1Department of Neurology, Heinrich-Heine-University, 40225 Duesseldorf, Germany; and 2Institute for Neurobiology, University of Amsterdam, 1098 SM Amsterdam, The Netherlands
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ABSTRACT |
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Bruehl, C.,
W. J. Wadman, and
O. W. Witte.
Concentration Dependence of Bicarbonate-Induced Calcium Current
Modulation.
J. Neurophysiol. 84: 2277-2283, 2000.
High-voltage-activated calcium currents (HVA) of
CA1 neurons are prominently attenuated following a switch from
HEPES-buffered solution to one buffered with
CO2/HCO3. In the present study
we investigated whether bicarbonate ions or the dissolved
CO2 induce this alteration in current
characteristic. The study was carried out on freshly isolated CA1
neurons using the whole cell patch-clamp technique. Maximal calcium
conductance and the mean peak amplitude of the currents showed a
concentration-dependent decrease when cells were consecutively bathed
in solutions containing increasing amounts of bicarbonate and
CO2. This decrease is best described by the Hill
equation, yielding a maximal attenuation of 69%, a half-maximal
concentration (EC50) of 7.4 mM
HCO3
, and a Hill coefficient of 1.8. In parallel, the
potentials of half-maximal activation
(Vh,a) and inactivation
(Vh,i) were linearly shifted in
hyperpolarizing direction with a maximal shift, in the 10%
CO2/37 mM HCO3
containing
solution of 10 ± 1 mV for Vh,a
(n = 23) and 17 ± 1.4 mV for
Vh,i (n = 18). When
currents were evoked in solutions containing equal concentrations of
bicarbonate but different amounts of CO2, only
nonsignificant changes were observed, while marked alterations of the
currents were induced when bicarbonate was changed and CO2 held stable. The experiments suggest that
bicarbonate is the modulating agent and not CO2.
This bicarbonate-induced modulation may be of critical relevance for
the excitation level of the CNS under pathological situation with
altered concentration of this ion, such as hyperventilation and
metabolic acidosis.
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INTRODUCTION |
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In a previous study we have
shown that CO2/HCO3-buffered
solutions have modulatory effects on whole cell calcium currents in
neurons from the hippocampal area CA1 of the rat (Bruehl et al.
1998
, 1999
). When current characteristics were
compared between a
CO2/HCO3
-free bathing medium
(HEPES buffering) and a
CO2/HCO3
-containing solution,
we could demonstrate that the so-called high-voltage-activated (HVA)
calcium current decreases in amplitude and maximal conductance in the
latter bath solution. Furthermore, the potentials for half-maximal
activation and inactivation were strongly shifted to more negative
values. This kind of modulation may be of importance for the
excitability of the single neuron and, more generally to the whole
neuronal network, especially under conditions when bicarbonate
concentrations are strongly altered, e.g., during hypocapnia and
increased metabolism accompanying enhanced neuronal activity.
Changes in the membrane potential and excitability of neurons induced
by the de novo introduction of
CO2/HCO3 or by varying the
concentration of both have been demonstrated in cell cultures and slice
preparations previously (Church 1992
; Church and
McLennan 1989
; Cowan and Martin 1995
,
1996
). Increasing the amounts of
CO2/HCO3
led to a switch of the
activity mode from a spike-train to a burst-generating mode. Moreover,
calcium spikes (sodium currents were blocked by TTX) could be evoked at
more negative potential than in solutions without
CO2 and bicarbonate. Both phenomena indicate a
modulation of calcium currents by bicarbonate ions or the gas
CO2.
Both, bicarbonate and CO2 are end products of metabolism. A direct interaction of bicarbonate with calcium currents may therefore act as a negative feedback mechanism between excitability and energy consumption. Strong neuronal activity increases CO2 and bicarbonate, which in turn may reduce or even stop this activity by attenuation of calcium currents.
The present study addressed the following questions. First, it was investigated whether CO2 or the bicarbonate ion or even both are the modulating agents. Second, we tested whether the modulation is dependent on the concentration of the modulator. And finally, it was examined whether this modulator acts preferentially on intra- or extracellular sites of the membrane. The investigations were carried out on freshly isolated pyramidal neurons from the hippocampal CA1 area of the rat using the whole cell voltage-clamp technique.
