Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This work was undertaken to
obtain a direct measure of the stoichiometry of
Na+-independent K+-Cl cotransport
(KCC), with rabbit red blood cells as a model system. To determine
whether 86Rb+ can be used quantitatively as a
tracer for KCC, 86Rb+ and K+
effluxes were measured in parallel after activation of KCC with N-ethylmaleimide (NEM). The rate constant for NEM-stimulated
K+ efflux into isosmotic NaCl was smaller than that for
86Rb+ by a factor of 0.68 ± 0.11 (SD,
n = 5). This correction factor was used in all other
experiments to calculate the K+ efflux from the measured
86Rb+ efflux. To minimize interference from the
anion exchanger, extracellular Cl
was replaced with
SO
efflux at 25°C under these conditions is ~100,000-fold smaller than
the uninhibited Cl
/Cl
exchange flux and is
stimulated ~2-fold by NEM. The NEM-stimulated 36Cl
flux is inhibited by okadaic acid and
calyculin A, as expected for KCC. The ratio of the NEM-stimulated
K+ to Cl
efflux is 1.12 ± 0.26 (SD,
n = 5). We conclude that
K+-Cl
cotransport in rabbit red blood cells
has a stoichiometry of 1:1.
red blood cells; N-ethylmaleimide; ion regulation
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE COUPLED
COTRANSPORT of Cl with alkali cations serves a
variety of cellular functions, including transepithelial fluid absorption and secretion, cell volume regulation, and regulation of
intracellular Cl
(21, 41, 47). The first
cation-Cl
cotransporter to be described functionally
(16) and at the molecular level (51, 59) is
known as NKCC and mediates the electroneutral cotransport of 1 Na+, 1 K+, and 2 Cl
. A related
family of transporters, the K+-Cl
cotransporters (KCC), mediate the Na+-independent
cotransport of K+ and Cl
. KCC was first
characterized functionally in erythrocytes (14, 38) and
epithelia (37, 53). The cDNA sequences and tissue distributions of several KCC isoforms have been determined (19, 24, 25, 48, 50, 52). The most widely distributed isoform is
KCC1, which is activated by cell swelling or
N-ethylmaleimide (NEM) (19). Another isoform,
KCC2, is expressed specifically in neurons (50) and has a
role in determining the intracellular Cl
concentration
and the excitatory or inhibitory action of Cl
channels
(42, 54).
It is very clear that KCC represents obligatory cotransport rather than
interdependent Cl and K+ channels. For
example, in the basolateral membrane of Necturus gall
bladder, the effects of K+ gradients on net
Cl
fluxes are as expected for cotransport, rather than
separate channels, and the Cl
conductance is too low to
account for the Cl
flux (53). In red blood
cells, net uphill K+ transport can be driven by a
Cl
gradient, under conditions in which the membrane
potential is clamped with NO
To understand the functional consequences of
K+-Cl cotransport, it is important to know
the stoichiometry of the process. For example, if the stoichiometry is
1:1, then the driving force for net ion flux is zero when
[K+]i[Cl
]i = [K+]o[Cl
]o (where
[K+]i and [Cl
]i
represent intracellular K+ and Cl
concentrations and [K+]o and
[Cl
]o represent extracellular
K+ and Cl
concentrations) irrespective of the
membrane potential.1 For the
K+ gradients commonly present in cells {i.e.,
([K+]in/[K+]out)
~25}, a 1:1 K+-Cl
cotransporter would be
at equilibrium when intracellular Cl
concentration is 4 mM and extracellular Cl
concentration is 100 mM.
Therefore, at any intracellular Cl
concentration >4 mM,
1:1 KCC would mediate net efflux. However, if the stoichiometry is
actually not equimolar, the driving force for transport and the
consequences of KCC for intracellular Cl
would be very different.
There are several kinds of evidence, in addition to the lack of
potential dependence of the flux (35), that, in fact, the stoichiometry of KCC is 1:1. Most studies of the K+ and
Cl concentration dependence of KCC show no sign of more
than one binding site for each ion (3, 11, 19, 38, 52).
Most data on the flux reversal point, i.e., the K+ (or
Rb+) and Cl
gradients that result in zero net
flux (38, 40), indicate 1:1 cotransport. Finally, the
magnitudes of the net K+ and Cl
fluxes in
osmotically swollen duck red blood cells (44) and the
basolateral membrane of Necturus gall bladder
(53) are approximately equal.
