1 Division of Biomedical Sciences, University of California, Riverside, California 92521; and 2 Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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Na-K-2Cl cotransporter (NKCC)
and K-Cl cotransporter (KCC) play key roles in cell volume
regulation and epithelial Cl transport. Reductions in
either cell volume or cytosolic Cl
concentration
([Cl
]i) stimulate a corrective uptake of
KCl and water via NKCC, whereas cell swelling triggers KCl loss via
KCC. The dependence of these transporters on volume and
[Cl
]i was evaluated in model duck red blood
cells. Replacement of [Cl
]i with
methanesulfonate elevated the volume set point at which NKCC activates
and KCC inactivates. The set point was insensitive to cytosolic ionic
strength. Reducing [Cl
]i at a constant
driving force for inward NKCC and outward KCC caused the cells to adopt
the new set point volume. Phosphopeptide maps of NKCC indicated that
activation by cell shrinkage or low [Cl
]i
is associated with phosphorylation of a similar constellation of
Ser/Thr sites. Like shrinkage, reduction of
[Cl
]i accelerated NKCC phosphorylation
after abrupt inhibition of the deactivating phosphatase with
calyculin A in vivo, whereas [Cl
] had no specific
effect on dephosphorylation in vitro. Our results indicate that NKCC
and KCC are reciprocally regulated by a negative feedback system dually
modulated by cell volume and [Cl
]. The major effect of
Cl
on NKCC is exerted through the volume-sensitive kinase
that phosphorylates the transport protein.
sodium-potassium-chloride cotransport; intracellular chloride; cell volume regulation; ionic strength; cell water content; sodium-potassium-chloride cotransporter phosphorylation
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INTRODUCTION |
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HOW ANIMAL CELLS DETECT osmotic perturbations and then activate ion transport processes that restore their fluid volume remains incompletely understood. Abrupt swelling typically triggers a corrective release of KCl via cotransport or separate channels, whereas shrinkage evokes ion accumulation via Na-K-2Cl cotransport, Na/H exchange, or Na+ channels (22, 51).
Studies of red blood cells suggested that swelling-activated and
shrinkage-activated ion transporters are coordinately controlled by a
common mechanism (40, 43). When duck red blood cells are
equilibrated in an isosmotic artificial medium, they adopt a relatively
stable fluid volume, termed the "volume set point," at which both
swelling-activated K-Cl cotransporters (KCC) and shrinkage-activated
Na-K-2Cl cotransporters (NKCC) are nearly inactive (16,
52). Osmotic shrinkage and swelling stimulate one cotransport
process and further suppress the other. It has been demonstrated that
this set point is modulated by numerous factors. -Adrenergic
stimulation via intracellular cAMP (37, 52),
Mg2+ loading (53), deoxygenation
(52), calyculin A, and fluoride (37) all
reprogram the set point to larger volumes and thereby stimulate NKCC in
normal cells and suppress KCC in swollen cells. In contrast, depletion
of cytoplasmic Mg2+ (53) or treatment with
N-ethylmaleimide or staurosporine (C. Lytle, unpublished
results) reduces the set point.
Information on cell volume appears to be transmitted to volume-regulatory ion transporters, at least in part, through protein phosphorylation. In duck red blood cells, osmotic perturbations that displace cell volume from the prevailing set point, or interventions that raise the set point above the prevailing cell volume, promote phosphorylation of NKCC at a similar constellation of serine and threonine sites (27). The degree to which these sites are phosphorylated appears to reflect a simple competition between an unknown volume-responsive protein kinase (28) and a type 1 protein phosphatase (PP1) that assembles with the transporter's NH2-terminal domain (10, 45).
Another factor that has emerged as a key negative feedback regulator of
NKCC is cytosolic Cl concentration
([Cl
]i) (3, 4, 12, 14, 15, 24, 29,
36, 46, 48, 49, 56, 58), yet the characteristics and mechanism
of this regulation remain poorly understood. The present experiments
assess whether Cl
ions act allosterically on the
transporter itself or on the upstream biochemical reactions that
determine its volume-dependent phosphorylation. The results indicate
that cytosolic Cl
shifts the set point for activation of
NKCC and KCC to smaller volumes. We used this characteristic of the set
point to confirm that it determines the steady-state volume of the
cell, and we discuss the possible physiological implications of its
modulation by Cl
. The results also suggest that the major
effect of Cl
on NKCC is exerted through the
volume-sensitive kinase that phosphorylates the transport protein.
