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The Journal of Neuroscience, June 15, 1999, 19(12):4695-4704
A Furosemide-Sensitive K+-Cl
Cotransporter Counteracts Intracellular Cl Accumulation
and Depletion in Cultured Rat Midbrain Neurons
Wolfgang
Jarolimek,
Andrea
Lewen, and
Ulrich
Misgeld
I. Physiologisches Institut der Universität Heidelberg,
D-69120 Heidelberg, Germany
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ABSTRACT |
Efficacy of postsynaptic inhibition through GABAA
receptors in the mammalian brain depends on the maintenance of a
Cl
gradient for hyperpolarizing
Cl currents. We have taken advantage of the
reduced complexity under which Cl regulation can
be investigated in cultured neurons as opposed to neurons in other
in vitro preparations of the mammalian brain. Tightseal whole-cell recording of spontaneous GABAA
receptor-mediated postsynaptic currents suggested that an outward
Cl transport reduced dendritic
[Cl]i if the somata of cells were
loaded with Cl via the patch pipette. We
determined dendritic and somatic reversal potentials of
Cl currents induced by focally applied GABA to
calculate [Cl]i during variation of
[K+]o and [Cl]
in the patch pipette. [Cl]i and
[K+]o were tightly coupled by a
furosemide-sensitive K+-Cl
cotransport. Thermodynamic considerations excluded the significant contribution of a
Na+-K+-Cl
cotransporter to the net Cl transport. We conclude
that under conditions of normal [K+]o
the K+-Cl cotransporter helps
to maintain [Cl]i at low levels,
whereas under pathological conditions, under which
[K+]o remains elevated because of
neuronal hyperactivity, the cotransporter accumulates
Cl in neurons, thereby further enhancing neuronal excitability.
Key words:
Cl homeostasis; K+-Cl cotransporter; furosemide; Cl depletion; Cl
accumulation; Donnan equilibrium; cultured neurons
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INTRODUCTION |
GABA is the main inhibitory
transmitter in the mammalian brain. The dominant effect of
GABAA receptor activation is a hyperpolarization caused by
Cl flux into the cell (for review, see Sivilotti
and Nistri, 1991
; Kaila, 1994; Thompson, 1994). However, the direction
of the Cl flux depends on the
Cl gradient across the membrane. Indeed,
GABAA receptor-mediated hyperpolarizing and/or depolarizing
postsynaptic potentials have been observed (for review, see Kaila,
1994; Thompson, 1994). Some findings suggest variations in
intracellular [Cl] between different neurons and
even a distinct Cl distribution in different
compartments of a single neuron (Misgeld et al., 1986). Depolarizing
GABAA responses, however, can be caused by bicarbonate
efflux in combination with Cl influx or combined
Cl and HCO3
efflux (Grover et al., 1993; Kaila, 1994; Thompson, 1994; Staley et
al., 1995; Perkins and Wong, 1996; Kaila et al., 1997). To be able to
predict the direction of Cl currents flowing
during GABAA receptor-mediated inhibition it is essential
to understand the regulation of Cl homeostasis
that provides the transmembrane gradient.
A recently cloned K+-Cl
cotransporter gene (KCC2) represents a perfect candidate for the
regulation of neuronal Cl homeostasis (Payne et
al., 1996). In contrast to the ubiquitous presence of the
K+-Cl cotransporter KCC1,
expression of the K+-Cl
cotransporter KCC2 is detected in CNS only and seems to be neuron specific. KCC2 is also distinct from KCC1 in that KCC2 is not involved
in cell volume regulation and not activated by osmotic changes.
Furthermore, KCC2 has a high affinity for extracellular K+ ions. The properties of KCC2 allow the regulation
of [Cl]i to maintain
Cl gradients for hyperpolarizing GABAergic
inhibition. Thermodynamic considerations predict that the
electroneutral K+-Cl
cotransporter KCC2 operates near equilibrium under physiological ionic
conditions. Depending on [Cl]i and
[K+]o (Payne, 1997), the transport
will extrude or accumulate Cl.
The functional role of a particular Cl transport
system in neuronal Cl regulation is difficult to
establish in studies using integral preparations such as brain slices.
One complicating factor is the presence of
HCO3 anions. The
HCO3 permeability of the
GABAA channel (Bormann, 1988; Fatima-Shad and Barry, 1993)
impedes conclusions toward actual
[Cl]i if they are calculated from
reversal potentials of GABAA receptor-mediated anion
currents. Furthermore, a
HCO3/Cl exchanger
(Raley-Susman et al., 1993) may well interfere (Chesler, 1990), and pH
changes that result from manipulations of
[HCO3]o strongly affect
neuronal excitability (Jarolimek et al., 1989; Chesler and Kaila,
1992).
To investigate the functional role of Cl
transporters we used neuronal cultures that allow measurements of
Cl reversal potentials under nominally
HCO3-free conditions. We used patch
pipettes to manipulate [Cl]i and
tested [Cl]i regulation in somatic
and dendritic compartments by varying [K+]o. The
K+-Cl cotransporter KCC2 is
furosemide sensitive (Payne, 1997), so we used furosemide to test the
inward or outward direction of the Cl transport in
cultured neurons. We found that a transport with properties of the
neuronal-specific K+-Cl
cotransporter KCC2 can fully account for Cl
regulation in cultured midbrain neurons.
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MATERIALS AND METHODS |
Cell culture. Pregnant Wistar rats were anesthetized
by ether inhalation and killed by decapitation. The embryos were
removed, placed in sterile, ice-cold Gey's buffered salt solution
containing (in mM): NaCl 137,KCl 5, MgSO4 0.3, NaH2PO4 1, CaCl2 1.5, NaHCO3 2.7, KH2PO4 0.2, MgCl2 1, glucose 5, at pH 7.4, and immediately decapitated.
