1Cellular and System Physiology and 2Otorhinolaryngology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan
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ABSTRACT |
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Kakazu, Yasuhiro,
Soko Uchida,
Takashi Nakagawa,
Norio Akaike, and
Junichi Nabekura.
Reversibility and Cation Selectivity of the
K+-Cl Cotransport in Rat Central Neurons.
J. Neurophysiol. 84: 281-288, 2000.
The reversibility and cation selectivity of the
K+-Cl
cotransporter (KCC), which normally
extrudes Cl
out of neurons, was investigated in
dissociated lateral superior olive neurons of rats using the gramicidin
perforated patch technique. Intracellular Cl
activity
(
[Cl
]i) was maintained well
below electrochemical equilibrium as determined from the extracellular
Cl
activity and the holding potential, where the pipette
and external solutions contained 150 mM K+
([K+]pipette) and 5 mM K+
([K+]o), respectively. Extracellular
application of 1 mM furosemide or elevated
[K+]o increased
[Cl
]i. When the pipette solution
contained 150 mM Cs+
([Cs+]pipette),
[Cl
]i increased to a value higher than
the passive
[Cl
]i. An increase of
[Cl
]i with the
[Cs+]pipette was not due to the simple
blockade of net KCC by the intracellular Cs+ since
[Cl
]i, with the pipette solution
containing 75 mM Cs+ and 75 mM K+, reached a
value between those obtained using the
[K+]pipette and the
[Cs+]pipette. The higher-than-passive
[Cl
]i with the
[Cs+]pipette was reduced by 1 mM furosemide,
but not by 20 µM bumetanide or Na+-free external
solution, indicating that the accumulation of
[Cl
]i in the
[Cs+]pipette was mediated by a KCC operating
in a reversed mode rather than by Na+-dependent,
bumetanide-sensitive mechanisms. Replacement of K+ in the
pipette solution with either Li+ or Na+
mimicked the effect of Cs+ on
[Cl
]i. On the other hand,
Rb+ mimicked K+ in the pipette solution. These
results indicate that K+ and Rb+, but not
Cs+, Li+, or Na+, can act as
substrates of KCC in LSO neurons.
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INTRODUCTION |
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The
K+-Cl cotransporter (KCC)
is one of the cation/chloride cotransporters (CCC) present in the
plasma membrane. The KCC can carry K+ and
Cl
in the same direction electroneutrally with
a stoichiometry of 1:1 (for review, see Alvarez-Leefmans
1990
; Kaila 1994
; Lauf and Adragna
1996
; Payne 1997
). The thermodynamic driving
force for net KCC is derived from the sum of the chemical potential
differences for K+ and
Cl
. Normally the KCC, driven in a net outward
direction by the K+ gradient produced by
Na+-K+ pump
(Alvarez-Leefmans 1990
), pumps
Cl
out of the cell. The
Cl
extrusion results in the maintenance of a
steady state low intracellular Cl
concentration
([Cl
]i), thus producing
a Cl
electrochemical gradient that favors
Cl
influx into the cell through GABA- or
glycine-operated Cl
channels. KCC2, a subtype
of KCC, is expressed in the brain with an exclusively neuronal location
(Payne et al. 1996
; Rivera et al. 1999
;
Williams et al. 1999
). This KCC2 most likely plays a major role in Cl
homeostasis in mature neurons
(Kakazu et al. 1999
; Payne 1997
; Rivera et al. 1999
; Thompson and Gähwiler
1989
). Until now, the kinetics of CCCs have been evaluated by
measuring radioactive Rb+ fluxes
(86Rb+) instead of the
physiologically relevant K+ fluxes (Gillen
et al. 1996
; Payne 1997
). The detailed
properties and kinetics of the KCC have been extensively studied by
this method in red blood cells (Gibson et al. 1998
;
Kelley and Dunham 1996
; Lauf and Adragna
1996
). Although this flux technique is appropriate to examine
the basic functions of the cotransporters in an aggregation of cells of
one type, it is difficult to evaluate directly the ionic equilibrium
potentials and to monitor the transport activities in a single neuron.
