1Department of Epileptology,
Beck, Heinz,
Ralf Steffens,
Uwe Heinemann, and
Christian E. Elger.
Ca2+-Dependent Inactivation of High-Threshold
Ca2+ Currents in Hippocampal Granule Cells of Patients With
Chronic Temporal Lobe Epilepsy.
J. Neurophysiol. 82: 946-954, 1999.
Intracellular
Ca2+ represents an important trigger for various
second-messenger mediated effects. Therefore a stringent control of the
intracellular Ca2+ concentration is necessary to avoid
excessive activation of Ca2+-dependent processes.
Ca2+-dependent inactivation of voltage-dependent calcium
currents (VCCs) represents an important negative feedback mechanism to limit the influx of Ca2+ that has been shown to be altered
in the kindling model of epilepsy. We therefore investigated the
Ca2+-dependent inactivation of high-threshold VCCs in
dentate granule cells (DGCs) isolated from the hippocampus of patients
with drug-refractory temporal lobe epilepsy (TLE) using the patch-clamp
method. Ca2+ currents showed pronounced time-dependent
inactivation when no extrinsic Ca2+ buffer was present in
the patch pipette. In addition, in double-pulse experiments,
Ca2+ entry during conditioning prepulses caused a reduction
of VCC amplitudes elicited during a subsequent test pulse. Recovery
from Ca2+-dependent inactivation was slow and only complete
after 1 s. Ca2+-dependent inactivation could be
blocked either by using Ba2+ as a charge carrier or by
including
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) or EGTA in the intracellular solution. The influence of
the cytoskeleton on Ca2+-dependent inactivation was
investigated with agents that stabilize and destabilize microfilaments
or microtubules, respectively. From these experiments, we conclude that
Ca2+-dependent inactivation in human DGCs involves
Ca2+-dependent destabilization of both microfilaments and
microtubules. In addition, the microtubule-dependent pathway is
modulated by the intracellular concentration of GTP, with lower
concentrations of guanosine triphosphate (GTP) causing increased
Ca2+-dependent inactivation. Under low-GTP conditions, the
amount of Ca2+-dependent inactivation was similar to that
observed in the kindling model. In summary, Ca2+-dependent
inactivation was present in patients with TLE and Ammon's horn
sclerosis (AHS) and is mediated by the cytoskeleton similar to rat
pyramidal neurons. The similarity to the kindling model of epilepsy may
suggest the possibility of altered Ca2+-dependent
inactivation in patients with AHS.
The application of modern
electrophysiological, morphological, and molecular biological tools to
tissue removed during epilepsy surgery has permitted the identification
of candidate mechanisms that may be responsible for chronically
enhanced excitability. Factors controlling levels of intracellular
Ca2+ are of particular interest because increases
in the intracellular Ca2+ concentration represent
an important trigger for a variety of second-messenger-mediated events
that include activity-dependent neuronal plasticity and neuronal cell
death. Therefore stringent control of the intracellular calcium
concentration is essential to avoid excessive activation of
calcium-dependent processes. For this reason, the distribution and
functional characterization of ionotropic glutamate receptors and
voltage-dependent Ca2+ channels (VCCs), two of
the main routes for Ca2+ entry into neurons, have
been at the focus of interest in the investigation of epilepsy-related
changes in animal models of epilepsy and in human epilepsy (Beck
et al. 1997b Neurons possess a variety of intracellular Ca2+
buffering and sequestration systems including mitochondria, endoplasmic
reticulum, and different calcium-binding proteins (Carafoli
1987 Patient data
Surgical specimens from 28 patients with pharmaco-resistent TLE
were obtained for electrophysiological analysis (average age at surgery
29.3 ± 6.5 yr). The mean duration of TLE in the adult patients