Pieces of ventral midbrain tissue from the 14-d-old embryos were
mechanically dissociated and plated on a primary culture of glial cells
from the same area. Cell culture conditions were as described
previously (Bijak et al., 1991; Jarolimek and Misgeld, 1992). Cells
used in this study were 4-8 weeks in culture. Immunocytochemistry
studies have shown that this culture preparation contains ~5%
dopaminergic neurons and <5% serotonergic neurons (Rohrbacher et al.,
1995) in addition to ~70% GABAergic neurons (our unpublished
results). The fact that EPSPs attributable to activation of
glutamatergic receptors comprise a significant proportion of the
observed postsynaptic activity (Bijak et al., 1991) suggests that most
non-GABAergic neurons possess a glutamatergic neurotransmitter phenotype.
Electrophysiological recordings. Recordings were performed
at room temperature (22-25°C) in the whole-cell voltage-clamp
configuration with a patch-clamp amplifier (L/M-EPC 7, List, Darmstadt,
Germany). The composition of the extracellular solutions and the patch
pipette solutions are given in Table 1.
No CO2 or O2 was added, and the pH of all
solutions was adjusted to 7.3. Glucuronate salts were used because the
permeability of the anion through GABAA channels should be
small because of its restricted, bulky conformation. 5-N-(2,6-dimethyl-phenylcarbamoylmethyl)-triethylammonium
bromide (QX314) was added to the intracellular solution to block
Na+ and K+ currents (Connors and
Prince, 1982; Nathan et al., 1990; Colling and Wheal, 1994). The
Br salt was used because in initial experiments
the block of action potentials was faster and more complete with
Br than with the Cl salt.
Br has a permeability similar to that of
Cl at GABAA channels (Bormann, 1988);
therefore, concentrations of permeable ions used for calculations were
the sum of Cl and Br
concentrations. The reliability of this approach was tested by replacement of 5 mM
[Cl]o by 5 mM
[Br]o to obtain an identical
[Br] in the extracellular solution and the patch
pipette. This change had no measurable influence on reversal potentials
of GABA-induced currents (n = 4). We did not replace
K+ by Cs+ in the patch pipette
because Cs+ could interfere with the presumed
K+-Cl cotransporter competing
with K+ for the internal binding site and/or
changing the driving force for the transporter. CsCl was added,
however, to the extracellular solution to reduce leak conductance. In
preliminary experiments we tested whether extracellular
Cs+ affected the
K+-Cl cotransporter.
Furosemide (0.1 mM) shifted the dendritic reversal potential (EGABA) of GABA-induced
currents (IGABA) in a positive direction
at 2 mM [K+]o and in a
negative direction at 10 mM
[K+]o and low
[Cl]pip (see Results). Neither
dendritic EGABA nor its shifts that were induced
by furosemide changed when we added 5 mM
Cs+ (n = 5), suggesting that
Cs+ was not a substrate for the transporter and did
not compete for the extracellular binding site of
K+. Therefore the extracellular solution always
contained 5 mM Cs+ to reduce leak
conductance when we measured reversal potentials for GABA currents.
Spontaneous synaptic activity in the embryonic midbrain culture
consists of EPSCs that are blocked by the AMPA-type glutamate
antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) and IPSCs sensitive
to antagonists for the GABAA receptors bicuculline or
picrotoxin (Bijak et al., 1991; Jarolimek and Misgeld, 1991). To block
excitation, DNQX (20 µM) and the NMDA receptor-type
antagonist DL-2-amino-4-methyl-5-phosphono-3-pentenoic acid
(1 µM) were present in all extracellular recording
solutions.
Patch pipettes were fabricated from borosilicate glass (Hilgenberg,
Malsfeld, Germany), and their resistances to bath ranged from 2 to 4 M
. The access resistance was estimated from the amplitude of the
capacitive current evoked by a hyperpolarizing 10 mV step. Access
resistances varied between 5 and 12 M and were routinely checked
during the recording. No series resistance or slow capacitance compensation was used during the experiment because currents were measured >15 sec after the membrane potential was changed. Given the
small amplitude (<200 pA) of the recorded currents, series resistance
error was <2.5 mV. The liquid junction potential between the patch
pipette and the extracellular solution was calculated according to
Barry and Lynch (1991). The calculation yielded 15.8 and 17.5 mV for 15 and 4.5 mM
[A]pip, respectively. When
measured according to the procedure described by Neher (1992), the
values were 14.0 and 15.5 mV, respectively, which are in good agreement
with the calculated values. Because it has been suggested that
calculated rather than measured values be used (Barry and Lynch, 1991),
all potentials reported in this paper have been corrected for the
calculated liquid junction potentials.
Recordings were started >5 min after the whole-cell configuration was
established to allow adequate time for QX314 to take effect and for
anions to equilibrate. After 5 min, no further change in dendritic or
somatic EGABA was observed. GABA (1 mM) was applied by pressure ejection (1-20 kPa, 20-40
msec) every 15 sec from a pipette with a <1 µm opening.
IGABA was measured in the presence of
tetrodotoxin (0.3 µM) to avoid superposition of
IGABA and action potential-dependent IPSCs as
well as the activation of fast Na+ currents in the
recorded cell. All inorganic salts were of analytical grade (Merck,
Darmstadt, Germany). Drugs were from Sigma (Deisenhofen, Germany)
except DNQX and tetrodotoxin (RBI, Köln, Germany).