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METHODS |
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Cell preparation
CA1 pyramidal neurons were isolated enzymatically from male
Wistar rats (75-85 g) as described in detail previously
(Vreugdenhil and Wadman 1992). From both hippocampi,
500-µm-thick slices were cut, and the CA1 area was dissected. These
tissue pieces were incubated 75 min at 32°C in dissociation solution
(in mM: 120 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 20 PIPES, and 25 D-glucose; pH 7.0) containing 1 mg/ml trypsin (Bovine Type XI), which was
equilibrated with oxygen. Following enzymatic treatment, tissue was
rinsed twice and kept in the dissociation solution without trypsin at 19°C. Directly before measurements, tissue pieces were dispersed in
HEPES-buffered bath solution by trituration through Pasteur pipettes
with decreasing tip diameter, and cells were allowed to settle in the
perfusion chamber.
To assure total solution exchange, we used a bath with a volume of
~120 µl, which was perfused with a constant flow rate of 1 ml per
minute. Bath solutions contained 110 mM NaCl, 5 mM KCl, 2.5 mM
CaCl2, 1 mM MgCl2, 5 mM
4-aminopyridine (4-AP), 25 mM TEA-Cl, 25 mM D-glucose 25, and 0.5 µM tetrodotoxin (TTX); pH was set at 7.4 (unless otherwise
stated). For seal formation all cells were patched in the
above-mentioned solution, plus 10 mM HEPES. Bath solutions containing
CO2/HCO3 as the pH-buffering
system were thoroughly gassed with different amounts of
CO2 (2.5; 5; 10%) before HCO3
was added. Special care was taken to assure that the solutions were
always equilibrated with CO2 throughout the
course of the experiment, since otherwise CaCO3
would have precipitated.
All chemicals were obtained from Sigma (Deisenhofen, Germany) and Merck (Darmstadt, Germany).
Current recording
Currents were measured under whole cell voltage-clamp conditions
at room temperature using patch pipettes of 2-4 M resistance. Electrode solution contained 80 mM CsF, 2 mM
MgCl2, 0.5 mM CaCl2, 15 mM
TEA-Cl, 10 mM EGTA, 5 mM phosphocreatine, 50 units/ml phosphocreatine kinase, 2 mM MgATP, 0.1 mM NaGTP, and 0.1 mM leupeptin; pH set at 7.3 (unless otherwise stated). The solution was strongly buffered by 50 mM
HEPES, to prevent intracellular pH changes following the introduction
of CO2/HCO3
-buffered solution.
The ATP regenerating system reduced the "run down" of the calcium
current; the gradual decrease in current amplitude never exceeded 10%
within the recording period of 10-15 min. Currents were measured with
an Axopatch 200A amplifier (Axon Instruments) and stored on an Atari ST
computer (1-kHz sample frequency). Capacitive transients and series
resistances were compensated on-line. Data were evaluated off-line
using a custom-made computer program. All current traces were corrected
for aspecific linear leak (reversal potential 0 mV) determined at
holding potential.
Experimental protocols
Calcium currents were activated using 200-ms voltage steps to
voltage levels between 40 and +40 mV. Holding potential was kept at
80 mV. The steady-state inactivation of the calcium current was
determined using a standard depolarization to +10 mV after the cell was
polarized for 3 s at various levels between
105 and 0 mV. During
solution changes a ramp protocol was carried out (from
100 to 50 mV;
within 150 ms) to monitor current changes and to ensure the stability
of the changes.
Neurons were bathed first in HEPES-buffered saline, and voltage
protocols that determine activation and inactivation properties were
performed. Next, the HEPES-buffered saline was replaced by a
CO2/HCO3-buffered saline while
the ramp protocol was applied. When a stable condition in the presence
of the CO2/HCO3
-buffered saline
was achieved, currents were examined again using the same set of
voltage-clamp protocols as during the HEPES condition.