Although the above data strongly suggest that KCC1 has 1:1
stoichiometry, there is one missing piece of information: a direct measurement of KCC-mediated net K+ and Cl
fluxes under conditions in which 1) the membrane potential
is clamped to remove the requirement that net K+ and
Cl
fluxes be equal to preserve global electroneutrality
and 2) the KCC-mediated fluxes are clearly distinguished
from those through other transport pathways for K+ and
Cl
. To obtain a direct estimate of the stoichiometry of
KCC, we have used the rabbit erythrocyte as a model system. The rabbit red blood cell has a comparatively large Cl
-dependent
K+ (or 86Rb+) flux that is
stimulated by cell swelling or treatment with NEM (2, 58).
This flux is very likely mediated by KCC1, although it is possible that
KCC3 is also expressed in red blood cells (52). The
KCC-mediated 86Rb+ flux, although large
compared with that in red blood cells of other mammalian species, is
still at least four orders of magnitude lower than the
36Cl
/Cl
exchange flux mediated
by the anion exchanger AE1 (band 3) (4). Nonetheless, we
have found that it is possible to inhibit the band 3-mediated flux by a
factor of ~105 by a combination of H2DIDS and
removal of extracellular Cl
and HCO
efflux, which is inhibited by okadaic
acid and calyculin A, as expected for KCC (32, 34, 57).
The NEM-stimulated K+ (or 86Rb+)
and 36Cl
effluxes indicate that the
stoichiometry of KCC in rabbit red blood cells is 1:1.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials.
Blood was drawn from an ear vein of a healthy New Zealand White rabbit
into heparin and stored for 4 days at 4°C as whole blood.
Radionuclides (86Rb+ as RbCl and
36Cl
as NaCl) were obtained from DuPont NEN
(Boston, MA). H2DIDS was synthesized from 4,4'
diaminostilbene-2,2'-disulfonic acid (DADS) by a modification
of the method used by Cabantchik and Rothstein (8) as
described previously (30). Okadaic acid and calyculin A
were purchased from Calbiochem. All other buffers, salts, and reagents
were obtained from Sigma (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA).
Cell preparation and NEM treatment. Cells were washed three times and incubated for 90 min at 37°C in HEPES-buffered physiological saline (HPS: 150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM sodium phosphate, 10 mM HEPES, pH 7.4) plus 10 mM glucose to try to establish a reproducible steady state. For 86Rb+ efflux measurements, the incubation in HPS included 1 µCi/ml 86RbCl. After the incubation in HPS, cells were washed once in HPS, suspended at 5% hematocrit in HPS, and chilled on ice before treatment with 2 mM NEM as described previously (28). The NEM treatment was at 0°C; treatment at low temperature avoids the inhibitory side reactions that take place during exposure of cells to high concentrations of NEM at 37°C (39). The suspensions were incubated for 15 min on ice and washed once, and the treatment was repeated. Finally, the cells were washed once in HPS, and the pellet was incubated for 20 min at 37°C to activate KCC; the details of the kinetics of activation of KCC after NEM pretreatment are described elsewhere (28). Calyculin A, if present, was added to a final concentration of 50 nM immediately before the 37°C activation incubation. After the 20-min incubation at 37°C, KCC is fully activated. The flux itself was measured at 25°C, because the KCC flux (when fully activated with NEM) has a relatively low temperature dependence at 25-37°C (31); therefore, the KCC flux is larger at 25°C relative to other transport pathways than it is at 37°C.
Comparison of 86Rb+ and
K+ efflux.
To evaluate 86Rb+ as a quantitative tracer for
K+, we measured net 86Rb+ efflux
and K+ efflux simultaneously in parallel suspensions of
cells from the same preparation. Cells were prepared exactly as
described above (with or without NEM treatment) and were suspended in
160 mM NaCl, 10 mM HEPES hemisodium (a 1:1 mixture of HEPES free acid
and Na+-HEPES, resulting in a pH equal to the pK
of HEPES), pH 7.45, and 104 M ouabain at 25°C. The
extracellular K+ concentration was measured at various time
points by immersion of a K+-selective electrode (Fisher
Scientific) in the suspension immediately after calibration of the
electrode in 160 mM NaCl, 10 mM HEPES, pH 7.45, and 0.01-10 mM
KCl. Total K+ in the suspension was measured by the same
method after lysis by repeated freezing and thawing. To allow
calculation of the volume of cells per volume of suspension, cells were
added to the medium from a loosely packed pellet with a positive
displacement pipette, and the hematocrit of an aliquot of the stock
packed suspension was measured. The intracellular K+
content determined in this manner was 111 ± 6.4 (SD) meq/l cells (n = 4 preparations), similar to published values
(10).
86Rb+ and
36Cl efflux measurements.