Extracellular solutions were applied by a multibarrel perfusion system
that was positioned ~250 µm away from the soma of the recorded cell
(Bijak et al., 1991; Jarolimek and Misgeld, 1992).
Data analysis. Recordings were filtered at 1.3-3 kHz with a
four-pole Bessel filter, acquired and analyzed with pClamp (Axon Instruments, Foster City, CA) hardware and software, and additionally stored on a DAT recorder. The time course and amplitude of spontaneous IPSCs (sIPSCs) were analyzed with a program written in our laboratory (Jarolimek and Misgeld, 1997; Rohrbacher et al., 1997). The reversal potential of IGABA was determined by linear
regression of the current-voltage relationship. Currents were recorded
at holding potentials (VH) on both sides
of EGABA. After 15-45 sec at a new VH, the current amplitudes induced by
three consecutive applications were averaged. Despite the rapid
exchange of the extracellular solution (~0.5 sec) around the neuron,
effects were determined >1 min after the start of the application to
achieve steady-state conditions. Theoretical
[Cl]i of the neuron was calculated
with the Nernst equation using measured EGABA
values and [Cl]o values given in
Table 1. Calculated values were not corrected for the presence of
Br in the pipette and in the neuron. The driving
force for the K+-Cl
cotransporter was estimated on the basis of the Nernst equation given
the calculated [Cl]i and
[K+]o from Table 1. Data are reported
as mean ± SEM.
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RESULTS |
Cl gradient between soma and dendrites
When tight-seal whole-cell recordings were performed under
conditions of 15 mM permeant anions in the patch pipette
([A]pip), 2 mM
[K+]o, and pharmacological
blockade of ionotropic glutamate receptors, sIPSCs did not reverse in
sign at a defined holding potential (VH).
Instead, at VH in a range near the reversal
potential (ECl = 
61 mV as determined from the
Nernst equation), inward and outward sIPSCs concurred in 46 of 51 cells
(Fig. 1A). All sIPSCs
were blocked by bicuculline (n = 5), suggesting that
they were mediated by GABAA receptors (Fig.
1B). Inward and outward sIPSCs appeared to be
separated (Fig. 1A1) or as sequences of inward
currents curtailed by outward currents (Fig. 1A2). As
shown in Figure 1A3, another pattern consisted of
clustered inward sIPSCs that were accompanied by a continuous barrage
of outward sIPSCs or vice versa. We analyzed the time courses of
apparently isolated inward and outward sIPSCs in a single cell at a
VH at which amplitudes of inward and outward
IPSCs were in a similar range (Fig. 1C,D). There was a clear
difference in the time courses. Outward sIPSCs consistently had a
slower rise time and time constant of decay than inward sIPSCs (rise
time, 1.9 ± 0.1 msec vs 5.3 ± 0.4 msec; decay time
constant, 16.2 ± 1.3 msec vs 38.6 ± 4.8 msec for inward and
outward sIPSCs, respectively; n = 5 cells). On the
basis of these observations, we assumed that outward sIPSCs were
generated in dendritic compartments but that inward sIPSCs were
generated near soma. Presynaptic GABAergic neurons of the network would generate inward and outward IPSCs if they formed synapses with all
parts of the somatodendritic surface. Presynaptic neurons contacting
restricted areas either near soma or in apical dendrites would generate
a continuous barrage or clusters of inward or outward IPSCs consistent
with their firing mode, continuous or in bursts. Assuming further that
under the given recording conditions in the culture (see Discussion)
soma and dendrites did not deviate considerably from isopotentiality,
we had to postulate [Cl]i to be
lower in dendritic than in somatic compartments.
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Figure 1.
GABAergic network neurons generated inward and
outward IPSCs in a single target neuron through GABAA
receptors. A1, Spontaneous IPSCs were inward and outward
current transients in a cell recorded at VH
63 mV. Inward and outward sIPSCs occurred in isolation
(1), or inward currents were curtailed by outward
sIPSCs (2). Note the difference in calibration
between 1 and 2. A3, In another cell inward sIPSCs
occurred in clusters interrupted by "silent" periods, whereas
outward sIPSCs occurred rather continuously
(VH 71 mV). B, The
GABAA antagonist bicuculline (bic)
reversibly blocked all inward and outward sIPSCs. a, b,
Expanded segments of the top trace where indicated. C,
D, Inward and outward sIPSCs differed in their time courses.
Inward (D1) and outward (C1) sIPSCs that
occurred in separation could be fitted by a single exponential function
(solid line; VH 63 mV). For
the sIPSCs shown, the time constant of decay was 26.2 msec (amplitude
coefficient 39.4) and 6.0 msec (amplitude coefficient 33.1). For
analysis, only sIPSCs that had no inflections in the rise or decay
phase were accepted. Rise time (10-90%) histogram of all inward
(D2) and outward (C2) sIPSCs showed that
rise time was slower for outward sIPSCs. Similarly, histograms of decay
time constant (C3, D3) revealed a much
slower decay for outward sIPSCs (C3). For the
histograms, 31 outward and 45 inward IPSCs (sampled in 1 min) were
analyzed. All recordings in this Figure were obtained in the presence
of ionotropic glutamate receptor antagonists and of 2 mM
[K+]o
([A]pip 15 mM).
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To examine compartmental differences in
[Cl]i we applied GABA (1 mM) focally to somatic and dendritic regions (Fig.