Current analysis
Peak amplitudes of the currents (I) evoked with the
activation protocol were plotted as a function of membrane potential
(V). The resulting I-V relations were fitted with
a combination of Boltzmann activation function and the
Goldman-Hodgkin-Katz (GHK) current-voltage relation (Hille
1992; Kortekaas and Wadman 1997
)
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(1) |
The voltage dependence of steady-state inactivation of the calcium
current was estimated from the relation of the amplitude of the current
versus the prepotential. This relation was well described by a
Boltzmann function, which also normalized the current
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(2) |
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(3) |
Statistics
Values are given as means ± SE. Statistical comparisons were made with Student's t-test. P < 0.05 was taken to indicate significant differences.
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RESULTS |
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Concentration-dependent alteration of HVA currents
In HEPES-buffered bath solution, HVA currents could be
evoked in 23 cells by voltage steps more positive than 35 mV (Fig. 1). They showed the typical fast
activation (time-to-peak at 0 mV: 9.2 ± 0.5 ms) and a slow and
incomplete inactivation (time constant at 0 mV
: 47.3 ± 3.3 ms). They reached their maximal peak amplitude at around 5 mV with a
value of
1.85 ± 0.1 nA (Fig. 1). When current amplitude was
plotted as a function of membrane potential, it gives the typical
I-V relationship, which could well be fitted by the
GHK-current-voltage equation (Eq. 1). In this way the
activation properties of the current could be described with only three
variables (Vh,
Vc, and
gmax). The mean value of the potential
of half-maximal activation (Vh) was
2.4 ± 0.9 mV and the mean value of the slope parameter
(Vc) was 6.5 ± 0.1 mV. The
maximal calcium conductance (gmax)
also obtained from the GHK fit was calculated to be 257 ± 17 nS
under these conditions.
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The voltage dependence of steady-state inactivation of these currents
were determined by holding the cells at different prepotentials followed by a voltage step to 10 mV. The amplitudes of the evoked calcium currents were then plotted as a function of prepotentials and
fitted for each individual cell with the Boltzmann equation (Eq. 2). The mean values of inactivation parameters in the Boltzmann equation (Vh,
Vc) were then calculated. The mean potential of half-maximal inactivation Vh was
32.0 ± 1.4 mV, and the slope parameter
(Vc) had a value of
11.0 ± 0.5 mV.
The cells were subsequently subjected to three bath solution changes,
in which the HEPES-buffered solution was replaced by solutions
containing 2.5% CO2/5.6 mM
HCO3, 5.0% CO2/18 mM
HCO3
, and 10% CO2/37 mM
HCO3
. All solutions had a pH value of 7.3. A 3-min
period was allowed to guarantee total solution exchange, before the
same protocols were applied as with HEPES-containing solution. The
introduction of CO2 and bicarbonate reduced the
current amplitude (Figs. 1 and 2) and the
calcium conductance (Fig. 3). The
decrease of current amplitude occurred within seconds after solution
exchange and reached its final value within the following minute. Since
the size of conductance and current amplitude were inversely related to
the amount of CO2 and HCO3
, we
plotted the relative reduction in gmax
as a function of the concentration of dissolved bicarbonate. The data
were then fitted with the Hill equation (Eq. 3), which gave
as parameters a maximal decrease of 69%, half-maximal concentration of
bicarbonate 7.4 mM and a Hill coefficient of 1.8. The potential of
half-maximal activation was shifted to more negative potentials, with
the largest shift in the 10% CO2/37 mM
HCO3
containing solution (
10.0 ± 1.0 mV),
while the slope parameter (Vc) at the
point Vh was not altered. The
concentration-dependent shift of Vh
appeared to be linear (Fig. 4) within the
concentration range tested. Steady-state inactivation was also
modulated in a concentration-dependent manner (Fig. 4). The potential
of half-maximal inactivation was shifted to more hyperpolarized
potentials, with a maximal shift of
17.0 ± 1.4 mV in the 10%
CO2/37 mM HCO3
-buffered
solution. The different shifts in Vh
for activation and inactivation widens the potential range in which a
"window current" can exist in the bicarbonate-containing solution,
i.e., a current that in that particular voltage range will
activate and not completely inactivate.