was 19.7 ± 9.0 yr (mean ± SE), and the mean age at the onset of seizures was 11.3 ± 6.1 yr. All adult patients suffered from complex partial seizures (CPS), with additional simple partial seizures (SPS) in 8 patients and additional secondary generalized seizures (SGS) in 14 patients. In all adult patients, the hippocampus was shown to be intimately involved in the generation of temporal lobe
seizures by noninvasive and invasive diagnostic procedures as described
elsewhere (Engel 1992 Preparation of acutely isolated dentate granule cells
Isolated hippocampal granule cells were prepared as described
previously (Beck et al. 1997a Patch-clamp whole cell recording
Patch pipettes were fabricated from borosilicate glass
capillaries (1.5 mm OD, 1 mm ID; Science Products, Hofheim, Germany) on
a Narishige P83 puller (Narishige, Tokyo, Japan). They usually had a
resistance of 2-3 M The decay of whole cell Ca2+ currents elicited with 100-ms
command pulses was fit with a monoexponential function with a
steady-state component
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
; Blümcke et al. 1996
;
Kamphuis et al. 1992
; Köhr and Mody
1991
; Vreugdenhil and Wadman 1994
). With respect
to ionotropic
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
(AMPA) and N-methyl-D-aspartate (NMDA)
receptors, Ca2+ influx is controlled by the
expression of different receptor isoforms or splice variants as well as
by kinase-dependent modification of the receptor protein (e.g.,
Köhr and Seeburg 1996
; Seeburg 1993
). Ca2+ entry through high-threshold
VCCs is controlled through a negative feedback mechanism via the
intracellular Ca2+ concentration (Eckert
and Chad 1984
; Imredy and Yue 1994
;
Köhr and Mody 1991
). In L-type channels, this
property seems to depend on a short sequence of the C terminus that
does not include a nearby sequence with homology to
Ca2+-binding domains (Zhou et al.
1997
). The transduction mechanism of this calcium-dependent
inactivation is not well understood, but the signaling pathway seems to
involve elements of the cytoskeleton (Johnson and Byerly
1993
, 1994
). In addition, the amount of
Ca2+ that can be buffered or sequestered
immediately after Ca2+ entry into the neuron
influences the amount of Ca2+-dependent inactivation.
; McBurney and Neering 1987
). High-affinity
Ca2+ buffering proteins such as calmodulin,
parvalbumin, calretinin, and calbindin-D28K
expecially have been suggested to play an important role in rapidly
sequestering Ca2+ at the site of
Ca2+ entry. In dentate granule cells, a reduction
of the calcium-binding protein calbindin-D28k has
been observed in the kindling model of epilepsy and in human patients
with temporal lobe epilepsy (TLE) (Baimbridge et al.
1985
; Magloczky et al. 1997
; Sloviter et
al. 1991
). This loss of buffering capacity has been suggested to lead to increased Ca2+ levels following
Ca2+ influx and increased calcium-dependent
inactivation of voltage-dependent Ca2+ currents
in rat dentate granule cells (Köhr et al. 1991
;
Köhr and Mody 1991
). In contrast, reduced
Ca2+-dependent inactivation together with a
persistent increase in the amplitude of high-threshold
Ca2+ currents has been observed in CA1 pyramidal
cells following kindling electrogenesis (Vreugdenhil and Wadman
1994
). Therefore we attempted to investigate the properties and
the mechanism of calcium-dependent inactivation in dentate granule
cells from human patients with TLE.
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
). The surgical removal of the hippocampus by selective amygdalohippocampectomy was clinically indicated in every case to achieve seizure control. All patients were
under a full antiepileptic drug regimen at the time of operation. To
reduce interpatient variability, only patients with a histopathological diagnosis of solitary Ammon's horn sclerosis (AHS) with severe neuronal loss in the CA1, CA3, and CA4 subfield and relative sparing of
CA2 (Margerison and Corsellis 1966
) were selected for
this study. Informed consent was obtained from all patients for
additional histopathological and electrophysiological evaluation. All
procedures were approved by the ethics committee of the University of
Bonn Medical Center and conform to standards set by the Declaration of
Helsinki (1989).