KCC2 has also been suggested to function as a buffer carrying excess
extracellular K+
([K+]o) into the cell
under conditions of elevated
[K+]o (Payne
1997
). To explain this bidirectionality of the net fluxes mediated by the KCC and its functional consequences in a single neuron,
the direct demonstration of the reversibility of KCC net fluxes in
native central neurons is necessary (Kaila 1997
;
Thompson and Gähwiler 1989
).
In addition, it has been reported that
EGABA and EIPSP recorded
with Cs+ filled microelectrodes are more
depolarized than those with K+ filled electrodes
(Thompson and Gähwiler 1989), which also suggests that intracellular cations, such as Cs+, affect
neuronal Cl
regulation (Kakazu et al.
1999
). However, the effects of various intracellular cations on
[Cl
]i regulation, i.e.,
CCC, have not been elucidated in detail in neurons.
In the present study, we employed the gramicidin perforated patch
recording technique on acutely dissociated lateral superior olive (LSO)
neurons to examine the mechanisms of
[Cl]i regulation. The
gramicidin technique applied on LSO neurons is particularly well suited
to study the KCC for two reasons. First, only monovalent cations, but
not Cl
ions, are permeant through the
gramicidin pore, thus allowing electrical recording without a direct
influence of Cl
in the pipette solution on
[Cl
]i (Akaike
1997
). With the gramicidin technique, the effects of various
monovalent cations perfused from the pipette solution on native
[Cl
]i regulation could
be investigated by observing the recovery of
[Cl
]i after its
alteration induced by the opening of glycine-operated Cl
channels. Second, KCC is well developed and
plays a major role in
[Cl
]i regulation in
mature rat LSO neurons (Kakazu et al. 1999
). In this
paper, we demonstrate the reversibility of the KCC and its cation
selectivity. Our results also strongly argue against using
Cs+ as an internal cation in perforated patch
experiments designed to examine neuronal Cl
regulation.
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METHODS |
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Acutely dissociated LSO neurons
The experimental protocol was approved by the Ethics Review
Committee for Animal Experimentation of our institution. The mechanical dissociation technique of CNS neurons was employed as described previously (Rhee et al. 1999). In brief, Wistar rats
aged postnatal day 13-15 (P13-15, either sex) were decapitated under
anesthesia with pentobarbitone sodium (i.p., 55 mg/kg), and 300 µm
transverse slices of the brain including the LSO were prepared with a
microslicer (VT-1000S, Leica, Nussloch, Germany). The slices
were incubated for 1 to 2 h in the incubation solution containing
(in mM): 124 NaCl, 5 KCl,1.2
KH2PO4, 1.3 MgSO4, 2.4 CaCl2, 10 glucose, and 24 NaHCO3. The pH was 7.4 oxygenated
with 95% O2-5% CO2 gas
mixture at room temperature (22-26°C). Thereafter, the hemi-slice
divided into the right and left LSO side was transferred into a Petri dish and a fire-polished glass pipette was lightly applied to the
surface of the LSO identified under a binocular microscope (Nikon,
SMZ-1, Japan). The pipette was vibrated horizontally at 5-10 Hz for
about 2 min, and then the slice was removed. The mechanically dissociated LSO neurons, prepared without using any enzyme, adhered to
the bottom of the dish within 30 min. Neuronal somata with intact
morphological features, such as the proximal dendritic processes, were
used in the present experiments.
Solutions
The standard external solution used for electrical recordings
contained (in mM): 150 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 hydroxyethylpiperazine-N-2-ethanesulphonic acid (HEPES), 10 glucose. For Na+-free experiments, NaCl in the
standard external solution was replaced with
N-methyl-D-glucosamine (NMDG)-OH, which was
first dissolved in water and titrated with HCl at pH 7.0 ± 0.1. The Cl activities of the extracellular
solutions (
[Cl
]o)
measured with Cl
-sensitive electrodes using a
liquid ion exchanger (WPI IE-170, WPI) according to the method reported
by Komune et al. (1993) were 114.5, 120, and 122 mM in
the standard solution, 20 mM
[K+]o and 30 mM
[K+]o, respectively.
Since
[Cl
]o in the
Na+-free solution (94.2 mM) was different from
that in the standard solution (114.5 mM), we prepared a modified
standard solution containing (in mM): 110 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, 10 glucose, 80 mannitol
(
[Cl
]o; 95.6 mM).