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Bicarbonate acts from outside
In the preceding experiments, several conditions were altered to
induce the described effects. First the concentration of CO2 and of HCO3 were
concomitantly increased, and second the amount of
CO2/HCO3
that occurs within the
cells by formation and dissociation according to the
Henderson-Hasselbalch equation, was different for each bath solution.
Therefore additional experiments were performed to unravel the
questions whether CO2 or HCO3
is the primarily modulating agent and whether this agent acts on the
outer or inner side of the cell membrane.
The latter question was investigated by superfusing cells with a bath
solution that contained 5.0% CO2/26 mM
HCO3 with a pH value set to 7.4, and intracellular pH
of the cells was preset by the electrode solution to three different
values (7.0, 7.3, and 7.6). Since the intracellular pH is heavily
buffered by 50 mM HEPES, the
CO2/H2CO3
that crosses the membrane has to form lower concentrations of
bicarbonate in a more acidic milieu and higher concentrations with a
more alkaline pH. For each intracellular pH value, 15 cells were
measured. In these experiments no relation between the
intracellular bicarbonate concentration and the previously observed
effects could be observed. There was neither a difference in the
reduction of the maximal calcium conductance (41 ± 8%, 38 ± 3%, and 41 ± 6%; Fig. 5), the
reduction of the peak amplitude (24 ± 5%, 24 ± 3%, and
31 ± 3%), nor a difference in the shift of the half-maximal
potential of activation (6.3 ± 1.6 mV, 4.7 ± 0.9 mV, and
5.1 ± 1.9 mV). Only the voltage shift of the inactivation showed
a slight tendency to change with the intracellular bicarbonate concentration (Vh was 6.0 ± 2.2 mV, 7.5 ± 1.1 mV, and 10 ± 2.8 mV), but this difference did
not reach significance.
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Bicarbonate, but not CO2 modulate HVA currents
To determine whether CO2 or the bicarbonate
ions modulate the HVA current, an additional series of experiments was
carried out. Neurons (6 cells tested in each solution) were bathed in solutions gassed by three different CO2
concentrations (2.5, 5.0, and 10%), while the concentration of
bicarbonate was kept at 26 mM (Fig. 5, left group). This
composition leads to different extracellular pH values (7.76, 7.41, and
7.17), which would introduce a second alteration when the superfusion
is switched from the HEPES-buffered solution to the
CO2/HCO3-buffered solution. The
pH values of the HEPES-buffered solution were therefore adjusted to
match the pH of the
CO2/HCO3
-buffered solution.
Under these conditions the reduction of maximal calcium conductance
showed a weak relation with increased CO2 values
(38 ± 5%, 41 ± 3%, and 48 ± 4%; Fig. 5, left
panel), but the trend did not reach significance. The same held
for the reduction in mean peak amplitude of the calcium currents
(21 ± 5%, 28 ± 3%, and 36 ± 6%). Also the
potential of half-maximal inactivation showed comparable negative
shifts for the three solutions tested (shift in
Vh was 8.8 ± 0.7 mV, 9.1 ± 1.0 mV, and 9.8 ± 1.5 mV). The potential of half-maximal
activation was shifted but showed no direct relation with the amount of
dissolved CO2
(Vh was 5.7 ± 1.0 mV, 3.7 ± 1.5 mV, and 6.7 ± 1.6 mV).
The experiments shown above suggest that bicarbonate ions alone
modulate the calcium current properties. To substantiate this assumption, a second set of experiments was carried out. Solutions containing different amounts of HCO3 ions (12, 26, 45 mM) and a constant concentration of CO2 (5%) were tested in respect to their modulating effect (Fig. 5; middle group). The pH of the HEPES-buffered solution was adjusted to the
pH of the bicarbonate-containing solution, like in the experiment above, to avoid effects related to a pH-change. Superfusion of neurons
(11 cells per solution) with these solutions resulted in a reduction of
the conductance (21 ± 4%, 39 ± 3%, 59 ± 5%) and of
mean peak amplitude (5 ± 4%, 24 ± 3%, 38 ± 5%) of
the measured currents (Fig. 5). Moreover a clear positive correlation
with increasing bicarbonate was observed for the negative shift of the
half-maximal potential of activation (4.8 ± 0.7 mV, 5.7 ± 0.6 mV, 11.2 ± 0.8 mV) and for the half-maximal potential of
inactivation (8.6 ± 0.7 mV, 9.1 ± 0.7 mV, 14.2 ± 2.5 mV). As with all other experiments, no changes in the slope parameters
of the Boltzmann curves (Vc) were
observed for activation or inactivation. Taken together, the lack of
effect with increasing concentrations of CO2 and
the prominent alterations induced by increased bicarbonate concentrations demonstrate that bicarbonate ions modulate the calcium
current properties.
