fatty acids
acetate, and butyrate, the ketone body
-hydroxybutyrate, and several amino acids (Jackson et al., 1994
).
These metabolites are major inputs into the tricarboxylic
acid cycle. Because of its high organic solute permeability, we have suggested previously that modulation of
ICl, swell channel activation by ATP levels may function as
an important feedback regulatory mechanism (Jackson et al., 1994
; Strange et al., 1996
). Unregulated loss of cellular metabolites would likely disrupt cellular energy production. Regulation of ICl, swell by cellular ATP levels could
therefore prevent depletion of energy-producing carbon
sources when cellular energy production is reduced.
ICl, swell activation has been invoked as an important
component of cellular volume regulation when cells are
swollen by anoxic and ischemic insults (Kimelberg, 1995;
Roy, 1995
). We have argued previously that the requirement for intracellular ATP would rule out or diminish such
a role (Jackson et al., 1994
; see also Patel et al., 1998
).
However, as we have demonstrated here, it is clearly possible to activate the channel in the absence of ATP.
In patch-clamped cells, channel activation via the ATP-independent pathway is controlled by the rate of volume increase. Other factors may control activation by this pathway as well. Whether the ATP dependence of ICl, swell is modulated in intact cells is uncertain and deserves further study. If such modulation occurs, it is interesting to speculate on its physiological significance. As noted above, activation of ICl, swell in a metabolically compromised cell may further compromise metabolism and lead to additional disruption of cellular functions. Thus, if swelling is not too severe, it may be beneficial to the cell to protect its metabolism at the expense of volume homeostasis. On the other hand, if swelling is extreme and membrane lysis is imminent, volume control may take precedence over energy metabolism. We suggest that the modulation of channel ATP dependence may be an adaptation that allows cells to cope with the potentially competing demands of preservation of energy metabolism and volume regulation.
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FOOTNOTES |
---|
Address correspondence to Dr. Kevin Strange, Department of Anesthesiology, Vanderbilt University Medical Center, 504 Oxford House, 1313 21st Avenue South, Nashville, TN 37232. Fax: 615-343-3916; E-mail: kevin.strange{at}mcmail.vanderbilt.edu
Original version received 5 October 1998 and accepted version received 13 January 1999.
Dr. Basavappa's current address is Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520.We thank Dr. Fred Quant for the generous gift of the N1E115 mouse neuroblastoma cells.
This work was supported by National Institutes of Health grants NS-30591 and DK-51610. Dr. Tamara Bond was supported by a Stroke Investigator Award from the Heart and Stroke Foundation of Ontario.
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Abbreviation used in this paper |
---|
NMDG, N-methyl-D-glucamine.
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