Finally, we present evidence that the volume set point is not
influenced by the shifts in cytosolic ionic strength (
i)
that accompany cell shrinkage and swelling.
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EXPERIMENTAL PROCEDURES |
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Materials. 86RbCl was obtained from Dupont NEN, staurosporine and calyculin A were from Biomol or LC Labs, protease inhibitors were from Boehringer-Mannheim, 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) was from Aldrich Chemical, and reagent grade chemicals, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), ouabain, and nystatin were from Sigma. Salts of methanesulfonic acid (>99%; Fluka) were prepared by neutralization with NaOH, KOH, or N-methyl-D-glucamine (NMDG). Monoclonal antibody T14, directed against the carboxy-terminal 310 amino acids of the human colonic Na-K-Cl cotransporter, was developed and used as described previously (27, 30). Hybridoma cells were grown in the ascites of pristane-primed severe combined immunodeficiency mice (Taconic CB-17 Fox-Chase SCID).
Preparation of red blood cells. Blood was drawn from the brachial vein of White Pekin ducks (Anas platyrhynchos) into heparinized syringes. After centrifugation, the plasma and buffy coat were discarded and the red blood cells were washed three times in ice-cold duck fluxing solution (DFS; 146 mM NaCl, 6 mM KCl, 0.1 mM Na2PO4, 10 mM glucose, 20 mg/l penicillin, 45 mg/l streptomycin, and 20 mM Na-TES, pH = 7.40 at 41°C, 320 mosmol/kgH2O). To achieve a steady state with respect to ion and water content, cells were routinely preincubated in DFS at 5% hematocrit for 1 h in a gyrating water bath at 41°C.
Alteration of cytosolic Cl.
Cells were incubated in DFS containing methanesulfonate
(MSA
) or sulfamate (SFM
) in place of
Cl
for 30 min at 41°C. These monovalent sulfonates
equilibrate rapidly (t1/2 < 30 s)
across the duck red blood cell membrane via AE1-mediated anion exchange
(26) without altering cell volume or transmembrane Na+, K+, or H+ distributions
(44). In circumstances in which Cl
was
replaced by MSA
or SFM
on one side of the
membrane only, anion gradients and cell pH were stabilized by
inhibition of anion exchange with 250 µM DNDS, which does not affect
NKCC or KCC activity.
Alteration of cytosolic ionic strength.
The electrolyte content of duck red blood cells was altered by
equilibrium dialysis with nystatin (7). Cells were
permeabilized by incubation in an ice-cold medium containing (in mM)
115 KCl, 50 sucrose, 7.5 Na-HEPES (pH 7.65 at 23°C), 5 glucose, 1 MgSO4, and 0.1 NaHPO4 with 45 µg/ml nystatin.
After 20 min, the cells were transferred to in an otherwise identical
"loading medium" containing either 45 KCl or 45 KCl plus 135 K-MSA
and incubated for 60 min at 4°C, yielding cells with half-normal or
twice-normal ionic strength, respectively, and physiological cell
[Cl] and water content. To restore native membrane
permeability, the cells were washed four times at room temperature in
the same loading medium containing 0.4% bovine serum albumin instead
of nystatin. The dependence of NKCC activity on cell volume was
measured by incubating the cells for 10 min at 41°C in flux media
containing (in mM) 40 NaCl, 5 KCl, 7.5 Na-TES (pH 7.4), 5 glucose, 1 MgSO4, 0.1 Na2HPO4, and 0.025 ouabain with trace 86Rb and various concentrations of
sucrose. For cells loaded with extra electrolyte (135 mM K-MSA), the
flux media were supplemented with 135 mM NMDG-MSA to preserve osmotic
balance. Bumetanide-sensitive 86Rb influx was plotted as a
function of intracellular water content, which was measured on paired
cell samples.
Cell volume and ion content.
Intracellular water was measured by a gravimetric method as described
previously (27). For measurement of
[Cl]i, cells were washed twice rapidly in
ice-cold isosmotic sodium-gluconate and then extracted with 3.6%
perchloric acid. Chloride in the extract was analyzed coulometrically
with a Radiometer CMT-10 chloride titrator, with a small correction,
amounting to ~3 mmol/l cell water, for other silver-complexing agents
in the extract (e.g., glutathione).