To compare NEM-stimulated 86Rb+ and
36Cl
effluxes, cells were pretreated with NEM
as described above, and the efflux of 86Rb+ or
36Cl
was measured at 25°C in a medium
consisting of 120 mM Na2SO4, 20 mM MOPS, pH
7.1, 20 µM H2DIDS, 40 µM 2,4-dinitrophenol (2,4-DNP), and 100 µM ouabain. All NEM pretreatments and subsequent washes can
be performed on 86Rb+-loaded cells with
negligible losses of tracer. For 36Cl
efflux
measurements, cells were preincubated exactly as described above (but
without 86Rb+). After the 20-min incubation at
37°C to activate KCC, cells were loaded with
36Cl
by incubating a 50% suspension in HPS
containing 1 µCi/ml Na36Cl for 5 min at 25°C. Cells
were then washed twice in chilled 120 mM
Na2SO4, 20 mM MOPS hemisodium, pH 7.1, and 10 µM H2DIDS and immediately resuspended in flux medium to
start the efflux measurement. The medium had been bubbled with
N2 to minimize the HCO
for SO
Cl contents and flux calculation.
Cl
contents were determined by mixing 0.5 ml of stock
suspension (80% hematocrit) with 0.3 ml of HPS containing a known
amount of 36Cl
. After the cells and medium
were allowed to equilibrate for a few minutes at 25°C, cells were
centrifuged, and the radioactivity in the supernatant was determined by
scintillation counting. The cellular Cl
was calculated
from the total 36Cl
, the final extracellular
36Cl
, the total extracellular water, and the
volume of cells. In a medium containing 160 mM Cl
, the
Donnan ratio
([Cl
]in/[Cl
]out)
at 25°C and pH 7.4 was 0.75, which is close to the known value for
human red blood cells under these conditions (22). The net
Cl
flux (meq · l
cells
1 · h
1) was calculated from
the rate constant for 36Cl
efflux
(h
1) times the cellular Cl
contents (meq/l cells).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Comparison of NEM-stimulated K+ and
Rb+ efflux.
These experiments were designed to measure NEM-stimulated net
K+ and Cl effluxes under conditions in which
the membrane potential is clamped at ~0 mV to remove electrical
constraints on the K+ and Cl
fluxes. In our
experience, the most accurate way to measure solute transport is with
radioactive tracers, and 86Rb+ is commonly used
as a tracer for K+ in the study of KCC (14,
38). To determine whether 86Rb+ is a
quantitative tracer for K+ with regard to rabbit red cell
KCC, cells were washed and loaded with 86Rb+,
and KCC was activated by pretreatment with NEM. Cells were then washed
and resuspended in 160 mM NaCl-10 mM HEPES, pH 7.45, at 25°C for
measurement of efflux of 86Rb+ and
K+ as described in MATERIALS AND
METHODS.
|
Clamping the membrane potential with 2,4-DNP.
The goal of these experiments is to measure NEM-stimulated
K+ (or 86Rb+) and Cl
effluxes under conditions of maximum inhibition of other transport pathways for these ions. However, if K+ and
Cl
are the main permeant ions, the constraint of
electroneutrality would require that net K+ and
Cl
effluxes be equal, even if the actual cotransport
itself were an electrogenic 1:2 or 2:1 process. To remove the
electrical constraint on the K+ and Cl
fluxes, the protonophore 2,4-DNP was added to the flux medium to
provide a conductive pathway for H+. Macey et al.
(45) showed that protonophores can increase the H+ permeability of the human red cell membrane sufficiently
to make the membrane potential close to the Nernst potential for
H+. This finding is in agreement with the earlier
observation of Harris and Pressman (23) that protonophores
increase the rate of valinomycin-mediated K+ efflux in
human red blood cells.
Inhibition of Cl exchange by H2DIDS and
removal of extracellular Cl
.
It is so difficult to measure a Cl
tracer flux associated
with red cell KCC because red blood cells have a very large
Cl
/Cl
exchange flux mediated by band 3 (AE1). In a medium containing 150 mM Cl
at pH 7.4, the
rate constant for 36Cl
/Cl
exchange in rabbit red blood cells at 0°C is ~3/min (unpublished data), which is similar to that in human red blood cells under comparable conditions (20). With the assumption that the
temperature dependence of red cell Cl
/Cl
exchange is the same in rabbits and humans (4), the rate
constant for Cl
/Cl
exchange at 25°C in
rabbit red blood cells should be ~180/min. The rate constant for
NEM-stimulated 86Rb+ efflux in rabbit red blood
cells at 25°C is ~0.0015/min (see below), which is a factor of
100,000 slower than the AE1-mediated Cl
/Cl
exchange flux. Accordingly, it is necessary to inhibit the AE1-mediated flux by a very large factor to have a chance of detecting a
Cl
flux associated with K+-Cl
cotransport.
Lack of effect of traces of CO2.
To minimize residual Cl/HCO
/HCO
efflux significantly.
Lack of effect of 20 µM H2DIDS on NEM-stimulated
86Rb+ efflux.