The pH in all external solutions were adjusted to 7.4 with
tris(hydroxymethyl)aminomethane (Tris)-OH. Osmolarities in all external
solutions were confirmed to be similar (317-326 mOsm/kg) by osmometric
measurements (OM802, Vogel, Germany). The patch pipette solution for
gramicidin perforated patch recording contained (in mM): 150 KCl and 10 HEPES. For Cs+, Li+,
Na+, and Rb+ containing
pipette solutions, KCl in the pipette solution was simply replaced with
an equimolar CsCl, LiCl, NaCl, and RbCl, respectively. All pipette
solutions were buffered to pH 7.2 with Tris-OH. Gramicidin was first
dissolved in methanol to prepare a stock solution of 10 mg/ml and then
diluted to a final concentration of 100 µg/ml in the pipette
solutions. The gramicidin-containing solution was prepared just before
the experiment.
Electrophysiological measurements
Ionic currents and voltages were measured with a patch clamp
amplifier (EPC-7, List-Electronic, Germany), low-pass filtered at 1 kHz
(FV-665, NF Electronic Instruments, Japan), monitored on an
oscilloscope (HS-5100A, Iwatsu, Japan), and recorded on both a pen
recorder (Recti-Horiz-8K21, Nihondenki San-ei, Japan) and a digital FM
tape recorder (RD-120TE, TEAC, Japan). In ramp experiments, 3 × 107 M tetrodotoxin (TTX)
and 10
5 M
LaCl3 were added to the extracellular solutions.
To measure the reversal potential of the glycine induced current
(Egly), ramp voltage steps from 0 to
100 mV of
2-s duration were applied before and during glycine by using a function
generator (MacLab, AD Instruments, Australia). Data were also
simultaneously collected using computer software (Scope v3.6, AD
Instruments, Australia). Patch pipettes were constructed of glass
capillary tubes with an outer diameter of 1.5 mm prepared using a
vertical puller (PB-7, Narishige Scientific Instruments, Japan). The
tip resistance of the electrodes was 4-8 M
. The junction potential
between the patch pipettes and bath solution was nulled before giga ohm
(G
) seal formation. After establishing contact with the cell
surface, a G
seal was established by applying gentle suction to the
patch pipette interior. After the cell attached configuration had been attained, the patch pipette potential was held at
50 mV, and
10 mV
hyperpolarizing step pulses of 300 ms duration were periodically delivered to monitor the access resistance. In the gramicidin perforated patch recording, the access resistance reached a steady level <30 M
within 40 min after making the G
seal. In the
conventional whole-cell configuration after rupturing gramicidin
perforated patch membrane by applying greater negative pressure to the
pipette interior, the reversal potential of Egly
moved to around 0 mV (+4.7 mV was obtained as ECl
from the Nernst equation using the Cl
activity
in the standard solution and 150 mM KCl pipette solution). Thus the
difference in Egly values between
gramicidin-perforated patch and conventional whole cell recordings is a
good indicator for monitoring the stability of the recording
configuration. All experiments were carried out at room temperature
(22-26°C).
Drugs
Rapid "square-wave" change of external solution was
performed with "Y-tube" method described previously
(Nakagawa et al. 1990). Using this method, the external
solution could be completely exchanged within 20 ms. Drugs used in the
experiments were gramicidin D, bumetanide (Sigma Chemical, St. Louis,
MO), glycine (Kanto, Tokyo), furosemide (Tokyo Kasei, Tokyo), and TTX
(Sankyo, Tokyo). The final dimethyl sulfoxide (DMSO) concentration in
the experiments did not exceed 0.1%.
Statistical analysis
Data were expressed as mean ± SE. Differences between groups were analyzed for statistical significance using the Student's t-test.
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RESULTS |
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Regulation of intracellular Cl by
K+-Cl
cotransporter in LSO neuron
Under voltage clamp conditions at a holding potential
(VH) of 50 mV, repetitive applications of
3 × 10
5 M glycine
at an interval of 3 min elicited outward currents with constant
amplitude in all LSO neurons examined. These experiments used a pipette
filled with 150 mM K+
([K+]pipette) and a
standard extracellular solution containing 5 mM K+
([K+]o, see Fig.