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DISCUSSION |
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The present study shows the modulatory action of bicarbonate ions on neuronal whole cell calcium currents. Current amplitude and maximal calcium conductance decreased in the presence of bicarbonate, and the potentials of half-maximal activation and inactivation were shifted to more negative values. The data show that bicarbonate alone, and not CO2, is capable of inducing these alterations. Furthermore, a concentration-dependent interaction between bicarbonate and calcium currents has been demonstrated. The outer membrane seems to be the site of action of bicarbonate, since changing the concentration of this ion intracellularly did not show any effect.
Bicarbonate modulates calcium currents
A main question of the present study was to decide whether CO2 or the bicarbonate ion is the molecule that induces the observed alterations of whole cell calcium current. Carbon dioxide has indeed the ability to bind with protein, thereby forming so-called carbamino complexes, of which the complex of CO2 and hemoglobin is one of the best known. Binding of a small molecule such as CO2 to a big protein structure often leads to conformational alterations, which could change the chemical and physical properties of such molecules. It therefore was conceivable that binding of CO2 to or close to calcium channel proteins may decrease calcium permeability of the channel pore and reduce the total calcium current. However, increasing the concentration of CO2 up to 10% by leaving the concentration of bicarbonate constant did not yield differences in the maximal whole cell calcium conductance (ref. Fig. 5). Instead, increasing the amount of dissolved bicarbonate, at constant CO2 concentration, had a marked and concentration-dependent effect on calcium conductance, current, and voltage dependence. Together these data demonstrate that bicarbonate is the modulatory agent and not CO2.
The reduction of maximal calcium conductance and of current amplitude followed a nonlinear pattern with a maximum of 69%, when cells where consecutively bathed with solutions containing increasing amounts of bicarbonate. To hold pHo at constant level (7.3), these solutions were also gassed with increasing concentrations of CO2. The resulting concentration response curve was best fitted with the Hill equation, which delivered three independent parameters (maximal effect, EC50, and the Hill coefficient). The EC50 (7.4 mM) is the amount of bicarbonate at which half of the maximal reductions could be achieved, but due to the steep slope, is also the point at which small changes of bicarbonate would have the biggest effect on the calcium currents.
Physiological and pathological relevance
In mammalian brain, extracellular bicarbonate concentrations are
in the range of 24-26 mM (Betz et al. 1989). At this
value the concentration-dependent reduction is almost at its saturation level, and only strong changes of bicarbonate concentration would induce additional reductions or increases of the currents. Therefore under normal physiological circumstances a noteworthy dynamic influence
of bicarbonate on neuronal calcium currents is unlikely.
During pathological situations, strong decreases of bicarbonate concentration may lead to prominent increases of calcium current amplitude and to an altered excitability pattern.
Hyperventilation induces a loss of dissolved CO2
in blood as well as in the extracellular space surrounding neurons.
This results in an increased pH and ends up in a so-called respiratoric alkalosis, since in the early stage bicarbonate is still at normal levels. To re-establish normal pH values bicarbonate has to be reduced,
which could be done by extruding HCO3 for example by
higher extrusion rates for this ion in the kidney. An increase of
pHo of only 0.1 units will therefore be followed by a down-regulation of bicarbonate to almost half the original level,
which in turn will increase calcium currents, according to our
concentration-response curve, by up to 20-30%. In this light, the
so-called hyperventilation test, a diagnostic tool to evoke epileptic
seizures, might cause increased calcium currents, which play a
prominent role in epileptogenesis (Speckmann and Walden
1986
; Speckmann et al. 1990
; Witte
1987
).