To use H2DIDS as an inhibitor of band 3 in these
experiments, it is necessary to determine whether H2DIDS
affects KCC under these conditions. In human red blood cells at 0°C,
H2DIDS inhibits Cl/Cl
exchange
half-maximally at 0.05 nM (56); 20 µM should therefore produce >99% inhibition of anion exchange. We found that 20 µM H2DIDS in the flux medium does not have a detectable effect
on either the basal or the NEM-stimulated 86Rb+
efflux in red blood cells (Fig. 2).
|
NEM-stimulated 36Cl efflux.
Figure 3 shows that pretreatment of cells
with 2 mM NEM stimulates 36Cl
efflux into an
Na2SO4 medium containing 2,4-DNP and
H2DIDS. The baseline flux is very consistent among
different cell preparations; we do not know the nature of this
Cl
transport pathway. We found a clear stimulation of the
36Cl
efflux by NEM in eight of eight
experiments. The NEM-stimulated flux is inhibited by the protein
phosphatase inhibitors calyculin A (Fig.
4) and okadaic acid (not shown), which
are known inhibitors of activation of K+-Cl
cotransport (32, 34). There is no measurable effect of
calyculin A on the 36Cl
in the absence of NEM
treatment, but a small KCC-mediated Cl
flux could be
present and not detected.
|
|
Stoichiometry of NEM-stimulated K+
(or 86Rb+) and
Cl efflux.
In five cell preparations, the tracer effluxes of
86Rb+ and 36Cl
were
measured in parallel in the same flux solution used in Figs. 3 and 4,
with or without pretreatment of the cells with NEM. The pretreatments
were the same for the 86Rb+ and
36Cl
efflux measurements; the only difference
was the radionuclide. The ratio of the NEM-stimulated K+
efflux to Cl
efflux ranged from 0.79 to 1.45 (Fig.
5), with an average of 1.12. The
variations among different experiments are considerable, because
experiments require measurement of differences. It is clear,
however, that the NEM-stimulated effluxes of K+ and
Cl
are very nearly equimolar.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The experiments described above have provided the most direct
evidence to date that the red blood cell
K+-Cl cotransporter (most likely KCC1)
mediates a Cl
flux and that the cotransport has a 1:1
stoichiometry of K+-Cl
cotransport. The
evidence for 1:1 stoichiometry is that NEM stimulates a
36Cl
efflux that is very nearly the same as
the NEM-stimulated K+ efflux (Fig. 5). The fact that
K+ and Cl
effluxes are equal is not a
consequence of the requirement for global electroneutrality, because
the protonophore 2,4-DNP was present to allow H+ flux to
neutralize any charge imbalance associated with KCC. These results are
discussed below in the context of previous work related to the
stoichiometry of KCC.
In most studies (3, 11, 19, 38, 52), the KCC-mediated flux
of K+ (or Rb+) has a simple hyperbolic
(Michaelis-Menten) dependence on the K+ and
Cl concentrations. Some data suggest a sigmoidal
Cl
dependence of the KCC-mediated K+ or
Rb+ flux (5, 14, 46), but the apparent
sigmoidicity could be a consequence of the fact that the fluxes are
more difficult to measure at low Cl
concentration, and
the relative errors are larger. The apparent sigmoidicity in the
Cl
dependence of KCC in these studies is less pronounced
than that exhibited by the
Na+-K+-2Cl
cotransporter
(26), which clearly transports two Cl
ions.
Certainly the bulk of the kinetic data on KCC is consistent with a 1:1 mechanism.
Another experimental approach to determine stoichiometry is to measure
the flux reversal point, i.e., the combination of K+ (or
Rb+) and Cl gradients at which the net flux
is zero. Lauf (38) originally showed that the flux
reversal point in LK sheep red blood cells is very close to that
expected for 1:1 K+-Cl
cotransport.
Subsequently, Delpire and Lauf (11) found that, at 37°C,
LK sheep red blood cells exhibit a net K+ flux under
conditions in which a 1:1 mechanism would predict no net flux; this
result was not in agreement with a 1:1 stoichiometry. More recently,
Lauf and Adragna (40) clarified this issue by determining
the flux reversal point of KCC in pH-clamped LK sheep red blood cells
in the presence of various Cl
gradients; the results are
entirely consistent with 1:1 electroneutral K+-Cl
cotransport. This work also provides
evidence against H+ or OH
cotransport
associated with KCC, because the flux reversal point is not dependent
on extracellular pH over a wide range.