,b
). Human hippocampal
specimens were placed in ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM) 125 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, 10 glucose, and 26 NaHCO3 (pH 7.4, 95%
CO2-5% O2) immediately following surgical removal. Coronal slices (400 µm) were prepared from the corpus of the hippocampus with a vibratome and transferred to
a storage chamber with warmed ACSF (95% CO2-5%
O2). After an equilibration period of 60 min.,
the first section was transferred to a conical polystyrene tube with 5 ml of incubation medium containing (in mM) 126 NaCl, 2 KCl, 1 CaCl2, 2 MgCl2, 26 PIPES,
10 glucose, and 1.25 NaHPO4 (pH 7.4, 100%
O2). Pronase (2-3 mg/ml; protease type XIV,
Sigma) was added to the oxygenated medium. After incubation for 25 min,
the slice was washed in ice-cold incubation medium. The dentate gyrus
was dissected under a binocular microscope (Zeiss, Oberkochem, Germany)
and triturated in 2 ml of ice-cold trituration solution with
fire-polished glass pipettes. The cell suspension was then placed in a
Petri dish for subsequent patch-clamp recordings. At least two
subsequent washes with extracellular recording solution (see
Patch-clamp whole cell recording) were performed before
starting whole cell recording. Only neurons with an ovoid soma and a
single dendrite reminiscent of granule cell morphology in situ were
included in the present study. The isolated cells were superfused with an extracellular solution containing 140 mM tetraethylammonium chloride
(TEA), 5 mM 4-aminopyridine (4-AP), 5 mM CaCl2, 10 mM glucose, 10 mM
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic
acid (HEPES), and 1 µM tetrodotoxin (TTX) (chemicals obtained from Sigma). In some experiments, BaCl2 was substituted for
CaCl2. The osmolarity was adjusted to 283 mosm with
sucrose. The different extracellular solutions were applied with a
superfusion pipette placed at a distance of 30-50 µm from the cell
body. The superfusion rate was adjusted by hydrostatic pressure.
. The pipettes were filled with an intracellular solution containing (in mM) 80 Cs-methanesulfonate, 20 TEA, 1 CaCl2, 5 MgCl2, 11 ethylene
glycol-bis-(2-aminoethyl)-tetraacetic acid (EGTA), 10 HEPES, 10 ATP,
and 0.5 guanosine triphosphate (GTP) (pH 7.4 CsOH). In some
experiments, 5 mM
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid
(BAPTA)/0.5 mM Ca2+ were used to buffer
intracellular calcium or the calcium buffer, and
Ca2+ was omitted altogether from the
intracellular solution. In further experiments, GTP was omitted from
the intracellular solution. The substances taxol (2, 20, and 100 µM),
phalloidin (20 µM), colchizine (20 µM), and cytochalasin B (20 µM) were added to the intracellular solution in some experiments.
These substances were aliquoted in 5 mM methanol stock solutions and
stored at
20°C until use. Methanol was evaporated from the aliquots
before addition of the intracellular solution and sonication. The
intracellular solution containing an ATP regenerating system was
composed of 110 mM CsCl, 3 mM TEA, 10 mM HEPES, 4 mM Mg-ATP, 25 mM phosphocreatine, and 50 U/ml creatinephosphokinase. The osmolarity
was adjusted to 275-280 mosm with sucrose in all intracellular
solutions. Recordings performed with different intracellular solutions
were interleaved in individual patients to minimize effects due to
interpatient variability. Tight-seal whole cell recordings were
obtained at room temperature (21-24°C) according to Hamill et
al. (1981)
. Membrane currents were recorded using a patch-clamp
amplifier (EPC9, HEKA Elektronik, Lambrecht/Pfalz, Germany) and
collected on-line with the "TIDA for Windows" acquisition and
analysis program (HEKA Elektronik, Lambrecht/Pfalz, Germany). The
membrane capacitance was measured using the EPC9 capacitance
cancellation according to Sigworth et al. (1995)
(12.3 ± 3.1 pF, mean ± SE). The input resistance of
the examined neurons was above 1 G
in most neurons with the
recording solution. The uncompensated series resistance RS estimated by the EPC 9 capacitance
cancellation technique (Sigworth et al. 1995
) was
6.7 ± 1.7 M
. Series resistance compensation was employed to
improve the voltage-clamp control (40-60%). The maximal residual
voltage error estimated by multiplying the maximal Ca2+
current amplitude with the effective series resistance after compensation did not exceed 3 mV. A liquid junction potential of
9.7
mV was calculated between the intra- and extracellular solution with
the generalized Henderson liquid junction potential equation as
described by Barry and Lynch 1991
.