Transport assays. Ion influx via NKCC or KCC was measured as described previously using 86Rb+ as a surrogate for K+ (27). Red blood cells were preincubated for 10 min (5% hematocrit, 41°C) in hypertonic DFS (plus 100 mM sucrose) to stimulate Na-K-2Cl cotransport or hypotonic DFS (minus 46 mM NaCl) to stimulate K-Cl cotransport. All flux media contained 50 µM ouabain to eliminate 86Rb+ uptake by the Na/K pump. Influx was initiated by the addition of 86Rb+ and terminated 1-10 min later by dilution with ice-cold "stop solution" (DFS containing 250 µM bumetanide). Extracellular 86Rb+ was removed by washing the cells three times in ice-cold stop solution. Intracellular 86Rb+ was quantified by gamma spectroscopy (Beckman). All influx assays were confined to an initial period during which changes in cell volume were negligible and intracellular 86Rb+ increased at a constant rate. Na-K-2Cl cotransport was estimated as the component of 86Rb+ influx inhibited by 10 µM bumetanide; this component constituted ~95% of the ouabain-insensitive 86Rb+ influx in osmotically shrunken cells. K-Cl cotransport was estimated as the 86Rb+ influx inhibited by 1 mM bumetanide; this component constituted ~90% of the ouabain-insensitive 86Rb+ influx in osmotically swollen cells.
Determination of Na-K-2Cl cotransport protein phosphorylation. Immunoprecipitation of NKCC from extracts of 32P-labeled cells was performed as described previously (27) with monoclonal antibody T14 (30). Immunoprecipitates were separated by SDS gel electrophoresis, and the 32P content of the 146-kDa cotransport protein band was analyzed by autoradiography with a storage phosphor screen (PhosphorImager, Molecular Dynamics).
Two-dimensional phosphopeptide analysis. Gel bands containing the 32P-labeled cotransport protein were excised, rinsed thoroughly with water, and equilibrated with 200 mM NH4HCO3. Gel slices were then rotated with 0.5 ml of 200 mM NH4HCO3 containing 100 µg of TPCK-treated trypsin (Sigma) for 24 h at 37°C, with the addition of 50 µg of freshly prepared trypsin after 17 h. The digest was subjected to three cycles of lyophilization and reconstitution in water (0.5 ml). The final residue was dissolved in 20 µl of electrophoresis buffer (10% acetic acid, 1% pyridine, pH 3.5) and spotted onto thin-layer cellulose sheets (20 × 20 cm, Eastman Kodak) along with marker dyes (xylene cyanol FF and phenol red, 2 µg each). Phosphopeptide maps were generated by electrophoresis at 500 V for ~3 h followed by crossed ascending chromatography in 1-butanol-pyridine-acetic acid-water (60:40:12:48 vol/vol/vol/vol). To ensure uniformity between samples, electrophoresis and chromatography were allowed to continue until the marker dyes had migrated fixed distances. Phosphopeptides were detected by autoradiography with a storage phosphor screen.
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RESULTS |
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The set point for osmotic activation of NKCC depended on
[Cl]i (Fig.
1). When extracellular
[Cl
] was maintained at 150 mM and cytosolic
Cl
was exchanged with MSA
, the relation
between NKCC activity and cell water content shifted to the right and
upward (Fig. 1A). Because this maneuver does not
significantly alter cytosolic
i, osmolarity,
[K+], [Na+], pH, or Donnan charge
(26, 44), the causative factor appeared to be the
Cl
anion itself. Experiments conducted on control cells
(in the absence of DIDS) indicated that the effect is rapidly
reversible (data not shown). Replacement of Cl
on both
sides of the membrane produced a similar shift in the relation between
cell water content and NKCC protein phosphorylation (Fig.
1B), whereas replacement of extracellular Cl
alone had no detectable effect (data not shown). Thus cytosolic Cl
influences the reactions that couple NKCC
phosphorylation to cell volume.
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Certain anions of the lyotropic series are known to perturb cellular
processes through their effects on protein charge, conformation, and
aggregation (9). In red blood cells, for example,
substitution of Cl with NO
, or SCN
, but not with MSA
or SFA
, increases the net negative charge on
intracellular protein, causing a loss of permeant anions, a decrease in
cell volume, and an acidic shift in cytosolic pH (44).