3A, bottom trace). Since the reversal potential
of the glycine responses (Egly) can be taken to
be ECl in our nominally bicarbonate-free in vitro
system, the intracellular Cl
activity
(
[Cl
]i) was directly
calculated from the extracellular Cl
activity
(
[Cl
]o, 114.5 mM)
and Egly (ECl = 58 log
[Cl
]i,
119.4 mV).
Thus to monitor the net activity of the neuronal Cl
regulatory system, which directly influences
[Cl
]i, we measured
Egly in the present study. Assuming that no
Cl
regulatory mechanism existed, an increase of
[Cl
]i by passive
Cl
influx through glycine channels should
degrade the electrochemical gradient. However, glycine at the intervals
of 3 min induced a constant outward current and the cell
[Cl
]i stayed
constant at 5.1 ± 0.2 to 11.2 ± 0.4 mM (n = 6, see Fig. 3B), significantly lower than passive
[Cl
]i (15.7 mM at a
VH of
50 mV). Therefore, a functional
Cl
extrusion mechanism must exist in the LSO neurons.
Effect of furosemide and bumetanide
In the presence of 1 mM furosemide, the outward glycine-induced
current (Igly) gradually decreased in amplitude
(Fig. 1A, top
trace) and
[Cl
]i, determined
from Egly (data not shown), gradually increased toward passive
[Cl
]i
(Fig. 1B). Following washout of furosemide, the amplitude of Igly and
[Cl
]i recovered
almost to those values before the application of the drug. The value of
[Cl
]i obtained at
the third glycine application in furosemide (13.5 ± 1.8 mM) was
significantly higher than that just before furosemide (9.3 ± 1.7 mM, n = 3, P < 0.05, paired
t-test). Furosemide at the concentration employed here
inhibits not only KCC, but also the
Na+-K+-Cl
cotransporter (NKCC; Gillen et al. 1996
), which drives
Cl
inward and accumulates
[Cl
]i (Kaila
1994
). However, bumetanide, a potent blocker of NKCC (Gillen et al. 1996
; Payne 1997
), at a
concentration of 20 µM did not alter the amplitude of outward
Igly and
[Cl
]i (Fig.
1A, bottom trace, and Fig. 1C,
n = 3). It is possible for the NKCC in the LSO neurons
to have a slow rate constant for the inhibition and thus require more
time. However, the outward Igly and
[Cl
]i were little
changed even after about 25 min of incubation (Fig. 1C).
These results suggest that the NKCC does not seem to be responsible for
Cl
regulation in the LSO neurons.
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[Cl]i regulation by the K+
chemical gradient
An increase of extracellular K+
concentration ([K+]o)
from 5 to 20 mM gradually reduced the amplitude of outward
Igly (Fig. 2A, top trace) and
increased [Cl
]i
close to the calculated passive
[Cl
]i at a
VH of
50 mV (Fig. 2B, filled circles
with asterisk; 15.6 ± 0.4 mM, n = 4). After the
return of [K+]o from 20 to 5 mM, Igly and
[Cl
]i gradually
recovered to the level prior to 20 mM
[K+]o. The ideal value of
[Cl
]i in 20 mM
[K+]o from the chemical
potentials alone (see DISCUSSION) is 16 mM, similar to the
passive
[Cl
]i. Then,
we next tried 30 mM [K+]o
to clarify whether the actual
[Cl
]i was determined
by VH or merely by the chemical potentials. A
change of [K+]o from 5 to
30 mM reversed the polarity of Igly from outward to inward (Fig. 2A, bottom trace); the actual
[Cl
]i attained a
higher value (19.0 ± 1.3 mM, n = 3) than the
passive
[Cl
]i (Fig.
2B, open squares). For experiments in which the
[K+]pipette was used,
[K+]o increased
[Cl
]i in a
concentration-dependent manner (Fig. 2C). These results indicate that
[K+]o-dependent
[Cl
]i regulation plays
a major role in Cl
extrusion in LSO neurons.
Together with the results from the furosemide and bumetanide
experiments, we conclude that the KCC is a likely candidate for the
mediation of Cl
extrusion and the maintenance
of an
[Cl
]i lower
than the passive
[Cl
]i in the LSO
neurons.