where A and B correspond to the amplitudes
of the noninactivating and inactivating component, respectively and
(1)
to the decay time constant. All results were expressed as means ± SE. Statistical analyses were performed with the program SPPS version
6.1.2. (SPSS, Munich, Germany). Differences were proven with the
Mann-Whitney U-Wilcoxon Rank test, with the significance
level set to 0.05 and denoted with asterisks in the figures.
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RESULTS |
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Recordings from >150 human neurons were obtained between 5 and 90 min after isolation from hippocampal coronal slices of the resected
hippocampus of 28 patients with drug-refractory TLE. Between 2 and 11 neurons were studied from each individual patient. As in a previous
study (Beck et al. 1997b), slowly activating and
inactivating high-threshold calcium currents could be elicited by
depolarizing command pulses from a holding potential of
50 mV.
Time-dependent inactivation of high-threshold VCCs in different Ca2+ buffering conditions
Ba2+ currents through VCCs were large and showed a slow inactivation during 100-ms voltage pulses. When the charge-carrying ion was changed to Ca2+ in the superfusion medium, the maximal current obtained at the peak of the current-voltage relation (see Fig. 1B) was markedly reduced. This effect was not influenced significantly by the presence or absence of BAPTA or EGTA, indicating that elevation of intracellular free Ca2+ does not influence the peak amplitude of VCCs.
|
A markedly faster inactivation of Ca2+ currents during the command pulse became apparent when no extrinsic Ca2+ buffer was present in the recording pipette (Ca2+ currents scaled to Ba2+ current amplitude; Fig. 1A1, top panel, calibration bar applies to Ba2+ current). In contrast, both Ca2+ and Ba2+ currents through VCCs showed a very similar, slow time course of decay when 5 mM BAPTA or 11 mM EGTA (not shown) were included in the patch pipette (Ca2+ currents scaled to Ba2+ current amplitude; Fig. 1A1, bottom panel). This observation was confirmed by calculating the ratio of the current amplitude at the end of the 100-ms command pulse to the peak current at the various command voltages. In the absence of extrinsic buffer, Ca2+ currents showed significantly more inactivation during the command pulse than Ba2+ currents (Fig. 1A2, top panel). In addition, the time constants of inactivation measured by fitting a monoexponential function (Eq. 1) to the decaying phase of the current showed a U-shaped dependence on the command potential (see inset, Fig. 1A2). The inactivation rate could be markedly reduced by including 5 mM BAPTA/0.5 mM Ca2+ in the pipette solution (Fig. 1A2, bottom panel).
Voltage dependence of high-threshold VCCs in different Ca2+ buffering conditions
The peak of the activation curve was shifted to more positive
potentials when Ca2+ was used as a charge carrier
(Fig. 1B), regardless of the presence of extrinsic
intracellular buffer. This suggests that this effect is probably not
due to an action of intracellular free Ca2+.
Similar to an earlier study in rats (Köhr and Mody
1991), addition of BAPTA to the intracellular solution shifted
the voltage dependence of VCCs toward more positive potentials (Fig.
1B) with either Ca2+ or
Ba2+ as a charge carrier.