Despite their disruptive nature, NO
have been commonly used as replacements for
Cl
in studies of volume-responsive ion transport. To
determine whether the effects we observed here are caused by removing
Cl
or by introducing the replacement anion, we compared
the effects of MSA
, NO
on the volume set point. Each of these anions
equilibrates within seconds across the red blood cell membrane via AE1,
but none is transported by NKCC (17, 26). When 75% of
cellular Cl
was exchanged with NO
, much greater degrees of shrinkage were required to
activate NKCC (Fig. 2). The effect of
anions on the set point (expressed as l cell water/kg cell solid)
followed a typical "lyotropic" sequence: MSA
(l.6) > Cl
(l.5) > NO
(l.1). Besides shifting the set point
to smaller volumes, NO
also
appeared to inhibit ion translocation via NKCC (bumetanide-sensitive 86Rb influx) in maximally shrunken cells.
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When duck red blood cells are equilibrated in an artificial isotonic
medium, they eventually adopt a water content at which both NKCC and
KCC are essentially inactive (16). In this "resting" state, NKCC units remain partially phosphorylated, presumably at sites
necessary but not sufficient alone to trigger ion translocation (27, 28). Replacement of Cl with
NO
(Figs. 1-3) or
SFA
(Fig. 3) promoted NKCC phosphorylation in resting
cells but evoked no further phosphorylation of NKCC units already
stimulated by cell shrinkage or norepinephrine. NKCC phosphorylation,
whether evoked by cell shrinkage, norepinephrine, or Cl
replacement, decreased to undetectable levels within 10 min after inhibition of kinase activity with 1 mM N-ethylmaleimide or
5 µM staurosporine (data not shown). These results indicate that Cl
, and to a greater degree, NO
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The relation between [Cl]i and spontaneous
NKCC activity is shown in Fig. 4. After
altering [Cl
]i, 86Rb influx via
NKCC was assayed in a Cl
medium containing DNDS (to
minimize AE1-mediated dissipation of the
Cl
/MSA
gradients). NKCC activity increased
as [Cl
]i decreased below ~60 mM. Because
Cl
participates directly in the cotransport reaction
as a transported substrate, part of its effect could reflect
trans-inhibition, a characteristic of many cotransport
mechanisms with ordered substrate binding (54). To
distinguish whether the inhibitory effect of [Cl
]i on inward cotransport is kinetic or
regulatory in nature, the experiment was repeated on cells treated with
calyculin A, a potent inhibitor of PP1 and PP2A (18). By
preventing NKCC dephosphorylation, this agent renders the cotransporter
maximally active and refractory to the deactivating influences of cell
swelling, N-ethylmaleimide, or staurosporine
(27). With NKCC fixed in its phosphorylated form, changes
in [Cl
]i had a much smaller influence on
bumetanide-sensitive 86Rb influx (Fig. 4). These results
substantiate earlier conclusions, based on ion flux studies with squid
axon, that the predominant effect of [Cl
]i
on inward cotransport is regulatory and not related to titration of the
internal Cl
transport sites (4, 50).
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If shrinkage-activated NKCC and swelling-activated KCC respond to the
same signal of volume change, alterations in
[Cl]i might exert tandem effects on their
respective set points. Increasing [Cl
]i
from 0 to 70 mM should reduce the set point from ~1.95 to ~1.55 l
water/kg cell solid (Fig. 1). To test this prediction, the relation between [Cl
]i and KCC activity was
determined in cells osmotically swollen to 1.95 l water/kg cell
solid. As predicted, KCC activity was high in Cl
-rich
cells (where the set point < cell volume) yet absent in Cl
-free cells (where the set point = cell volume)
(Fig. 5). Comparable results were obtained with SFM
rather than MSA
in place of Cl
. Although
the [Cl
]i supporting half-maximal KCC
activity (~10 mM) appeared to be somewhat lower than that yielding half-maximal NKCC activity (~30 mM;
Fig. 4), the two values cannot be compared directly because they were
obtained under different experimental conditions. The fact that KCC
activity was measured in swollen cells whereas NKCC activity was
measured at normal volume could be especially important, because work
on the squid axon (3) demonstrated that cell swelling shifts the relation between NKCC activity and
[Cl
]i to lower concentrations (see
DISCUSSION). These results add [Cl
]i to the list of physiological factors
(norepinephrine, deoxygenation, [Mg2+]i)
known to produce concerted shifts in the volume dependence of NKCC and
KCC in duck red blood cells.