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To examine the effect of intracellular K+
concentration ([K+]i) on
[Cl]i regulation, the
150 mM K+ in the
[K+]pipette was replaced
with 150 mM Cs+
([Cs+]pipette). Just
after the access resistance dropped to <30 M
and became stable, as
confirmed by rectangular pulses from
50 to
60 mV of 300 ms
duration, we started to measure Igly and
Egly. For the
[K+]pipette, the
amplitude of outward Igly at a
VH of
50 mV was almost constant throughout the
experiment (Fig. 3A,
bottom trace, n = 6). On the other hand, the
amplitude of outward Igly gradually decreased
when recording with the
[Cs+]pipette, and finally
Igly reversed to an inward current (Fig. 3A, top trace). Although
[Cl
]i in the
[Cs+]pipette at the first
application of glycine was almost equal to that in the
[K+]pipette (Fig.
3B, arrow),
[Cl
]i in
measurements with the
[Cs+]pipette gradually
increased and reached a significantly higher value than that seen with
the [K+]pipette (Fig.
3B, **; 18.3 ± 1.0 mM, n = 4). We also
used a pipette solution containing 75 mM K+ and
75 mM Cs+ ([K+ and
Cs+]pipette). Although the
initial
[Cl
]i in
[K+ and
Cs+]pipette were similar
to that in the
[K+]pipette,
[Cl
]i in experiments
with the [K+ and
Cs+]pipette also gradually
increased, similar to that seen with the [Cs+]pipette. However,
[Cl
]i finally
attained a value between those measured with the two types of the
pipette after several applications of glycine (Fig. 3B, *;
10.8 ± 0.1 mM, n = 3). These results indicate
that the KCC activity depends on
[K+]i and that
Cs+ cannot substitute for
K+ to drive KCC.
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Reversibility of KCC
The data obtained from the experiments changing extra- and
intracellular K+ concentrations implies the
possible reversibility of the KCC. The outward
Igly in [K+ and
Cs+]pipette in 5 mM
[K+]o was reversed to an
inward direction in 20 mM
[K+]o (Fig.
4A). In addition,
[Cl
]i in experiments
with the [K+ and
Cs+]pipette in 20 mM
[K+]o attained a value
over passive
[Cl
]i
(Fig. 4B), unlike the case with the
[K+]pipette and 20 mM
[K+]o (Fig.
3A). This finding suggests that the net direction of KCC is
determined by the K+ chemical gradient across the
membrane. To demonstrate the involvement of KCC operating in the
reverse mode as the cause for the elevated
[Cl
]i in recording
with [Cs+]pipette as
compared with the passive
[Cl
]i at a
VH of
50 mV (Fig. 3B), furosemide
was applied and the inward Igly in
[Cs+]pipette was
monitored. In the presence of 1 mM furosemide, the amplitude of inward
Igly and
[Cl
]i gradually
decreased for the
[Cs+]pipette (Fig.
5A, top trace, and
B, n = 6). In contrast, 20 µM bumetanide
did not affect the inward Igly or the
[Cl
]i even after an
incubation period of over 20 min (Fig. 5A, middle trace, and C, n = 3). In addition,
treatment with Na+-free extracellular solution in
[Cs+]pipette to block
NKCC activity by depriving Na+ substrate from
both intra- and extracellular sides did not change the inward
Igly or the
[Cl
]i (Fig.
5A, bottom trace, and D,
n = 3). Together with the results of Fig. 1, these
results indicate that: 1) the decrease of
[K+]i, by replacing
K+ with Cs+ in the pipette
solution, reversed the direction of KCC, thus resulting in an
accumulation of Cl
in the cell and the
maintenance
of
[Cl
]i higher
than passive
[Cl
]i;
and 2) the NKCC appears to have little effect on
[Cl
]i homeostasis in
our acutely dissociated LSO neuron at postnatal day 13-15, as
previously demonstrated (Kakazu et al. 1999
).
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|
Selectivity of intracellular monovalent cations on KCC function
We also investigated the selectivity of intracellular monovalent
cations other than K+ and
Cs+ on KCC function. As in the case with the
[Cs+]pipette, the
amplitudes of outward Iglys gradually decreased and finally reversed their polarities when using a pipette solution containing 150 mM Li+
([Li+]pipette) or 150 mM
Na+
([Na+]pipette) (Fig.
6A, top and middle
traces). In addition, the time courses of the increases of
[Cl
]i when using the
[Li+]pipette or the
[Na+]pipette (Fig.
6B) were also similar to those in recordings with the
[Cs+]pipette (Fig.