2). To provide comparable conditions of
origin for sIPSCs and GABA-evoked currents
(IGABA), application was adjusted so that
the amplitude of IGABA was in the range of
amplitudes of sIPSCs. When tested in a voltage range around the
reversal potential (EGABA ± 25 mV), slope
conductances of dendritic and somatic IGABA were linear (Fig. 2). However, slope conductance was smaller for dendritic than for
somatic applications, and often pressure or application time for the
focal application had to be increased to obtain a measurable dendritic
IGABA. The difference was probably caused by
different density of receptors and/or surface size facing the application pipette if GABA was applied near soma or near dendrites. As
expected from the observation of inward and outward sIPSCs, dendritic
IGABA (application >100 µm away from soma)
was outward at a VH at which somatic
IGABA of the same cell was inward (Fig. 2). In a
singe cell, EGABA differed by up to 25 mV (mean
12.9 ± 2.3 mV; n = 9) between soma and dendrites
(Table 2).
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Figure 2.
Dendritic and somatic GABA applications generated
Cl currents with different reversal potentials. In
the same cell, currents induced by focal application of GABA (1 mM) to a dendrite ( ) or the soma ( ) had different
reversal potentials. Application sites as shown for a cell in the
inset (s, somatic; d,
dendritic). Recording conditions were 2 mM
[K+]o and 15 mM
[A]pip. Letters mark
the points on the current-voltage relationship at which the sample
currents shown above had been recorded. Symbols
represent the mean of three consecutive applications. Note the voltage
range at which GABA evoked an outward current in the dendrite and an
inward current at the soma.
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Dependence of the somatodendritic Cl gradient
on a furosemide-sensitive transport
Calculation of inorganic [A]i
from EGABA values reported above revealed that
dendritic [A]i was lower than
somatic [A]i. If dendritic
[Cl]i was lowered by a
furosemide-sensitive transport, it should be possible to reduce the
somatodendritic [Cl] gradient by furosemide
application. Therefore, we tested the effects of furosemide on sIPSCs
and IGABA. Furosemide altered the driving force
for sIPSCs. At VH near the expected
EIPSC, amplitudes of inward sIPSCs
increased and those of outward sIPSCs decreased or they reversed in
sign (Fig. 3A). A new apparent
steady state was achieved after maximally 1 min, and the effect of
furosemide was fully reversible in a similar time. Effects of
furosemide were observed at concentrations as low as 10 µM (Fig. 3B; n = 4 of 5 cells)
and increased in a concentration-dependent manner (n = 9). The maximal concentration we used in all further experiments was
0.1 mM to avoid unspecific effects of furosemide
(Cabantchik and Greger, 1992). To quantify the effect of furosemide on
EGABA, current-voltage relationships for
IGABA were determined in the presence and
absence of furosemide (0.1 mM). In one series of experiments (n = 15), the position of the application
pipette was guided by the current direction of sIPSCs. First, the range of VH was determined at which inward and outward
sIPSCs were measurable. Thereafter, a VH at the
positive side of this range was chosen to record predominantly outward
sIPSCs that were likely generated in dendritic compartments (Fig.
4A1). In this situation
the position of the application pipette was chosen so that
IGABA was inward. In most instances the
compartment reached from the soma up to 100 µm away from the somatic
patch pipette for smoothly tapering dendrites (Fig. 2,
inset). At a distance from soma of ~200 µm, current
direction of IGABA and sIPSCs concurred (Fig.
4A2). Furosemide only slightly increased the
amplitude of somatic IGABA (Fig.
4A1), whereas dendritic IGABA
reversed in sign (Fig. 4A2). As shown in Figure
4B, a new steady-state for dendritic
IGABA was achieved within 30-45 sec. The effect
of furosemide was fully reversible. The data indicate a pronounced
shift of EGABA under furosemide in dendrites but
not at the soma.
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Figure 3.
Furosemide changed the reversal potential of
sIPSCs. A, Spontaneous IPSCs were inward and outward at
VH of 61 mV. Furosemide
(furo; 0.1 mM) increased the
frequency and amplitude of inward sIPSCs (recorded in 2 mM
[K+]o, 15 mM
[A]pip), whereas outward
sIPSCs disappeared. The onset of the furosemide effect is already seen
after 10 sec, and the effect is reversible. Letters mark
time points that are shown at higher sweep speed in the lower traces.
B, In another cell under otherwise identical recording
conditions as in A, VH was
set to record outward sIPSCs. Application of increasing concentrations
of furosemide first reduced the amplitude of outward sIPSCs and then
reversed sign of sIPSCs.
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Figure 4.
Furosemide affected the reversal potential of
dendritic IGABA more strongly than of
somatic IGABA. A, Pressure
ejection of GABA ( ) (distance to the soma is given in
parentheses) induced an inward current when applied
close to the soma (A1) but an outward current in
dendrites (A2). Under control recording conditions (15 mM [A]pip; 2 mM [K+]o;
VH 66 mV), sIPSCs were recorded at a
VH at which outward sIPSCs predominated. In
the presence of tetrodotoxin (TTX) to eliminate
sIPSCs, furosemide reversed the current direction of dendritic
IGABA (A2), whereas the
amplitude of somatic IGABA was increased
only slightly (A1). Both effects were completely
reversible. To obtain a clear signal in presence of sIPSCs, slightly
larger ejection pressure was used than was needed in TTX.
B, Chart recording of the time course of the
furosemide-induced shift of EGABA. In the
same cell as in A, dendritic
IGABA measured in TTX reversed current
direction on application of furosemide. This effect readily reversed on
washout.