Moreover, metabolic acidosis, following ischemic events, may also result in an increase of calcium current and conductance, since the elevation of proton concentration stimulates the medulla oblongata followed by an increased ventilation rate and finally by a loss of CO2. This loss of CO2 again starts the same regulatory mechanism following respiratoric alkalosis as mentioned above.
Relation between HCO3, pH, and membrane
potential
The observed effects will be strengthened in normal brain, since
pHi is not as heavily buffered as in our
experiments, in which pH of intracellular solutions (i.e., electrode
solution) were strongly buffered with the artificial proton buffer
HEPES (50 mM). This high concentration of HEPES has been demonstrated to avoid acidification of the intracellular space following
superfusion with weak acids containing solutions, comparable to our
CO2/HCO3-buffered solution
(Tombaugh and Somjen 1997
). Furthermore, it has been
shown that acidification of the soma plus superfusion with bicarbonate
enhances the reduction of current amplitude by almost 30%
(Bruehl et al. 1998
, 1999
).
In invertebrate preparations (leach and snail) a superfusion with
CO2/HCO3-buffered solution
leads not only to an acidification of the intracellular pH, but also to
a negative deflection (~2-5 mV) of the membrane potential
(Schlue and Thomas 1985
; Thomas 1976
).
This may be explained by the bicarbonate-dependent attenuation of
calcium currents, which are small but present at resting potential,
especially when cells frequently generate action potentials. This
assumption is further substantiated since the negative shifts of
resting potential in leach neurons could be diminished by adding the
potent calcium channel blocker Cd2+ during
superfusion with
CO2/HCO3
-buffered solution
(unpublished observation).
Artificially induced hypercapnia (i.e., increased
pCO2 plus decreased pHo)
can induce hyperpolarizations but also depolarizations on rat CA3
neurons (Lehmenkuhler et al. 1989). The variability between membrane responses was related to the distance of the neurons
from the bath fluid: cells in the innermost layers of the slice
hyperpolarized while cells close to the bath solution depolarized. The
concentration of bicarbonate in the tissue close to the bath fluid
resembles more or less the concentration in the surrounding saline
(usually 26 mM), while in the inner portions of the slice, additional
bicarbonate will be formed from the increased pCO2. With the concentration dependence of the
HCO3
effects on the calcium currents it is unlikely
that this alteration of membrane potential is due to changes of calcium
currents. It is, however, conceivable that HCO3
ions
affect also potassium or sodium conductances, and this may cause such
alterations. Alternatively, these effects may be attributed to
pH-related current modulation (Tombaugh and Somjen 1996
,
1997
).
In the present study hippocampal CA1 neurons were taken for the
measurements simply as a test probe, since they are easily prepared and
well characterized. Nevertheless this should not limit the present
findings to the hippocampus, and in particular to the CA1 area, but may
be of relevance for all neurons in areas throughout the brain, as in
cortical and subcortical structures. This is likely because the
affected HVA calcium current is present and equals the one of CA1
neurons, in all those cells (Bruehl and Wadman 1999;
Hille 1992
).
At present we cannot determine whether the observed processes are due
to a direct binding of the bicarbonate ion to the outer membrane, which
interferes with calcium channels, or whether it is based on surface
charge screening effects. The latter may cause a shielding of the
calcium-attractive site in the pore mouth, which would decrease the
permeability of calcium ions. Such a screening has been postulated for
protons (Hille 1992), which compete with calcium ions
for the docking site on the outer cell surface and can change the
electric field around the channel pore that attracts calcium ions.
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ACKNOWLEDGMENTS |
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The authors thank D. Steinhoff for perfect technical assistance.
The investigations were supported by Sonderforschungsbereich 194 B2.
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FOOTNOTES |
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Address for reprint requests: C. Bruehl, Heinrich-Heine-University, Dept. of Neurology, Geb.: 22.22/TVA, 40225 Duesseldorf, Germany (E-mail: bruehl{at}uni-duesseldorf.de).
Received 16 March 2000; accepted in final form 28 July 2000.
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REFERENCES |
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