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If the coordinate operation of NKCC and KCC actually determines the
physiological volume of the cell, increasing the set point by reducing
[Cl]i should bring about a corresponding
increase in steady-state water content. To test this hypothesis, duck
red blood cells were incubated in a synthetic, isotonic medium similar
to plasma for 1 h
long enough for them to adopt and maintain the
so-called "lower steady-state" volume (47) or
"volume set point" (40) at which both NKCC and KCC are
minimally active (31, 16). To remove potential
thermodynamic and kinetic constraints on ion uptake via NKCC,
extracellular K+ concentration
([K+]o) was raised from 6 mM to 10 mM. This
maneuver, by itself, does not affect the volume of resting cells but
promotes rapid swelling if NKCC is made active by hormonal
(norepinephrine) or osmotic stimulation (31). Thus under
these experimental conditions, the extent to which NKCC affects cell
volume is determined by regulatory rather than thermodynamic or kinetic
factors. Cells were then incubated for 3 h in isotonic media
containing different concentrations of Cl
, 156, 78, or 31 mM. Because partial substitution of Cl
with
MSA
does not alter the distribution ratios
([ion]i/[ion]o) of Na+,
K+, or Cl
(44), the net chemical
potential for inward NKCC should be equivalent in all three batches of
cells, as should that for outward KCC. As predicted, reprogramming the
set point to larger volumes by reducing
[Cl
]i caused the cells to seek
correspondingly larger steady-state volumes (Fig.
6). In two other experiments we noted
that adding calyculin A, which elevates the set point to extreme
degrees, caused all three batches of cells to swell rapidly to a very
large volume and that inhibition of both NKCC and KCC with furosemide (1 mM) prevented these volume shifts. These observations suggest that
the steady-state water content of the duck red blood cell is determined
by a controlled uptake or release of salt via NKCC or KCC, as dictated
by the volume set point.
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An immediate effect of osmotic perturbation is dilution or
concentration of cytosolic salt. It has been asserted that changes in
i initiate or modulate volume regulatory responses
(6, 13, 57), and in red blood cells there is evidence that
i influences the volume set point for swelling-activated
K-Cl cotransport (43, 13) and shrinkage-activated Na/H
exchange (43). To evaluate this possibility, we compared
the relation between NKCC activity and cell volume in cells enriched
twofold or depleted twofold in electrolyte by the technique of nystatin
dialysis. To avoid ancillary effects of anions,
[Cl
]i was held constant at 30 mM and
i was varied by adding or omitting the inert ion pair
K+-MSA
. Changing
i over a very
broad range had no significant effect on the volume set point for
activation of NKCC (Fig. 7).
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Two-dimensional phosphopeptide maps of NKCC protein isolated from
osmotically shrunk 32P-labeled cells showed a distinctive
pattern of tryptic phosphopeptides similar to those obtained previously
(27), consistent with heterogeneous phosphorylation at
multiple sites. NKCC units phosphorylated by cell shrinkage and by
Cl removal yielded qualitatively indistinguishable maps
(Fig. 8). Twelve discrete spots,
designated spots 1-12 in Fig. 8, were detected with
both stimuli. The relative intensity of the major spots was consistent
between experiments. One exception was spot 11, which appeared in some but not all samples of NKCC isolated from shrunken cells. These results suggest that Cl
removal and cell
shrinkage promote phosphorylation of a similar constellation of
regulatory sites, and by inference, act ultimately through the same
protein kinase on NKCC. This view is consistent with our finding that
one form of stimulation precludes further NKCC phosphorylation by the
other (Fig. 3) and that both stimuli are equipotently blocked
(IC50
0.5 µM) by staurosporine (data not shown).
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NKCC is deactivated by a PP1 that associates with its
NH2-terminal domain (45, 10). Because the
phosphorylation state of NKCC reflects a competition between concurrent
kinase and phosphatase activities (45, 27), net
phosphorylation could result from kinase stimulation, phosphatase
inhibition, or a combination of both. To determine whether
Cl depletion stimulates the kinase, as does cell
shrinkage (27, 28), we compared the rate at which NKCC
becomes active in cells containing either Cl
or
MSA
after adding calyculin A (Fig.