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In another set of experiments, furosemide (0.1 mM)-induced
shifts in EGABA were quantified for dendritic
(application ~200 µm apart from soma) and somatic
IGABA. As shown in Figure
5A, furosemide shifted
dendritic and somatic EGABA in positive
direction but did not reduce slope conductance. This suggests that
furosemide changed [A]i but had no
blocking action on GABAA receptors. Furosemide (0.1 mM) shifted somatic EGABA less than
dendritic EGABA (4.2 ± 1.0 mV vs 11.8 ± 1.4 mV; n = 8) (Fig. 5B), with the result
that furosemide reduced the somatodendritic inorganic
[A] gradient. These findings showed that at 15 mM [A]pip and 2 mM [K+]o the somatic
inorganic [A]i was mainly governed
by A diffusion from the patch pipette into the
cytoplasm of the cell body. Dendritic inorganic
[A] was significantly lowered with respect to
somatic [A]i by the operation of a
furosemide-sensitive Cl outward transport.
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Figure 5.
Furosemide reduced the difference of reversal
potentials of IGABA between dendrites and
soma. A, Current-voltage relationship of somatic
(A1) and dendritic (A2)
IGABA could be fitted by a linear regression
line (15 mM [A]pip and 2 mM [K+]o).
Furosemide shifted the current-voltage relationship to more positive
values, whereas the slope conductance was unchanged. The effect was
reversible on washout. EGABA in the absence
of furosemide was more negative for dendritic
IGABA than for somatic
IGABA. In furosemide, the difference between
dendritic and somatic EGABA was small,
indicating that the change of EGABA was more
pronounced at the dendrites than at the soma. B,
Bar charts summarize mean
EGABA (and SEM) for the different recording
conditions. The number of cells is nine for somatic
IGABA and six for dendritic
IGABA.
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Dependence of the direction of the Cl
transport on [K+]o and
[Cl]i
The concentrations of furosemide used in our study inhibit the
K+-Cl cotransporter (KCC2) in
heterologous expression systems. At concentrations of
[Cl]i (4-10 mM) and
[K+]o (2-10 mM) that can
be expected to occur in the mammalian brain, KCC2 operates near its
thermodynamic equilibrium. The direction of Cl
transport depends on [Cl]i and
[K+]o (Payne, 1997). We tested whether
these characteristics also apply to the regulation of
Cl homeostasis in neurons.
IGABA at 15 mM
[A]pip
Increasing [K+]o from 2 to 10 mM shifted dendritic EGABA to
positive values (Table 2). An apparent, new steady state was attained only after the inward current elicited by
[K+]o had peaked (Fig.
6A1). When measured at
2 and 10 mM [K+]o in the
same cells, the shift of dendritic EGABA mounted
to 16.5 ± 3.3 mV (n = 3). In 10 mM
[K+]o, furosemide (0.1 mM) shifted dendritic EGABA to even
more positive values (3.3 ± 0.7 mV; n = 4), but
the shift was consistently smaller than it had been at 2 mM
[K+]o (11.8 ± 1.4 mV;
n = 8) (Fig. 6A2-A4). This
result corresponds to the expected diminution of the driving force for
the K+-Cl cotransporter at
high [K+]o. Assuming that somatic
inorganic [A] was dominated by
[A]pip, elevation of
[K+]o should not result in large
changes in somatic EGABA. As expected, increasing [K+]o from 2 to 10 mM hardly changed somatic EGABA
(1.0 ± 0.5 mV; n = 3) when tested in the same
cells, and furosemide had no effect on somatic
EGABA at 10 mM
[K+]o (Table 2). Consequently, in 10 mM [K+]o and furosemide,
there was little if any somatodendritic gradient for inorganic
[A].
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Figure 6.
The furosemide-sensitive
K+-Cl cotransporter extruded
or accumulated Cl depending on
[K+]o and
[Cl]i. A, The shift
of dendritic EGABA induced by furosemide was
larger in 2 mM [K+]o than
in 10 mM [K+]o when
[A]pip was high (15 mM).
A1, Elevating [K+]o
from 2 to 10 mM shifted EGABA of
dendritic IGABA ( marks application of
GABA) to positive values, which resulted in a reversal of current
direction (VH is indicated below chart
records). Washout of furosemide had only a small effect on
IGABA in 10 mM
[K+]o (A2) but a strong
effect in 2 mM [K+]o.
A4, Current-voltage relationship for the cell shown in
A1-A3. For clarity, control and recovery data were
pooled. Arrows indicate direction of the shift in
dendritic EGABA. B, In
another cell recorded with low
[A]pip (4.5 mM),
furosemide induced a small positive shift at 2 mM
[K+]o but a small negative shift of
dendritic EGABA at 10 mM
[K+]o.
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IGABA at 4.5 mM
[A]pip
In the experiments presented above, the patch pipette was used to
load the somatic compartment with moderate inorganic
[A]. To evaluate the role of
[Cl]i, we lowered
[A]pip (4.5 mM). With
4.5 mM [A]pip,
dendritic IGABA was more negative than with 15 mM [A]pip (Table 2).
According to the smaller driving force for the K+-Cl cotransporter at 4.5 mM [A]pip and 2 mM [K+]o, the
furosemide (0.1 mM)-induced shift of dendritic
EGABA was small (7.0 ± 0.9 mV;
n = 6) (Fig. 6B). Increasing
[K+]o from 2 to 10 mM
strongly shifted dendritic EGABA to positive values (27.8 ± 2.8 mV; n = 6). At 10 mM [K+]o,
furosemide induced small shifts of dendritic
EGABA; however, they were in the
direction opposite to the shifts in 2 mM
[K+]o (5.0 ± 0.7 mV;
n = 7) (Fig. 6B). These data indicate
that at low [A]pip the direction of
the Cl transport in the dendrites had been
reversed by elevating [K+]o. At 2 mM [K+]o,
[A]pip and the furosemide-sensitive
Cl outward transport determined
dendritic [Cl]i, whereas at
10 mM [K+]o,
dendritic [Cl]i was governed by the
Gibbs-Donnan equilibrium and the furosemide-sensitive inward transport.