Reuss (53), in one of the early studies defining the
existence of KCC, measured net K+ and Cl
fluxes across the basolateral membrane of Necturus gall
bladder. The uncertainties in the measurements did not allow
calculation of a stoichiometry, but the data are consistent with a 1:1
mechanism. In contrast, Larson and Spring (37) presented
evidence that the stoichiometry of K+-Cl
cotransport is 3:2 in Necturus gall bladder, although the
authors pointed out that parallel transport processes could affect the calculated stoichiometry. Vascular smooth muscle cells exhibit a
K+-Cl
cotransport process (1)
that has an apparent stoichiometry of as many as 25 Cl
ions per K+ ion, but this unusual stoichiometry may be
related to exchange processes (55).
Zeuthen (60) has estimated the stoichiometry of
K+-Cl cotransport through the ventricular
membrane of Necturus choroid plexus; the estimate was based
on microelectrode determinations of changes in cytoplasmic
K+ and Cl
concentrations after changes in the
extracellular ion concentrations. Estimates of the ion fluxes were
complicated by the large water fluxes that accompanied the solution
changes, but the data are consistent with a 1:1
K+-Cl
cotransport. It would be of interest to
know whether red cell K+-Cl
cotransport is
associated with an obligatory water flux, as found by Zeuthen in
Necturus choroid plexus. However, because of the high water
permeability of the mammalian red cell membrane mediated by water
channels, we cannot tell whether there is a water flux obligatorily
coupled with K+-Cl
cotransport in rabbit red
blood cells.
To our knowledge, there has been only one report of a
36Cl flux associated with KCC in any cell.
Lytle and McManus (43) showed that, in duck red blood
cells treated with DIDS, there is a 36Cl
efflux that is stimulated by cell swelling and is dependent on intracellular K+. This work provided excellent evidence
that KCC does, in fact, mediate a Cl
flux. Lytle and
McManus did not report the stoichiometry of K+ (or
86Rb+) and 36Cl
effluxes. In related work, Lytle (44) demonstrated very
clearly that the net efflux of K+ and Cl
from
swollen duck red blood cells (with band 3 inhibited) has a
stoichiometry that is indistinguishable from 1:1, as does the net
uptake of Rb+ and Cl
into swollen,
K+-free cells. The main differences between the present
results and those of Lytle, other than the species difference (rabbit vs. duck), are that KCC was activated by NEM, rather than cell swelling, and a protonophore was included in our experiments. Lytle's
work on duck red blood cells is completely consistent with our
experiments with rabbit red blood cells.
Possible role of parallel pathways.
In these experiments, we have assumed that the NEM-stimulated
Rb+ and Cl fluxes are attributable to KCC.
This assumption is based on the fact that no other transporter for
K+ or Cl
in red blood cells is known to be
stimulated by NEM. Moreover, the NEM-stimulated Cl
flux
(Fig. 4) is inhibited by calyculin A, as was previously known for
KCC-mediated Rb+ fluxes (32, 34). Nonetheless,
it is useful to consider the possibility that stimulation of KCC by NEM
also causes stimulation of either a K+ or Cl
flux through a parallel pathway and thereby introduces error in the
stoichiometry estimate.
86Rb+ as tracer for
K+.
Our calculation of the K+ flux from the
86Rb+ tracer flux depends on the assumption
that the KCC-mediated (NEM stimulated) K+ efflux rate
constant is 0.68 times the 86Rb+ efflux rate
constant. This ratio was determined in five experiments at the same
temperature and pH as the other experiments (Fig. 1), but the
Rb+-to-K+ comparisons were performed in an
NaCl, rather than an Na2SO4, medium. The reason
for using an NaCl medium is that the K+ efflux measurements
with microelectrodes require longer times for reasonable accuracy
(~100 min vs. 30 min for tracer efflux); at such long times, the
intracellular Cl concentration would have been lowered
significantly in the Na2SO4 medium. The initial
intracellular conditions are identical in all experiments (whether in
NaCl or Na2SO4), and we do not see any reason
to suspect that the ratio of 86Rb+ to
K+ efflux rate constants would be any different in
Na2SO4 vs. NaCl medium. Nonetheless, our
calculation of stoichiometry does depend on the relative K+
and Rb+ efflux rate constants measured in an NaCl medium.
Role of O2.
In these experiments, the flux media were bubbled with N2
to minimize CO2 and HCO
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Carlo Brugnara, Phil Dunham, and Peter Lauf for
helpful discussions regarding the issue of whether
86Rb+ is a quantitative tracer for
K+ in the study of K+-Cl
cotransport. We are also grateful to Chris Lytle for sending material
from his PhD dissertation.
![]() |
FOOTNOTES |
---|
This work was supported by National Institute of General Medical Sciences Grant R01-GM-26861-21.
1 The membrane potential is not part of the driving force for an electroneutral process, but the rate constants for individual kinetic events in the process could, in principle, be dependent on the membrane potential.