Double-pulse experiments
To further investigate the Ca2+ dependence
of VCCs, we tested the dependence of VCC amplitudes on prior entry of
Ca2+. In double-pulse experiments, a varying
amount of Ca2+ entry was obtained by
systematically varying the voltage of a prepulse, and the effect on the
peak Ca2+ current during a subsequent test pulse
was measured (Fig. 2A1; sample
traces with prepulse voltages of 60 and 0 mV). The reduction of
Ca2+ current during the test pulse is indicative
of a Ca2+-dependent inactivation process, because
it directly measures the level of inactivation produced by previous
Ca2+ entry (Eckert and Chad
1984
). The inactivation of the peak Ca2+
current elicited during the test pulse was then plotted as a function
of the prepulse voltage (Fig. 2A2). The prepulse voltage at which maximal inactivation occurred was usually around 0 mV, coinciding with voltage at which a peak Ca2+ current can be
elicited (see Fig. 1B). The degree of test pulse current
inactivation shows a U-shaped relation to prepulse potential, being
maximal at potentials that produce maximal Ca2+ entry, and
showing a pronounced reduction as the prepulse potential approaches the
Ca2+ equilibrium potential (Fig.
2A2). The inactivation of Ca2+
current during the test pulse was clearly larger when no extrinsic buffer was dialyzed into the cell (Fig. 2, A1 and
A2). When measurements were performed with either BAPTA
or EGTA present in the patch pipette, Ca2+-dependent
inactivation was not significantly different from that observed with
Ba2+ as a charge carrier (Fig. 2A2). These
data are summarized in Fig. 2B (
, no extrinsic
buffer;
, EGTA;
, BAPTA). The values of the bars signify the
average Ca2+ current amplitudes during the test pulse
showing the largest amount of inactivation normalized to the maximal
amplitude of the test current.
|
To test whether patients showed a high variability with respect to Ca2+-dependent inactivation, we investigated neurons from different patients using the double pulse protocols described above (100-ms prepulses) using intracellular solutions without Ca2+ buffers and Ca2+ as a charge carrier. Data gathered from two to seven neurons from each of five patients showed no significant interpatient variability (Fig. 2C). However, individual neurons from each patient showed a relatively high variability with respect to the amount of Ca2+-dependent inactivation.
The amount of Ca2+-dependent inactivation was related
to the amount of Ca2+ influx during the conditioning pulse.
A reduction in the duration of the conditioning prepulse led to a
reduction in Ca2+-dependent inactivation (Fig.
3A). To more accurately
describe the dependence of Ca2+ current inactivation on
prior Ca2+ influx, we attempted to quantify the amount of
Ca2+ influx during the various conditioning prepulses by
integrating the Ca2+ current during the prepulse. The
amount of Ca2+-dependent inactivation obtained as in Fig. 2
was then plotted versus the charge carried by Ca2+ during
the prepulse (Fig. 3B, ). The inactivation ratio
increased with the amount of Ca2+ entry during the
prepulse. In contrast, when the intracellular Ca2+ increase
during the prepulse was prevented by including 5 mM BAPTA in the
recording pipette, the increase in the inactivation ratio could be
markedly reduced (Fig. 3B,
).
|
Removal of Ca2+-dependent inactivation
Next, we investigated the removal from Ca2+-dependent inactivation following Ca2+ influx into the neuron during a conditioning prepulse. We determined the removal of Ca2+-dependent inactivation by gradually increasing the interval between a 50-ms conditioning prepulse and a test pulse in double pulse experiments (Fig. 4A, inset). Recovery from inactivation occurred gradually in the absence of extrinsic Ca2+ buffers with Ca2+ as a charge carrier within an interpulse interval of 1 s (Fig. 4, A and B). When intracellular Ca2+ was strongly buffered with 5 mM BAPTA or 11 mM EGTA (not shown), little inactivation could be observed with the shortest interpulse interval tested (10 ms). This small, putatively Ca2+-independent inactivation showed a somewhat more rapid recovery to baseline levels within 300-400 ms interpulse interval (Fig. 4B).
|
Role of the cytoskeleton in Ca2+-dependent inactivation
The role of the cytoskeleton in
Ca2+-dependent inactivation was investigated by
applying substances that stabilize or destabilize microtubules and
microfilaments, respectively, via the patch pipette. None of these
agents changed the voltage dependence and steady-state inactivation
properties of Ba2+ currents through VCCs under
conditions of strong intracellular Ca2+ buffering
(not shown). Likewise, neither taxol nor phalloidine altered the
Ca2+-independent portion of inactivation in
double-pulse experiments with 5 mM intracellular BAPTA (see Fig.