We next tested the effects of [K+]o
changes on somatic EGABA at low
[A]pip. At 2 mM
[K+]o, somatic
EGABA was more positive than dendritic
EGABA (Table 2). However, elevation of
[K+]o and application of furosemide
(0.1 mM) had effects similar to those on dendritic
EGABA (Table 2). At 2 mM
[K+]o, furosemide shifted
somatic EGABA to more positive values (4.2 ± 0.5 mV; n = 8). Elevating
[K+]o from 2 to 10 mM
shifted somatic EGABA in the same direction (7.0 ± 0.1 mV; n = 4) and reversed the shift of
somatic EGABA under furosemide (3.4 ± 0.6 mV; n = 6). In 10 mM
[K+]o, therefore, no
somatodendritic gradient for inorganic
[A]i existed in either the presence
or absence of furosemide.
sIPSCs
Finally, we tested whether inward and outward sIPSCs were altered
by [Cl]i,
[K+]o, and furosemide as
expected from the data reported on IGABA. At 2 mM [K+]o and low
[A]pip, the mean amplitude of
sIPSCs measured at different VH could not be
fitted by a linear regression because sIPSCs appeared as inward and
outward currents near EIPSC (n = 4) (Fig. 7A). Increasing [K+]o from 2 to 5 mM while
recording from the same cell allowed the current-voltage relation to
be fitted by a first order regression because the sIPSCs reversed in
sign at a defined VH (Fig. 7B). At 5 mM [K+]o, the ratio
of
[Cl]o/[Cl]i
approximately equaled
[K+]i/[K+]o;
hence no driving force for an electroneutral
K+-Cl cotransport existed,
leading to a collapse of the somatodendritic [A]
gradient. Furthermore, the time course of inward IPSCs measured near
the reversal potential became slower in 5 mM
[K+]o than apparent inward IPSCs had
been in 2 mM [K+]o in the
same cell (rise time, 1.48 ± 0.04 msec vs 1.00 ± 0.04 msec;
time constant of decay, 16.5 ± 0.1 msec vs 7.1 ± 0.3 msec for 59 inward sIPSCs in 5 mM
[K+]o and 37 inward sIPSCs in 2 mM [K+]o at
VH 77 and 87 mV, respectively). As expected
from the thermodynamic properties of the
K+-Cl cotransporter,
increasing [K+]o to 8 mM
at 4.5 mM [A]pip
reversed the transport direction of the Cl
transport. Under these conditions, furosemide decreased the amplitude of inward sIPSCs and increased the amplitude of outward sIPSCs (n = 3) (Fig. 8). The
changes were the opposite of the changes induced by furosemide
at 15 mM [A]pip and low
[K+]o (Fig. 3A).
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Figure 7.
Near the thermodynamic equilibrium of the
K+-Cl cotransporter, GABAergic
network neurons generated sIPSCs with the same reversal potential at
the soma and the dendrites of the target neuron. A, In a
cell (4.5 mM
[A]pip), inward and outward
sIPSCs occurred at a VH 80 to 90 mV.
Current-voltage relationship for the sIPSCs was determined by sampling
peaks of inward or outward currents, respectively (A1).
A2, Traces show representative examples for inward and
outward sIPSCs at two VH values that were 5 mV apart. B1, In the same cell as in A,
the current-voltage relationship for peaks of all sIPSCs could be well
fitted by a linear regression line (r = 0.96) at 5 mM [K+]o. In the plots,
mean values and SE bars are shown unless SE bars remained within symbol
sizes. Minimum to maximum of sIPSC numbers sampled at each
VH ranged from 48 to 132.
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Figure 8.
Under conditions of high
[K+]o and low
[A]pip, furosemide shifted
EIPSC to a more negative value.
A, Furosemide reduced the amplitude of inward sIPSCs
(recorded with 4.5 mM
[A]pip and 8 mM
[K+]o). B, In
the same cell recorded under identical conditions as in
A but at a more positive
VH, furosemide increased the
amplitude of outward sIPSCs. Letters mark segments on
the chart plots that were shown at higher sweep speed in the lower
traces.
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DISCUSSION |
Our data show that a furosemide-sensitive transport can accumulate
or extrude Cl from embryonic midbrain neurons in
long-term culture. In terms of sensitivity to furosemide and to changes
in [Cl]i and
[K+]o, the cotransporter
exhibits functional characteristics described for the neuronal
K+-Cl cotransporter isoform
(KCC2) expressed in human embryonic kidney cell lines (Payne, 1997).
Because of the properties of the electroneutral K+-Cl cotransporter,
[K+]o determines the driving force for
Cl fluxes across GABAA receptor
channels and hence regulates synaptic inhibition.
Reduction of a [Cl]i gradient
between dendrites and soma by furosemide or
[K+]o
We observed a striking difference in driving forces for
GABAA currents between cell soma and dendrites. Both,
bicuculline-sensitive sIPSCs and focal GABA applications revealed the
gradient. At a VH near the expected equilibrium
potential for Cl, GABAA currents were
inward near soma and outward in dendritic parts. Furosemide reduced the
somatodendritic differences in the driving forces, inducing a shift in
EGABA that was more pronounced for dendritic
currents than for somatic currents. A reduction of the somatodendritic
gradient was also observed if [K+]o
was increased or [A]pip was reduced.