9). This approach takes advantage of the
fact that calyculin A enters the duck red blood cell within seconds and
blocks ongoing dephosphorylation of NKCC at all volume-sensitive Ser/Thr residues (27), presumably by inhibiting the
transporter's PP1 subunit. As we found previously (28),
calyculin A evoked a progressive increase in both NKCC activity and
NKCC protein phosphorylation (Fig. 9), which conformed to a logistic
function describing a positive feedback model. In earlier work
(28) we showed that the process includes an initial phase
(between 0 and 2 min) that depends strongly on cell volume and a
secondary phase that does not. Like cell shrinkage, replacement of
intracellular Cl
with MSA
at a normal
volume had only a minor influence on the ultimate level of NKCC
activity evoked by calyculin A (Fig. 4) whereas it markedly increased
the initial rate at which this level was reached. If the initial phase
of activation by calyculin A accurately reflects the activity of the
volume-sensitive protein kinase that phosphorylates NKCC, the data
indicate that this kinase is much more active in cells containing
MSA
in place of Cl
with the same volume,
osmolarity, Donnan charge, ionic strength, pH, and protein
concentration.
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To assess whether Cl stimulates the PP1 subunit, we
measured the influence of various anions on NKCC dephosphorylation in vitro. Cells were equilibrated with 32P and osmotically
shrunk to label NKCC. When these cells were lysed in 5 volumes of warm
permeabilization buffer, the cotransport protein lost 32P
rapidly (Fig. 10A).
Dephosphorylation could be prevented by adding either 0.1 µM
calyculin A (Fig. 10A) or 7 µg/ml inhibitor-2 (an endogenous inhibitor of PP1) or by chilling the permeabilization buffer
to 2°C. Rinsing the lysed cells free of cytosolic residue with an
ice-cold buffer of physiological ionic strength did not alter the
subsequent rate of dephosphorylation in warm permeabilization buffer.
We surmise that the observed dephosphorylation reflects the activity of
bound PP1. Adding 150 mM NaCl increased this activity ~40% (Fig.
10B). Similar increases were also observed with sodium salts
of MSA
and NO
itself. As expected, the fluoride anion, which is
known to inhibit PP1, blocked dephosphorylation at 10 mM almost as
effectively as calyculin A. Thus whereas Cl
strongly
inhibits phosphorylation of NKCC, it has no apparent effect on
dephosphorylation.
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DISCUSSION |
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Our results substantiate the concept that NKCC and KCC are
reciprocally regulated by a negative feedback system that is dually modulated by cell volume and [Cl]. The data demonstrate
that intracellular Cl
alters the volume dependence of
NKCC by inhibiting the shrinkage-stimulated protein kinase that
converts the cotransport protein into an active form. Anions with
greater chaotropic character (Cl
< NO
) cause the cell to behave as
if increasingly swollen, apparently by inhibiting the same
volume-sensitive kinase.
Since its original description in squid axon (49),
modulation by [Cl]i has become recognized
as a common if not universal property of NKCC in animal cells (2,
4, 12, 14, 15, 24, 29, 36, 46, 48, 56, 58). The present work
demonstrates that [Cl
]i exerts tandem
effects on shrinkage-induced NKCC and swelling-induced KCC by shifting
their shared volume set point. A growing body of evidence suggests that
other volume-responsive ion transport pathways are likewise affected by
Cl
and foreign anions. For example, like NKCC, activation
of Na/H exchange by cell shrinkage is repressed if Cl
is
replaced with NO
(1, 11,
19, 39, 42, 43), whereas substitution with less disruptive or
impermeant anions like gluconate has the opposite effect
(48). Replacing Cl
with
NO
also reduces the degree of
swelling required to activate K-Cl cotransport (23, 42)
and Na/Ca exchange (38) in red blood cells. These
observations reinforce the concept that volume-activated transporters are coordinately regulated by a common system of sensors
and transducers (40).