Address for reprint requests and other correspondence: M. L. Jennings, Dept. of Physiology and Biophysics, University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, AR 72205 (E-mail: JenningsMichaelL{at}uams.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.
Received 1 February 2001; accepted in final form 17 April 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Adragna, NC,
White RE,
Orlov SN,
and
Lauf PK.
K-Cl cotransport in vascular smooth muscle and erythrocytes: possible implication in vasodilation.
Am J Physiol Cell Physiol
278:
C381-C390,
2000
2.
Al-Rohil, N,
and
Jennings ML.
Volume-dependent K+ transport in rabbit red blood cells: comparison with oxygenated human SS cells.
Am J Physiol Cell Physiol
257:
C114-C121,
1989
3.
Bergh, C,
Kelley SJ,
and
Dunham PB.
K-Cl cotransport in LK sheep erythrocytes: kinetics of stimulation by cell swelling.
J Membr Biol
117:
177-188,
1990[ISI][Medline].
4.
Brahm, J.
Temperature-dependent changes of chloride transport kinetics in human red cells.
J Gen Physiol
70:
283-306,
1977
5.
Brugnara, C.
Characteristics of the volume- and chloride-dependent K transport in human erythrocytes homozygous for hemoglobin C.
J Membr Biol
111:
69-81,
1989[ISI][Medline].
6.
Brugnara, C,
Van Ha T,
and
Tosteson DC.
Role of chloride in potassium transport through a K-Cl cotransport system in human red blood cells.
Am J Physiol Cell Physiol
256:
C994-C1003,
1989
7.
Cabantchik, ZI,
and
Greger R.
Chemical probes for anion transporters of mammalian cell membranes.
Am J Physiol Cell Physiol
262:
C803-C827,
1992
8.
Cabantchik, ZI,
and
Rothstein A.
Membrane proteins related to anion permeability of human red blood cells. I. Localization of disulfonic stilbene binding sites in proteins involved in permeation.
J Membr Biol
15:
207-226,
1974[ISI][Medline].
9.
Canessa, M,
Fabry ME,
and
Nagel RL.
Deoxygenation inhibits the volume-stimulated, Cl-dependent K+ efflux in SS and young AA cells: a cytosolic Mg2+ modulation.
Blood
70:
1861-1866,
1992[Abstract].
10.
Coldman, MF,
and
Good W.
The distribution of sodium, potassium, and glucose in the blood of some mammals.
Comp Biochem Physiol A Physiol
21:
201-206,
1967.
11.
Delpire, E,
and
Lauf PK.
Kinetics of Cl-dependent K fluxes in hyposmotically swollen low K sheep erythrocytes.
J Gen Physiol
97:
173-193,
1991[Abstract].
12.
Delpire, E,
and
Lauf PK.
Kinetics of DIDS inhibition of swelling-activated K-Cl cotransport in low K sheep erythrocytes.
J Membr Biol
126:
89-96,
1992[ISI][Medline].
13.
Dorup, I,
and
Clausen T.
86Rb is not a reliable tracer for potassium in skeletal muscle.
Biochem J
302:
745-751,
1994[ISI][Medline].
14.
Dunham, PB,
and
Ellory JC.
Passive potassium transport in low potassium sheep red cells: dependence upon cell volume and chloride.
J Physiol (Lond)
318:
511-530,
1981[Abstract].
15.
Gasbjerg, PK,
Funder J,
and
Brahm J.
Kinetics of residual chloride transport in human red blood cells after maximum covalent 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid binding.
J Gen Physiol
101:
715-732,
1993[Abstract].
16.
Geck, P,
Pietrzyk C,
Burckhardt B-C,
Pfeiffer B,
and
Heinz E.
Electrically silent cotransport of Na, K, and Cl in Ehrlich cells.
Biochim Biophys Acta
600:
432-447,
1980[ISI][Medline].
17.
Gibson, JS,
Speake PF,
and
Ellory JC.
Differential oxygen sensitivity of the K+-Cl cotransporter in normal and sickle human red blood cells.
J Physiol (Lond)
511:
225-234,
1998
18.
Giebel, O,
and
Passow H.
Die Permeabilität der Erythrocytenmembran für organischen Anionen. Zur Frage der Diffusion durch Poren.
Pflügers Arch
271:
378-388,
1960.
19.
Gillen, CM,
Brill S,
Payne JA,
and
Forbush B, III.
Molecular cloning and functional expression of the K-Cl cotransporter from rabbit, rat, and human.
J Biol Chem
271:
16237-16244,
1996
20.
Gunn, RB,
Dalmark M,
Tosteson DC,
and
Wieth JO.
Characteristics of chloride transport in human red blood cells.
J Gen Physiol
61:
185-206,
1973
21.