5B, ). The following experiments were then carried out without extrinsic
Ca2+ buffers and using Ca2+
as a charge carrier. Representative current traces in individual neurons following intracellular dialysis of the different cytoskeletal agents are shown in Fig. 5A. Phalloidin (20 µM), a
substance that stabilizes actin filaments, slowed the decay of VCCs
markedly. This effect could be prevented by additional inclusion of the microfilament-destabilizing agent cytochalasin B (20 µM) into the
patch pipette solution. Cytochalasin B administered alone had no
significant effects on Ca2+-dependent
inactivation. In contrast to phalloidine, taxol, which causes most of
the tubulin molecules in cells to aggregate into microtubules, had no
significant effect in the concentration range tested (2-100 µM).
When Ca2+-dependent inactivation was investigated
using double-pulse protocols (100-ms prepulses, 50-ms prepulses, not
shown) as described above, comparable effects could be observed, with
20 µM phalloidine (
) strongly affecting
Ca2+-dependent inactivation and taxol (
)
yielding no significant effects (Fig. 5B). In contrast to
the microfilament disruptor cytochalasin B, inclusion of colchizine
alone, which disaggregates microtubules, showed a slight increase in
Ca2+-dependent inactivation in double-pulse
experiments.
|
As the removal of inactivation has been shown to depend on
Ca2+-dependent processes, we hypothesized that
the time course of removal of inactivation in the presence of
phalloidin should be similar to that observed in the presence of
intracellular Ca2+ buffers. We therefore
investigated the removal of inactivation following intracellular
application of phalloidine. Indeed, the removal of inactivation with
phalloidine present in the recording pipette was similar to that
observed with strong intracellular Ca2+ buffering
(Fig. 6, , data from Fig. 4 shown for
comparison).
|
Effects of intracellular GTP
The present data seemed to suggest that in contrast to rat
hippocampal neurons (Johnson and Byerly 1994), it is
mainly microfilaments that interact with VCCs, because microtubular
stabilizers did not significantly reduce
Ca2+-dependent inactivation. However, this lack
of effect might be due to a preexisting high level of polymerization of
individual tubulin molecules in our experimental setup. Because GTP is
a necessary prerequisite for polymerization of tubulin dimers, we hypothesized that the relatively high concentration of GTP (500 µM)
in our intracellular solutions might cause most of the tubulin present
in the neuron to aggregate into microtubules with a high degree of
stability. In this case, we would not necessarily expect a large effect
of taxol in the presence of GTP. Conversely, the absence of GTP should
result in 1) increased Ca2+-dependent
inactivation and 2) sensitivity of this increased
Ca2+-dependent inactivation to intracellular
application of taxol. Therefore we omitted GTP from our intracellular
solutions in a first series of experiments. In addition, a set of
experiments with intracellular solutions identical to those used by
Köhr and Mody (1991)
, i.e., lacking GTP and with
an ATP regenerating system, was performed. In these recording
configurations, Ca2+-dependent inactivation was
significantly increased compared with recordings performed with 500 µM intracellular GTP (Fig. 7). When 20 µM taxol were included in the intracellular solution in the absence
of GTP, the Ca2+-dependent inactivation could now
be reduced significantly. Additional inclusion of 20 µM colchizine in
the recording pipette reduced the effects of taxol (Fig. 7). Similar
experiments in the absence of ATP could not be performed, because
omitting ATP accelerated rundown of VCCs.
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DISCUSSION |
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The electrophysiological properties of human (Isokawa et
al. 1991, 1993
; Williamson et al.
1993
) and rat dentate granule cells (see Fricke and
Prince 1984
; Stanton et al. 1989
) have been
investigated in a number of studies employing intracellular recording
with sharp microelectrodes. In addition, some groups have analyzed voltage-dependent ionic currents in isolated human hippocampal (Beck et al. 1997a
,b
; Reckziegel et al.
1998
) and cortical neurons (Sayer et al. 1993
).