Our finding that [Cl]i modulates the volume
dependence of NKCC is fully consistent with observations made by
Breitweiser and colleagues (3, 4). Working on the
internally dialyzed squid giant axon, they demonstrated that ion influx
via NKCC is tonically suppressed by physiological levels of
[Cl
]i and that cell shrinkage relieves this
inhibition by shifting the relation between
[Cl
]i and ion influx. Our data, when
plotted in the same manner, indicate a similar effect. Thus in both
squid axon and duck red blood cell, the regulation of NKCC by cell
volume and [Cl
] is mutually interdependent, i.e., each
signal influences the set point of the other. This linkage is
consistent with our evidence that the two signals converge on a protein
kinase that phosphorylates NKCC.
The effect of Cl substitution depends on the replacement
anion used. Relatively inert anions like MSA
and
SFM
(44) cause the duck red blood cell to
perceive itself shrunken at normal volume, whereas chaotropic anions
like NO
have the opposite
effect. The sequence of anion effects on the set point regulating NKCC
and KCC corresponds to a typical lyotropic series:
MSA
< Cl
< NO
. Besides affecting its
biochemical activation, NO
seem to interfere with ion translocation by NKCC as their presence reduces the rate of inward cotransport even after changes in
phosphorylation are prevented with calyculin A.
How anions affect the generation or the transmission of the volume
signal to NKCC and KCC remains unclear. Both Cl and
NO
]i is reduced (2). Our
evidence that low [Cl
]i and cell shrinkage
promote phosphorylation of NKCC in a nonadditive manner at common sites
implicates a single protein kinase modulated jointly by
[Cl
]i and cell volume. If osmotic
activation of this kinase involves macromolecular crowding or molecular
confinement, anions like Cl
and NO
ions discourage phosphorylation of
NKCC by interacting with the cotransporter itself. For example,
titration of one or both internal Cl
transport sites
could induce a conformation that is less accessible to the
shrinkage-stimulated kinase.
A curious feature of NKCC in most cells is that it responds much more
strongly to an episode of shrinkage caused by a loss of KCl and water
(isosmotic shrinkage) than to one caused by a loss of water alone
(hypertonic shrinkage). As noted by O'Neill (35), this
can be explained by differences in [Cl]i;
the decrease in [Cl
]i that attends
isosmotic shrinkage would act to elevate the volume set point and
thereby amplify the perceived degree of shrinkage, whereas the increase
in [Cl
]i that attends hypertonic shrinkage
would blunt the volume signal.
Cl may affect cotransport activity beyond its influence
on thermodynamic driving force (36, 35) and NKCC
phosphorylation (20). In endothelial cells, shrinkage
stimulates K+ influx via NKCC (36, 33) along
with NKCC phosphorylation (21, 34). Curiously, net salt
uptake is not evident, even though energetically favorable, unless
[Cl
]i is decreased (36, 20). A
similar phenomenon has been noted in Ehrlich ascites tumor cells
(25). O'Neill (35) proposed that
physiological [Cl
]i impedes reorientation
of the unloaded transporter, compelling the transporter to engage in an
unproductive exchange of extracellular for intracellular ions instead
of net salt uptake. Reducing [Cl
]i would
eliminate this kinetic barrier and permit net salt movement. The
proposed action of [Cl
]i is analogous to
that of aerobic metabolism on the antiport/uniport switch mechanism of
glucose transporters in avian red blood cells (8). If this
form of regulation by [Cl
]i exists, it is
not apparent in duck red blood cells, where the net uptake and the
Na/Na exchange modes of cotransport change in fixed proportion as
[Cl
]i is varied from 6 to 90 mM
(26).
Animal cells seem capable of detecting not only the severity of a
volume perturbation but also its cause (13, 32, 35, 55).
For example, trout red blood cells engage different volume-regulatory mechanisms depending on whether swelling is caused by a gain of KCl
plus water or of water alone (13, 32). Hypotonic swelling, which lowers [Cl]i, evokes a pathway for
efflux of organic osmolytes that resembles the volume-sensitive organic
anion channel VSOAC (6, 57). In contrast, isosmotic
swelling, which raises [Cl
]i, triggers only
K-Cl cotransport. Although the differential response has been
attributed to changes in
i, the experimental findings do
not exclude the possibility that the discriminating parameter is
instead [Cl
]i. Our finding that the volume
set point varies inversely with [Cl
]i could
explain why KCC is more responsive to isotonic vs. hypotonic swelling.