Dendritic [Cl]i resulting under our
experimental conditions (Fig.
9A) can be explained from
Gibbs-Donnan equilibrium and the thermodynamic profile of an
electroneutral K+-Cl
cotransport (Fig. 9B). K+ and
Cl can be expected to be nearly in equilibrium
across the neuronal membrane. [Cl]i
equal to [K+]o approximately satisfied
the Donnan relationship as well as the principle of electroneutrality.
[Cl]i was actually increased above
[Cl]i predicted from the Donnan
relationship or reduced below that concentration by a higher or lower
[Cl]pip, respectively. Near
soma, [Cl]pip determined
[Cl]i more closely than in the
dendrites. Depending on [K+]o,
dendritic [Cl]i was lower (Fig.
9Aa-Ac) or higher (Fig. 9Ad) than somatic
[Cl]i, indicating that
Cl movement across conductivity pathways exceeded
Cl diffusion from the patch pipette. The
electroneutral K+-Cl
cotransporter contributed to the conductivity pathways according to its
energy profile (Fig. 9B), as revealed by the shift in
dendritic [Cl]i resulting from
furosemide application. When [K+]o was
low and [Cl]pip was high (Fig.
9Aa), the driving force for the
K+-Cl cotransporter was
sufficiently large to generate a pronounced somatodendritic gradient.
The somatodendritic gradient was built up by the solution in the patch
pipette, which served as a Cl source, and the
K+-Cl cotransporter, which
counteracted that Cl load. The development of a
Cl gradient allowed us to study
Cl regulation, but the experiments did not
determine under which conditions such a gradient may exist in intact
neurons.
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Figure 9.
Cl homeostasis in
cultured neurons can be explained by the operation of an electroneutral
K+-Cl cotransporter and the
Gibbs-Donnan equilibrium. A,
[Cl]i was calculated from
EGABA listed in Table 2. Concentrations of
the inorganic anions in the patch pipette were as indicated.
Arrows indicate changes of
[A]i calculated from changes in
EGABA of somatic or dendritic
IGABA induced by furosemide.
Letters correspond to driving forces for the
K+-Cl cotransporter derived
from the energy profiles shown in B. B,
Plot of the driving force for the
K+-Cl cotransporter as a
function of [K+]o for different
[Cl]i (taken from
EGABA calculated for dendrites in
A). Calculations are as described in Material and
Methods. Positive values indicate outward transport.
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[Cl]i calculated from somatic
EGABA was always somewhat higher than
[Cl]pip. The small deviation was
likely attributable to injected currents carried by anions because we
set VH negative to the membrane potential.
Supporting this suggestion, the relative increase of [Cl]i above
[Cl]pip was larger with
[Cl]pip of 4.5 mM than
with [Cl]pip of 15 mM.
The low mobility of the main anion glucuronate promoted an increase of
[Cl]i above
[Cl]pip. Other possible factors such
as production of endogenous CO2 or the use of
Br were less likely contributors. An equal pH
inside and outside the cells probably prevented buildup of a
HCO3 gradient, and removal of the
gradient for Br had no measurable effect on
EGABA (see Materials and Methods).
The observed fast and slow sIPSCs could represent an artifact of an
imperfect space clamp. A similar finding was reported for
GABAA responses in CA1 neurons of hippocampal slices
(Pearce, 1993). In that study, arguments were put forward indicating
that a space-clamp artifact did not account for the different time courses of synaptic currents generated at different locations of the
neuron. In comparison, the analysis of IGABA in
cultured neurons that have high input resistances (
1 G) and short
dendrites (
300 µm) recorded in the presence of TTX, QX314, and
Cs+ should be much less hindered by geometrical
factors (Müller and Lux, 1993; Draguhn et al., 1997).
Furthermore, the slowing of the time course of inward sIPSCs when
[K+]o was increased (Fig. 7) indicated
that the time course of inward sIPSCs was shortened by superimposed
small outward sIPSCs as long as the somatodendritic
[Cl]i gradient existed.
The neuronal-specific K+-Cl
cotransporter is inhibited by furosemide (Ki 25 µM) (Payne, 1997). Furosemide has been used to establish
the involvement of a K+-Cl
cotransporter in synaptic inhibition (Misgeld et al., 1986; Thompson, 1994). Furosemide, however, has various disadvantages. In non-neuronal preparations, furosemide at concentrations >0.1 mM
inhibits not only cation-anion cotransporters but also other transport
proteins and Cl channels (Cabantchik and Greger,
1992). Here we could observe immediate effects of furosemide at
concentrations below 0.1 mM. Furosemide has been reported
to block GABAA channels that are composed of subunits
including 
4 and 6 (Wafford et al., 1996). These subunits are uncommon in midbrain areas (Wisden et al., 1992). In
neither this nor a previous study (Jarolimek et al., 1996) on midbrain
culture did we obtain evidence that furosemide reduced
GABAA currents. Depending on
VH, the amplitudes of IPSCs increased
when neurons were exposed to furosemide, and slopes of current-voltage
relationships for GABAA currents remained unaltered. As
shown in the previous study (Jarolimek et al., 1996), shifts of
reversal potentials of the same order of magnitude as those of
EGABA can be observed for glycine currents.
Furosemide blocks
Na+-K+-Cl
cotransporters more effectively than it blocks the
K+-Cl cotransporter
(Cabantchik and Greger, 1992). Thus, the furosemide effects described
here do not allow discrimination between the two transporters. Our
data, however, exclude a significant contribution of a
Na+-K+-Cl
cotransporter in the net Cl transport of cultured
midbrain neurons. Because of the large inwardly directed driving force
for Na+, the driving force of the net
Cl transport (Fig. 9Ac,d) would
not reverse near [K+]o and
[Cl]i predicted from the driving
force of a K+-Cl cotransporter
(Fig. 9Bc, d).