Haas, M,
and
Forbush B, III.
The Na-K-Cl cotransporter of secretory epithelia.
Annu Rev Physiol
62:
515-534,
2000[ISI][Medline].
22.
Harris, EJ,
and
Maizels M.
Distribution of ions in suspensions of human erythrocytes.
J Physiol (Lond)
118:
40-53,
1952[ISI].
23.
Harris, EJ,
and
Pressman BC.
Obligate cation exchanges in red cells.
Nature
216:
918-920,
1967[ISI][Medline].
24.
Hiki, K,
D'Andrea RJ,
Furze J,
Crawford J,
Woolatt E,
Sutherland GR,
Vadas MA,
and
Gamble JR.
Cloning, characterization, and chromosomal location of a novel human K+-Cl cotransporter.
J Biol Chem
274:
10661-10667,
1999
25.
Holtzman, EJ,
Kumar S,
Faaland CA,
Warner F,
Logue PJ,
Erickson SJ,
Ricken G,
Waldman J,
and
Dunham PB.
Cloning, characterization, and gene organization of K-Cl cotransporter from pig and human kidney and C. elegans.
Am J Physiol Renal Physiol
275:
F550-F564,
1998
26.
Isenring, P,
Jacoby SC,
Payne JA,
and
Forbush B, III.
Comparison of Na-K-Cl cotransporters NKCC1, NKCC2, and the HEK cell Na-K-Cl cotransporters.
J Biol Chem
273:
11295-11301,
1998
27.
Jennings, ML.
Proton fluxes associated with erythrocyte membrane anion exchange.
J Membr Biol
28:
187-205,
1976[ISI][Medline].
28.
Jennings, ML.
Volume-sensitive K+-Cl cotransport in rabbit erythrocytes. Analysis of the rate-limiting activation and inactivation events.
J Gen Physiol
114:
743-757,
1999
29.
Jennings, ML,
and
Adams-Lackey M.
A rabbit erythrocyte membrane protein associated with L-lactate transport.
J Biol Chem
257:
12866-12871,
1982
30.
Jennings, ML,
Adams-Lackey M,
and
Denney GH.
Peptides of human erythrocyte band 3 protein produced by extracellular papain cleavage.
J Biol Chem
259:
4652-4660,
1984
31.
Jennings, ML,
and
Al-Rohil N.
Kinetics of activation and inactivation of swelling-stimulated K+-Cl cotransport. Volume-sensitive parameter is the rate constant for inactivation.
J Gen Physiol
95:
1021-1040,
1990[Abstract].
32.
Jennings, ML,
and
Schulz RK.
Okadaic acid inhibition of KCl cotransport. Evidence that protein dephosphorylation is necessary for activation of transport by either cell swelling or N-ethylmaleimide.
J Gen Physiol
97:
799-817,
1991[Abstract].
33.
Joiner, CH,
Jiang M,
Fathallah H,
Giraud F,
and
Franco RS.
Deoxygenation of sickle red blood cells stimulates KCl cotransport without affecting Na+/H+ exchange.
Am J Physiol Cell Physiol
274:
C1466-C1475,
1998
34.
Kaji, D,
and
Tsukitani Y.
Role of protein phosphatase in activation of KCl cotransport in human erythrocytes.
Am J Physiol Cell Physiol
260:
C178-C182,
1991.
35.
Kaji, DM.
Effect of membrane potential on K-Cl transport in human erythrocytes.
Am J Physiol Cell Physiol
264:
C376-C382,
1993
36.
Knauf, PA.
Erythrocyte anion exchange and the band 3 protein: transport kinetics and molecular structure.
Curr Top Membr Transp
12:
249-363,
1979.
37.
Larson, M,
and
Spring KR.
Volume regulation by Necturus gallbladder: basolateral KCl exit.
J Membr Biol
81:
219-232,
1984[ISI][Medline].
38.
Lauf, PK.
Thiol-dependent passive K-Cl transport in sheep red cells. I. Dependence on chloride and external K+ (or Rb+) ions.
J Membr Biol
73:
237-246,
1983[ISI][Medline].
39.
Lauf, PK,
and
Adragna NC.
Temperature-induced functional deocclusion of thiols inhibitory for sheep erythrocyte K-Cl cotransport.
Am J Physiol Cell Physiol
269:
C1167-C1175,
1995
40.
Lauf, PK,
and
Adragna NC.
A thermodynamic study of electroneutral K-Cl cotransport in pH- and volume-clamped low K sheep erythrocytes with normal and low internal magnesium.
J Gen Physiol
108:
341-350,
1996[Abstract].
41.
Lauf, PK,
Bauer J,
Adragna NC,
Fujise H,
Zade-Oppen AM,
Ryu KH,
and
Delpire E.