Parker et al. (43) reported that the set point common to
swelling-activated KCC and shrinkage-activated Na/H exchange in dog red
blood cells varies inversely with i. Analogous effects of
i were not apparent in duck red blood cells: raising
or lowering
i twofold produced no significant change in
the volume set point. The discrepancy might be explained by their use
of NO
, to elevate
i. In duck red blood cells, NO
itself through a mechanism that is entirely
independent of
i (Fig. 2).
The relevance of volume-activated transport processes in vitro to
volume homeostasis in vivo remains uncertain (35, 40). Two
observations suggest that duck red blood cells seek their volume set
point through the controlled operation of NKCC and KCC. First, when
cells from freshly drawn blood are incubated in a synthetic, isotonic
medium similar to plasma, they gradually shrink ~5% to the so-called
lower steady-state volume (47, 16). The loss of salt and
water is mediated by KCC (16) and is associated with a
corresponding shift in the set point to the lower steady-state volume
that occurs when the cells are removed from the influence of endogenous
plasma catecholamines (52). Second, when the set point is
experimentally altered by manipulating
[Cl]i, the cells adopt and maintain the new
set point volume (Fig. 6).
The effect of Cl on the volume set point for NKCC and KCC
in duck red blood cells is especially powerful in the range of
[Cl
] ordinarily found in other vertebrate animal cells
(10-50 mM). If other cells employ a cognate system of volume
sensors and effectors, physiological fluctuations of
[Cl
]i could have an important influence on
their volume set point and hence water content. Cells would perceive
their volume as lower in circumstances in which
[Cl
]i is reduced. This modulation could
enable the cell to gauge not only the severity of the volume
perturbation but also its underlying cause so as to implement a
corrective strategy that restores both volume and
[Cl
]i (35, 55). For example,
if the cell loses both KCl and water (isosmotic shrinkage), the
attendant decrease in [Cl
]i would
potentiate phosphorylation of NKCC and corrective salt uptake. On the
other hand, if the cell loses only water (hypertonic shrinkage), the
increase in [Cl
]i would desensitize NKCC to
the shrunken state and avert further enrichment of cytosolic
Cl
. In a similar manner, modulation of the response to
cell swelling by [Cl
]i would help restore
ionic balance, e.g., swelling due to Cl
uptake would
potentiate activation of KCC. This modulation could function to
stabilize [Cl
]i and defend against
potentially deleterious changes in cytosolic
i.
Cl could play a special role in red blood cells, which
are much richer in Cl
(~74 mM) and protein (~8 mM
hemoglobin) than most other animal cells. At this high concentration,
the effect of Cl
on the volume set point is near maximal
and flat. Although its effect is clearly not modulatory in the red
blood cell, high [Cl
]i could act in a
continuous manner to maintain the set point at a smaller volume. This
could create the permissive environment necessary for red blood cells
to package exceptionally high concentrations of soluble protein
(hemoglobin). Thus, following the macromolecular crowding theory of
cell volume perception (40), high
[Cl
]i might serve to offset the effect of
inordinate macromolecular crowding on the volume set point, which if
unopposed, might make the red blood cell seek a swollen state with
adverse rheological consequences. This teleological rationalization
assumes that volume-sensing mechanisms based on macromolecular crowding
are shared by red blood cells and other cells alike, which may not be so.
In summary, these results suggest that in duck red blood cells water
content and [Cl]i are maintained at
interdependent set points by the coordinate control of separate
KCl-loading (NKCC) and KCl-extruding (KCC) transporters. The cell's
perception of its fluid volume is influenced by cytosolic
[Cl
], and vice versa, but not ionic strength.
Information on cell volume and [Cl
]i
appears to be transduced to NKCC by the same protein kinase. This
regulation could enable the cell to gauge not only the severity of the
volume perturbation but also its underlying cause so as to implement a
corrective strategy that restores both volume and [Cl
]i.
![]() |
ACKNOWLEDGEMENTS |
---|
The technical assistance of Rui Liu is appreciated.
![]() |
FOOTNOTES |
---|
This work was supported by the National Science Foundation (NSF-MCB9904605).
Address for reprint requests and other correspondence: C. Lytle, Div. of Biomedical Sciences, 2226 Webber Hall, Univ. of California, Riverside, Riverside, CA 92521 (E-mail: christian.lytle{at}ucr.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00130.2002
Received 19 March 2002; accepted in final form 5 July 2002.
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