Regulation of the neuronal Cl homeostasis by
the furosemide-sensitive K+-Cl
cotransporter
The electroneutral K+-Cl
cotransporter operates near equilibrium under conditions of normal
internal and external ion concentrations (Jensen et al., 1993; Payne,
1997). If [K+]o is kept at a high
level (Fig. 9Ad), the
K+-Cl cotransporter
accumulates Cl in the cell. Under physiological
conditions, Cl accumulation would be diminished by
the concomitant removal of K+ from the extracellular
space (Payne, 1997). During epileptiform activity, however,
[K+]o has been found to remain at high
levels (10-12 mM) (Heinemann and Lux, 1977), with the
result that the K+-Cl
transport actually accumulates Cl and hence
intensifies epileptiform activity. Therefore, furosemide may have some
antiepileptic potency under such an experimental condition (Hochman et
al., 1995). In contrast, in neuronal culture we observed that
furosemide induced burst activity (Jarolimek et al., 1996). In these
experiments, [K+]o was kept constant
at 5 mM. In the presence of antagonists for ionotropic
glutamate receptors, we observe frequent sIPSCs, suggesting that
membrane potentials of the majority of cells are near to firing
threshold, e.g. around 60 mV. The expected lower limit of
[Cl]i is around 5.6 mM
in 5 mM [K+]o
([Cl]o = 169 mM;
[K+]i assumed to be 150 mM). There is, however, an electromotor force (EM ECl)
driving Cl into the cell. The hyperpolarizing
gradient for synaptic inhibition will be maintained by the outwardly
directed K+-Cl cotransport.
The upper limit of [Cl]i is 20 mM if the K+-Cl
cotransporter is blocked by furosemide. Hence, in furosemide, the
inhibitory potency of the GABAA postsynaptic potentials is diminished or abolished.
CONCLUSIONS
In cultured neurons, the regulation of
[Cl]i and the gradients for IPSCs is
determined by the presence and activity of the K+-Cl cotransporter. The
driving force for the net Cl transport is set by
[K+]o and
[Cl]i. Values determined in this
study indicate that [Cl]i is kept
well below 10 mM as long as
[K+]o is 2 mM. Increases
in [K+]o will increase
[Cl]i, but uptake of
K+ into neurons on reversal of the transport will
buffer [K+]o changes (Payne, 1997).
However, if [K+]o remains high,
the transport will accumulate Cl in the cells.
In vivo additional factors determine gradients for IPSCs
through GABAA or glycine channels, complicating the analysis of their regulation. Despite this fact, a furosemide-sensitive K+-Cl cotransporter is likely
to operate in central neurons because the KCC2 mRNA is expressed in
hippocampal neurons (Payne et al., 1996). The permeability for
HCO3 shifts
EGABA by 10-15 mV (depending on
pHi) in a positive direction (Alvarez-Leefmans,
1990; Kaila, 1994). A regulation of GABAergic inhibition through
[HCO3]i (Kaila, 1994) is
less likely given the low permeability ratio of GABAA
receptors for
HCO3/Cl (Bormann,
1988; Fatima-Shad and Barry, 1993). Furthermore, an inwardly rectifying
chloride conductance (Smith et al., 1995), which does not appear to
have a major influence in our cultured neurons (Jarolimek et al.,
1996), may come into play. For an excitatory effect of GABA postnatally
as opposed to an inhibitory effect in adult neurons (Cherubini et al.,
1991), a transient dominance of a
Na+-K+-Cl
cotransporter (LoTurco et al., 1995; Plotkin et al., 1997) probably coincides with a delayed expression of the
K+-Cl cotransporter (Zhang et
al., 1991).
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FOOTNOTES |
Received Jan. 20, 1999; revised March 11, 1999; accepted March 16, 1999.
This work was supported by the Sonderforschungsbereich 317/B13 of the
Deutsche Forschungsgemeinschaft (to U.M.). We thank Drs. F. Kuenzi and
K. Wafford for comments on this manuscript and C. Heuser for excellent
technical assistance.
Correspondence should be addressed to Dr. Ulrich Misgeld, I. Physiologisches Institut, Universität Heidelberg, Im Neuenheimer Feld 326, D-69120 Heidelberg, Germany.
Dr. Jarolimek's present address: Merck Sharp & Dohme Research
Laboratories, Neuroscience Research Centre, Terlings Park, Harlow, Essex CM 20 2QR, UK.
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REFERENCES |
-
Alvarez-Leefmans FJ
(1990)
Intracellular Cl regulation and synaptic inhibition in vertebrate and invertebrate neurons.
In: Chloride channels and carriers in neurons (Alvarez-Leefmans FJ,
Russel JM,
eds), pp 109-158. New York: Plenum.
-
Barry PH,
Lynch JW
(1991)
Liquid junction potentials and small effects in patch-clamp analysis.
J Membr Biol
121:101-117[ISI][Medline].
-
Bijak M,
Jarolimek W,
Misgeld U
(1991)
Effects of antagonists on quisqualate and nicotinic receptor-mediated currents of midbrain neurones in culture.
Br J Pharmacol
102:699-705[ISI][Medline].
-
Bormann J
(1988)
Electrophysiology of GABAA and GABAB receptor subtypes.
Trends Neurosci
11:112-116[ISI][Medline].
-
Cabantchik ZI,
Greger R
(19
TOP
ABSTRACT
INTRODUCTION