3B). The initial value of
[Cl
]i for the
[Li+]pipette (10.2 ± 0.3 mM, n = 3, Fig. 6B, arrow) measured
at the first application of glycine was somewhat higher than those seen with the [Cs+]pipette
(8.5 ± 0.8 mM, n = 4, Fig. 6B, arrow)
or the [Na+]pipette
(8.5 ± 1.9 mM, n = 3, Fig. 6B, arrow).
However, there were no statistically significant differences between
two of three initial values (P > 0.1, unpaired
t-test).
[Cl
]is in the
[Li+]pipette,
[Na+]pipette, and
[Cs+]pipette measured at
the eighth glycine applications were 18.8 ± 1.0, 17.4 ± 1.2, and 18.3 ± 1.0 mM, respectively. Significant difference was
observed in
[Cl
]is
between the first and the eighth glycine applications in each pipette
solution (P < 0.01, paired t-test). There
was no significant difference between each two of three values obtained
at the eighth glycine application (P > 0.1, unpaired
t-test). On the other hand, in the pipette solution
containing 150 mM Rb+
([Rb+]pipette),
Igly, and
[Cl
]i at the initial
values were well maintained, similar to those in
[K+]pipette (Fig.
6A, bottom trace, and B). There was no
significant difference in
[Cl
]is at the eighth
glycine application between
[K+]pipette (8.0 ± 0.9 mM, n = 6, Fig. 3B) and
[Rb+]pipette (9.4 ± 1.3 mM, n = 3, Fig. 6B, P > 0.1, unpaired t-test). These results suggest that
1) neither intracellular Li+,
Na+, nor Cs+ can substitute
for K+ in the activation of the KCC; and
2) the potency of Rb+ to drive KCC is
similar to that of K+.
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DISCUSSION |
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An inwardly directed Cl electrochemical
gradient drives Cl
influx through ligand-gated
Cl
channels located in postsynaptic membranes
and thus induces a hyperpolarization in the majority of mature CNS
neurons. In the absence of active Cl
transport,
[Cl
]i will be passively
distributed according to the
[Cl
]o and the resting
membrane potential, preventing any net Cl
flux
following Cl
channels activation
(Alvarez-Leefmans 1990
; Zhang 1991
).
Under these conditions, only shunting inhibition would be effective (Funabiki et al. 1998
). In the present study, a constant
outward Igly and
[Cl
]i below
electrochemical equilibrium were maintained in the LSO neurons (Fig.
3A, bottom trace, and B), indicating
the presence of an active Cl
extrusion
mechanism, which works during the "resting interval" when the
glycine-gated channels are closed. This Cl
extrusion mechanism was apparently mediated by KCC, because furosemide, but not bumetanide, inhibited the Cl
extrusion
(Fig. 1), because a reduction of K+ chemical
gradient by manipulating
[K+]i and/or
[K+]o increased
[Cl
]i (Figs. 2-4),
and last, because the extrusion mechanism operated normally in
sodium-free media (Fig. 5).
Reversibility of KCC direction
The KCC carries K+ and
Cl in the same direction in an electroneutral
fashion (Alvarez-Leefmans 1990
; Lauf and Adragna
1996
). In recent studies, KCC2, a subtype of KCC, has been
identified exclusively in the brain (Payne et al. 1996
;
Williams et al. 1999
) and more specifically, KCC2 showed
a specific neuronal location with a central role in
Cl
extrusion as demonstrated in hippocampal
pyramidal neurons (Rivera et al. 1999
). Its net
transport activity is inhibited with furosemide at a concentration of 1 mM (Gillen et al. 1996
; Payne 1997
) and depends on the chemical gradients of K+ and
Cl
(Payne 1997
). Under
physiological conditions, the difference between
[K+]i and
[K+]o generated by
Na+-K+ pump produces a net
outward driving force on the KCC, resulting in a net
Cl
extrusion along with
K+ (Alvarez-Leefmans 1990
). In the
present study, the polarity of Igly was reversed
and the
[Cl
]i was in
excess of the passive
[Cl
]i (as determined
from the repetitive application of glycine) in both 30 mM
[K+]o (Fig. 2) and when
making recordings with the
[Cs+]pipette (Fig. 3).