In this study, we have taken advantage of the possibility to
investigate dentate granule cells isolated from the hippocampus of
patients with drug-refractory TLE using the whole cell patch-clamp
method, enabling us both to record VCCs in the voltage-clamp mode and
to control the composition of the intracellular solution via the patch
pipette. We have used these techniques to characterize
Ca2+-dependent inactivation of VCCs and the
involvement of the cytoskeleton in its mechanism.
Voltage- and Ca2+-dependent processes of
inactivation are not easily dissected in most neurons, because many
manipulations that alter Ca2+-dependent
inactivation also alter voltage-dependent inactivation and vice versa.
However, a number of criteria have been proposed by Eckert
and Chad (1984) to demonstrate the presence of
Ca2+-dependent in addition to
voltage-dependent inactivation of Ca2+ currents.
First, Ca2+-dependent inactivation should depend
on the species of ion carrying current through the membrane,
with Ca2+ being more effective than
Sr2+ or Ba2+. Indeed, the
inactivation rate as well as inactivation measured in double-pulse
experiments were considerably enhanced when Ca2+
as opposed to Ba2+ was used as a charge carrier.
Second, Ca2+-dependent inactivation should be reduced by
any means diminishing the intracellular Ca2+ increase
following influx of Ca2+. Indeed, introduction of different
Ca2+ buffers (BAPTA or EGTA) into dentate granule cells via
the patch pipette resulted in markedly slowed inactivation and reduced
inactivation measured in double-pulse experiments. Finally, we find
that, in double-pulse experiments, the degree of inactivation during
the test pulse has a U-shaped relation to prepulse potential, being maximal at potentials that produce maximal Ca2+ entry, and
showing a pronounced reduction as the prepulse potential approaches the
Ca2+ equilibrium potential (see Fig. 2). In addition,
plotting inactivation versus the time integral of Ca2+
entry during prepulses of varying duration and voltage yields a tight,
approximately linear relationship (see Eckert and Chad 1984
). These experiments show that an inactivation that is
dependent on Ca2+ entry inducing an increase in
[Ca2+]i is present in these neurons in
addition to voltage-dependent inactivation. Voltage-dependent
inactivation is probably most accurately reflected by the recordings
performed with high concentrations of Ca2+ buffers included
in the recording pipette.
Mechanism of Ca2+-dependent inactivation: involvement of the cytoskeleton
In control experiments, we have shown that this putative
voltage-dependent inactivation component is not influenced by agents that affect the cytoskeleton. In contrast, the mechanism of
Ca2+-dependent inactivation in human dentate
granule cells seems to be similar to that described in hippocampal
pyramidal neurons in the rat (Johnson and Byerly 1994),
involving microfilaments as well as microtubular elements of the
cytoskeleton. The mechanism of inactivation presumably involves
Ca2+-dependent destabilization of cytoskeletal
elements that have a structural relationship with the channel. This
destabilization possibly leads to channel inactivation. Including
microfilament stabilizers in the recording pipette would, according to
this model, reduce the effects of Ca2+ on
microfilaments, thereby also reducing
Ca2+-dependent inactivation. Agents that
stabilize microtubules were effective only in the absence of
intracellular GTP. Because aggregation of tubulin dimers into
microtubular structures requires GTP, one possible explanation for
these results may be that high (500 µM) concentrations of GTP present
intracellularly cause most of the tubulin dimers in the neuron to
aggregate into functionally stable microtubules. In this case, taxol
would not be expected to have an additional effect. In addition,
omitting GTP from the intracellular solution would be predicted to have
the two effects that were experimentally observed, namely that
Ca2+-dependent inactivation should be increased
and that taxol should then be effective in reducing the increased
Ca2+-dependent inactivation. Thus lowered
concentrations of GTP during periods of energy depletion might cause
increased Ca2+-dependent inactivation of
Ca2+ currents. Whether the actual free GTP
concentration in the cytosol of vertebrate CNS neurons is in a range
where it can contribute to a regulation of VCCs is presently unknown.
Nevertheless, this mechanism may be considered potentially
neuroprotective by reducing the Ca2+ influx into
these neurons.