Erythrocyte K-Cl cotransport: properties and regulation.
Am J Physiol Cell Physiol
263:
C917-C932,
1992
42.
Lu, J,
Karadsheh M,
and
Delpire E.
Developmental regulation of the neuronal-specific isoform of K-Cl cotransporter KCC2 in postnatal rat brains.
J Neurobiol
39:
558-568,
1999[ISI][Medline].
43.
Lytle, C,
and
McManus TJ.
Effect of loop diuretics and stilbene derivatives on swelling-induced K-Cl cotransport (Abstract).
J Gen Physiol
90:
28A-29A,
1987.
44.
Lytle, CY.
[Na-K-2Cl] and [K-Cl] Co-Transport in Duck Red Cells (Ph.D. dissertation). Durham, NC: Duke University, 1988.
45.
Macey, RI,
Adorante JS,
and
Orme FW.
Erythrocyte membrane potentials determined by hydrogen ion distributions.
Biochim Biophys Acta
512:
284-295,
1978[ISI][Medline].
46.
Mercado, A,
Song L,
Vazquez N,
Mount DB,
and
Gamba G.
Functional comparison of the K+-Cl cotransporters KCC1 and KCC4.
J Biol Chem
275:
30326-30334,
2000
47.
Mount, DB,
Hoover RS,
and
Hebert SC.
The molecular physiology of electroneutral cation-chloride cotransport.
J Membr Biol
158:
177-186,
1997[ISI][Medline].
48.
Mount, DB,
Mercado A,
Song L,
Xu J,
George AL, Jr,
Delpire E,
and
Gamba G.
Cloning and characterization of KCC3 and KCC4, new members of the cation-chloride cotransporter gene family.
J Biol Chem
274:
16355-16362,
1999
49.
Omachi, A,
Scott CB,
and
Glader BE.
2,4-Dinitrophenol inhibition of P32 release from human red cells.
Experientia
24:
244-245,
1968[ISI][Medline].
50.
Payne, JA,
Stevenson TJ,
and
Donaldson LF.
Molecular characterization of a putative K-Cl cotransporter in rat brain.
J Biol Chem
271:
16245-16252,
1996
51.
Payne, JA,
Xu J-C,
Haas M,
Lytle CY,
Ward D,
and
Forbush B, III.
Primary structure, functional expression, and chromosomal localization of the bumetanide-sensitive Na-K-Cl cotransporter in human colon.
J Biol Chem
27:
17977-17985,
1995.
52.
Race, J,
Makhlouf F,
Logue P,
Wilson F,
Dunham P,
and
Holtzman E.
Molecular cloning and functional characterization of KCC3, a new K-Cl cotransporter.
Am J Physiol Cell Physiol
277:
C1210-C1219,
1999
53.
Reuss, L.
Basolateral KCl co-transport in a NaCl-absorbing epithelium.
Nature
305:
723-726,
1983[ISI][Medline].
54.
Rivera, C,
Voipio J,
Payne JA,
Ruusuvuori E,
Lahtinen H,
Lamsa K,
Pirvola U,
Saarma M,
and
Kaila K.
The K+-Cl co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation.
Nature
397:
251-255,
1999[ISI][Medline].
55.
Saitta, M,
Cavalier S,
Garay R,
Cragoe EJ,
and
Hannaert PA.
Evidence for a DIOA-sensitive (K+,Cl) cotransport system in cultured vascular smooth muscle cells.
Am J Hypertens
3:
939-942,
1990[ISI][Medline].
56.
Shami, Y,
Rothstein A,
and
Knauf PA.
Identification of the Cl transport site of human red blood cells by a kinetic analysis of the inhibitory effects of a chemical probe.
Biochim Biophys Acta
508:
357-363,
1978[ISI][Medline].
57.
Starke, LC,
and
Jennings ML.
K-Cl cotransport in rabbit red cells: further evidence for regulation by protein phosphatase type 1.
Am J Physiol Cell Physiol
264:
C118-C124,
1993
58.
Stewart, GW,
and
Blackstock EJ.
Potassium transport in rabbit erythrocytes.
Exp Biol
48:
161-165,
1989[ISI][Medline].
59.
Xu, J-C,
Lytle C,
Zhu TT,
Payne JA,
Benz E, Jr,
and
Forbush B, III.
Molecular cloning and functional expression of the bumetanide-sensitive Na-K-Cl cotransporter.
Proc Natl Acad Sci USA
91:
2201-2205,
1994[Abstract].
60.
Zeuthen, T.
Cotransport of K+, Cl, and H2O by membrane proteins from choroid plexus epithelium of Necturus maculosus.
J Physiol (Lond)
478:
203-219,
1994[Abstract].