These results are most likely due to the reversed driving force on the
net transport of the KCC induced by an increase of
[K+]o and/or a decrease
of [K+]i. In both cases,
Cl
would accumulate in the cell. Conditions
where [K+]o increases
include an increase of the rate of neuronal action potentials during
which delayed K+ channels are activated
(Hounsgaard and Nicholson 1993
), a large glutamate
release opening nonselective cation channels and activating the
excitatory amino acid transporter (Levy et al. 1998
;
Szatkowski and Attwell 1994
), a high-frequency
stimulation of GABAergic inputs (Kaila et al. 1997
), or
application of a large concentration of exogenous GABA (Barolet
and Morris 1991
). Furthermore,
[K+]o reaches
approximately 60 mM during ischemia (Hansen 1985
), which
reduces intracellular ATP, resulting in an opening of ATP-sensitive K+ channels and deactivating the
Na+-K+ ATPase
(Koyama et al. 1999
), and nearly 100 mM in some forms of
epileptic discharge (Avoli et al. 1996
).
The driving force for net KCC (µKCC) is the
sum of the chemical potential differences of K+
(
µK) and Cl
(
µCl). When net KCC reaches thermodynamic
equilibrium,
µKCC is zero (i.e.,
µKCC =
µK +
µCl = 0). As
µK = RT ln
[K+]i/[K+]o
and
µCl = RT ln
[Cl
]i/[Cl
]o,
the forces driving KCC simplify to
[K+]i × [Cl
]i = [K+]o × [Cl
]o. In the case of
[K+]i = 150 mM and
[K+]o = 5, 20, and 30 mM,
the ideal
[Cl
]i are
calculated to be 3.8, 16, and 24.4 mM, respectively. Actually,
[Cl]i in 20 mM
[K+]o solution (*;
15.6 ± 0.43 mM, Fig. 2B) was very close to the ideal
value calculated above (16 mM). We also confirmed the actual
[Cl
]i values
scarcely changed even after the eighth glycine application (~21 min)
in 20 mM [K+]o solution.
Thus, it could be concluded that
[Cl
]i obtained after
the fourth glycine application in 20 mM
[K+]o solution nearly
reaches a stable state, suggesting that the thermodynamic driving force
for KCC is zero. However, in 30 mM [K+]o solution,
[Cl
]i after the
fourth glycine application (19.4 ± 1.3 mM) was different from the
value calculated (24.4 mM). Also the 3.8 mM theoretical
[Cl
]i in 5 mM
[K+]o was actually found
to be 5-11 mM. One possible reason for these discrepancies might be
the difficulties in the maintenance of the higher or the lower
[Cl
]i compared with the
passive [Cl
]i (i.e.,
from ECl) for the membranes having appreciable
conductance. Also, other Cl
transport pathways
might be in the system under study.
Several mechanisms could explain the net increase of
[Cl]i induced by the
replacement of [K+]i with
[Cs+]i. The chloride
actually overshoots the expected passive
[Cl
]i, whereas
simple blockade of the transporter should result in the passive
distribution. Therefore, Cs+ does not seem to
directly block the KCC function. From the observation of
[Cs+ and
K+]pipette, one
possibility is that the Cs+ binds with very low
affinity to the same transport site as K+. Thus,
replacement of [K+]i with
[Cs+]i will drive most of
the transporters into an inwardly facing state in the transport cycle.
This would greatly reduce the ability of KCC to mediate net flux.
Another possibility is that the depletion of
[K+]i could prevent KCC
function directly. We could not distinguish between these possibilities
in our experimental protocols.
A decrement in the K+ chemical gradient would
result in a change of net fluxes from the KCC (Kaila et al.
1997; Payne 1997
). In this paper, we directly
demonstrated the reversibility of KCC transport in function in native
mammalian neuron. We note that
[Cl
]i in the
[Cs+]pipette should be
infinite using the above formula. However, the experimental values of
[Cl
]i in
[Cs+]pipette was about
17-23 mM. This discrepancy might be explained by residual
[K+]i remaining even in
the [Cs+]pipette
condition. K+ might enter the cell on the KCC
faster than it can diffuse up the gramicidin pipette. At some steady
state value of
[Cl
]i
in [Cs+]pipette,
[K+]i would presumably be
constant, but unknown. In any case, intracellular perfusion of
Cs+ by the
[Cs+]pipette might not
exclude [K+]i completely,
so that the driving force of the KCC would, in fact, be finite. Also,
other Cl
regulators might be present on the
membrane. It is conceivable that accumulated Cl
may escape from the inside of the cell by other
Cl
regulators including leak pathways.