The data presented here are of considerable interest in relationship to
data on the Ca2+-dependent inactivation of
currents through the pore-forming 1C subunit,
in which Ca2+-dependent inactivation has been
shown to depend critically on a short sequence in the C-terminal end of
the channel (Zhou et al. 1997
). Interestingly, this
sequence is not identical to an immediately adjacent
Ca2+-binding EF hand motif, and
disruption of the Ca2+-binding ability of the EF-hand
domain does not interfere with Ca2+-dependent
inactivation. Whether this critical sequence might possibly interact
with proteins that are related to the cytoskeleton is hitherto unknown.
Comparison to animal models of epilepsy
The maximal Ca2+-dependent inactivation we
could observe, i.e., using low concentrations of GTP, were very similar
to data obtained in the kindling model of epilepsy (Köhr
and Mody 1991). This group used intracellular solutions lacking
GTP. In our own control experiments performed with solutions identical
to those employed by Köhr and Mody, we have obtained results
quantitatively very similar to our measurements without GTP. This may
be due to the substantial loss of the
Ca2+-binding protein
Calbindin-D28K from dentate granule cells that is
present both in kindled rats (Sloviter 1989
) and human
hippocampus from patients with TLE (Magloczky et al.
1997
). The high variability observed between neurons obtained
from an individual patient may correlate with the finding that
Calbindin-D28K is present at considerable levels
in some individual dentate gyrus granule cells in the specimens studied
electrophysiologically in this study (unpublished observations). However, caution must be exercised in the interpretation of
electrophysiological data from human specimens, because human control
tissue is not available for comparison with TLE tissue. Nevertheless,
the similarity between the kindling model and human TLE specimens may
suggest a pathophysiologically relevant increase in
Ca2+-dependent inactivation that is associated
with kindling in rats and TLE in humans.
In summary, we demonstrate that Ca2+-dependent inactivation in granule cells isolated from patients with TLE shows properties similar to the kindling model in rats. The mechanism of Ca2+-dependent inactivation involves microfilaments as well as microtubules, with the microtubule-dependent pathway being modulated by intracellular GTP. Interesting questions to be clarified by future studies include the nature of the transduction mechanism involved in Ca2+-dependent destabilization of cytoskeletal elements and the functional consequences of Ca2+-dependent inactivation for Ca2+ influx into dendrites following synaptic stimulation.
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ACKNOWLEDGMENTS |
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We thank Prof. Schramm, Prof. Zentner, and Dr. van Roost for providing neurosurgical specimens.
This research was supported by a grant from the Ministry of Science and Education, Northrhine-Westfalia; University of Bonn Center Grant BONFOR 111/2, DFG EL 122/7-1; and the Sonderforschungsbereich SFB 400 of the Deutsche Forschungsgemeinschaft.
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FOOTNOTES |
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Address for reprint requests: H. Beck, Dept. of Epileptology, University of Bonn Medical Center, Sigmund-Freud Str. 25, D-53105 Bonn, Germany.
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 18 May 1998; accepted in final form 9 April 1999.
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NOTE ADDED IN PROOF |
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After submission of this work, several studies have appeared showing that Ca2+/calmodulin binds to and modulates L-type and P/Q-type Ca2+ channels. These are Yui, N., Olcese, R., Bransby, M., Lin, T., and Birnbaumer, L. Proc. Natl. Acad. Sci. USA 96: 2435-2438, 1999; Lee, A., Wong, S. T., Gallagher, D., Li, B., Storm, D. R., Scheuer, T., and Catterall, W. A. Nature 399: 155-159, 1999; Zühlke, R. D., Pitt, G. S., Deisseroth, K., Tsien, R. W., and Reuter, H. Nature 399: 159-162, 1999; and Peterson, B. Z., DeMaria, C. D., and Yue, D. T. Neuron 22: 549-558, 1999. It remains to be determined how the cytoskeleton and calmodulin might interact to cause Ca2+-dependent inactivation of human voltage-dependent Ca2+ channels.
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