Cation selectivity of KCC function
Since only monovalent cations, but not Cl,
can pass the gramicidin pore (Akaike 1997
), this method
allowed the internal dialysis of various monovalent cations simply by
adding them to the pipette solution (Figs. 3 and 6). The
[Rb+]pipette did not
increase
[Cl
]i,
suggesting that Rb+ could substitute for
K+ for KCC function (Fig. 6), as well as for the
Na+-K+ pump
(Munakata et al. 1997
) and K+
channel (Jansen et al. 1998
). In contrast, internal
perfusion of Cs+, Li+, or
Na+ decreased the amplitude of the initially
outward Igly and finally reversed its polarity to
inward (Figs. 3 and 6). The effects on the
[Cl
]i observed using
a [Cs+]pipette,
[Li+]pipette, or
[Na+]pipette are probably
based on the same mechanism, because of the similarity of the time
courses of
[Cl
]i
increase (Figs. 3 and 6). Moreover, our studies, together with the
report that EGABA and EIPSP
recorded with Cs+ filled microelectrodes are more
depolarized than those with K+ filled electrodes
(Thompson and Gähwiler 1989
), strongly suggest that one should not perfuse intracellularly with monovalent cations other than K+ or Rb+ to
examine native neuronal Cl
regulation.
There is a possibility that an increase of
[Na+]i resulting from
diffusion from the
[Na+]pipette might affect
some other [Cl]i
regulating transporters, such as the
Na+-dependent
HCO3
-Cl
exchanger
or the
Na+-(K+)-Cl
cotransporter (N(K)CC) (Kaila 1994
). In the present
experiment, however, we employed the standard extracellular solution
buffered with HEPES, in the nominal absence
HCO3
, and thus
Na+-dependent
HCO3
-Cl
exchanger should have an insignificant role. The N(K)CC transports in
an electroneutral fashion. Its net direction is determined by the
chemical gradients of Na+,
(K+), and Cl
, and
stoichiometry of the transporter (Haas 1994
;
Kaila 1994
). Intracellular perfusion of
Na+ could be expected to drive the N(K)CC in a
net outward direction and thus to expel intracellular
Cl
. However, our results showed an increase of
[Cl
]i when using the
[Na+]pipette (Fig. 6). In
addition, bumetanide and an extracellular solution lacking
Na+ did not affect the Igly
or
[Cl
]i for either
the [K+]pipette or the
[Cs+]pipette (Figs. 1 and
5). These results suggest that the Na+-dependent
[Cl
]i regulators,
including N(K)CC, seem to be insignificant in
[Cl
]i regulation in LSO
preparation. Thus LSO neurons examined in the present study would seem
to be suitable to study KCC function in native CNS neurons because of
its constitutive expression of KCC and lack of NKCC (Figs. 1 and 5).
The LSO initially integrates the auditory information from the two
ears. The probability of action potentials in the LSO is determined by
the time differences of arrival from the ipsilateral (glutamatergic)
and contralateral (glycinergic) inputs. This process, which encodes
interaural intensity differences, supplies the cues to localize sound
in space (Koyano and Ohmori 1996; Sanes and Rubel
1988
). Therefore, the maintenance of the chloride gradient mediated by the KCC, with its consequent hyperpolarizing IPSP as well
as shunting inhibition (Funabiki et al. 1998
), is likely to be very important for LSO neurons in auditory information processing.
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ACKNOWLEDGMENTS |
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We thank Prof. Kai Kaila and M. Brodwick for discussions and helpful comments on this manuscript.
Grant-in-Aids (Nos. 1170240 and 11670044 to J. Nabekura, and Nos. 10470009 and 10044301 to N. Akaike) from the Ministry of Education, Science and Culture, Japan, supported this work.
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FOOTNOTES |
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Address for reprint requests: J. Nabekura, Cellular and System Physiology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan (E-mail: nabekura{at}mailserver.med.kyushu-u.ac.jp).
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 13 September 1999; accepted in final form 30 March 2000.
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REFERENCES |
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