From the Department of Biomedical Sciences and Consiglio Nationale delle Rícerche Center of Biomembranes, University of Padova, 35121 Padova, Italy
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Single channel patch-clamp recordings show that embryonic rat spinal motoneurons express anomalous L-type calcium channels, which reopen upon repolarization to resting potentials, displaying both short and
long reopenings. The probability of reopening increases with increasing voltage of the preceding depolarization
without any apparent correlation with inactivation during the depolarization. The probability of long with respect
to short reopenings increases with increasing length of the depolarization, with little change in the total number
of reopenings and in their delay. With less negative repolarization voltages, the delay increases, while the mean
duration of both short and long reopenings decreases, remaining longer than that of the openings during the
preceding depolarization. Open times decrease with increasing voltage in the range 60 to +40 mV. Closed times
tend to increase at V > 20 mV. The open probability is low at all voltages and has an anomalous bell-shaped voltage dependence. We provide evidence that short and long reopenings of anomalous L-type channels correspond
to two gating modes, whose relative probability depends on voltage. Positive voltages favor both the transition
from a short-opening to a long-opening mode and the occupancy of a closed state outside the activation pathway within each mode from which the channel reopens upon repolarization. The voltage dependence of the probability of reopenings reflects the voltage dependence of the occupancy of these closed states, while the relative probability of long with respect to short reopenings reflects the voltage dependence of the equilibrium between modes. The anomalous gating persists after patch excision, and therefore our data rule out voltage-dependent block by
diffusible ions as the basis for the anomalous gating and imply that a diffusible cytosolic factor is not necessary for
voltage-dependent potentiation of anomalous L-type channels.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Among the different types of neuronal voltage-gated
calcium channels, L-type channels play a specific role
in regulating activity-dependent gene expression (Murphy et al., 1991; Bading et al., 1993
; Deisseroth et al.,
1998
), neuronal survival and differentiation (Collins et al., 1991
; Ghosh et al., 1994
; Galli et al., 1995
; Finkbeiner and Greenberg, 1996
; Shitaka et al., 1996
; Brosenitsch et al., 1998
; Kirsch and Betz, 1998
), and some
forms of synaptic plasticity (Grover and Teyler, 1990
;
Aniksztejn and Ben-Ari, 1991
; Johnston et al., 1992
;
Kullmann et al., 1992
; Bolshakov and Siegelbaum, 1994
).
Multiple functionally and structurally different neuronal L-type channels have been described (Snutch et al.,
1991; Williams et al., 1992
; Forti and Pietrobon, 1993
;
Kavalali and Plummer, 1994
; Soldatov et al., 1995
; Ferroni et al., 1996
). In addition to classical cardiac-type
channels, anomalous L-type channels, which reopen
upon repolarization to resting potentials after a depolarization, have been reported in cerebellar, hippocampal, and sensory neurons (Fisher et al., 1990
; Slesinger
and Lansman, 1991
, 1996
; Forti and Pietrobon, 1993
;
Thibault et al., 1993
; Kavalali and Plummer, 1994
, 1996
;
Ferroni et al., 1996
; Cloues et al., 1997
). The probability of reopening of these L-type channels is voltage dependent, increasing with increasing voltage of the previous depolarization.
Different authors have proposed and supported different mechanisms accounting for voltage-dependent
reopenings of L-type channels. Forti and Pietrobon
(1993) have proposed that long reopenings of anomalous L-type channels are a manifestation of a voltage- dependent equilibrium between gating modes, whereby
increasing voltage drives the channel from a short- to a
long-opening mode (Pietrobon and Hess, 1990
), and
also increases the occupancy of a closed state outside
the activation pathway, which is connected to the open
state through a voltage-dependent transition within each mode. This additional closed state accounts for
both the delay with which long openings occur upon
repolarization of the membrane and the anomalous
voltage dependence of the mean open/closed times
and of the open probability, found by the same authors
(Forti and Pietrobon, 1993
). Alternatively, reopenings
have been interpreted as a manifestation of recovery
from voltage- and/or current-dependent inactivation
(Slesinger and Lansman, 1991
; Thibault et al., 1993
), resulting from voltage-dependent block of the channel
pore by a positively charged cytoplasmic particle (Slesinger and Lansman, 1996
). Kavalali and Plummer
(1994
, 1996
) have proposed that reopenings reflect a
particular form of voltage-dependent potentiation
(LVP) in which the conditioning depolarization essentially reduces the voltage necessary to activate the channel. The different interpretations might reflect real differences in the L-type channels under study or simply
derive from the emphasis of different aspects of the functional properties of essentially similar anomalous
L-type channels. The data presented in this paper favor
the second hypothesis.
As in most other neurons, the high-voltage activated
whole-cell calcium current of embryonic and neonatal
motoneurons can be dissected into four (L-, N-, P-, and
R-type) pharmacological components (Mynlieff and
Beam, 1992; Umemiya and Berger, 1994
; Hivert et al.,
1995
; Magnelli et al., 1998
). It is not known whether motoneurons express anomalous L-type channels. In
the only single channel characterization of calcium
channels in motoneurons (Umemiya and Berger, 1995
),
a classical L-type channel has been described.
Here we show that embryonic rat spinal motoneurons express L-type channels that reopen at negative repolarization voltages and display anomalous gating
properties similar to those of anomalous L-type channels of cerebellar granule cells. We have investigated the mechanism giving rise to the anomalous gating in
motoneurons. Our data are consistent with the model
proposed by Forti and Pietrobon (1993). They are also
consistent with reversible block of the open pore by a
positively charged cytoplasmic particle, but exclude block by diffusible ions, and imply that a diffusible cytosolic factor is not necessary for voltage-dependent potentiation of anomalous L-type channels.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Culture
Spinal motoneurons from embryonic day 15 (E15) Wistar rat embryos were grown in primary culture after purification by a two-step metrizamide-panning method according to the procedure
of Camu et al. (1993). In brief, ventral spinal cords were dissociated after trypsin digestion, and centrifuged over 6.5% metrizamide (Serva) cushions to eliminate the four-plate cells, which are dense enough to sediment through the cushion. The large cells were further enriched by immunopanning on Petri dishes coated with the IgG-192 antibody specific for the p75 neurotrophin receptor, which is specifically expressed by motoneurons at this
stage. The hybridoma was generously provided by Dr. C.E. Henderson (University Mediterrane, Marseille, France). Routinely,
90% of purified neurons express p75 immunoreactivity (Camu et
al., 1993
). The cells were plated on polyornithine- (Sigma Chemical Co.) and laminin- (GIBCO BRL) coated glass coverslips, and
cultured in Dulbecco's modified Eagle medium (GIBCO BRL)
supplemented with 17 mM glucose, 0.87 µM insulin, 0.99 mM
putrescine, 0.93 µM sodium selenite, 1.32 µM transferrin, 0.19 µM progesterone, 0.51 µM triiodothyronine, 0.45 µM tiroxine,
1.32 µM BSA (all purchased from Sigma Chemical Co.), 2 mM
glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin (all purchased from GIBCO BRL). 12 h after plating, the culture medium was supplemented with 25% muscle-conditioned medium, obtained from myotube cultures of newborn rat. Experiments were performed on motoneurons kept in culture from
1 to 5 d.
Patch-Clamp Recordings and Data Analysis
Single channel patch-clamp recordings followed standard techniques (Hamill et al., 1981). Currents were recorded with a
DAGAN 3900 patch-clamp amplifier, low-pass filtered at 1 kHz
(
3 dB; eight-pole Bessel filter), sampled at 5 kHz and stored for
later analysis on a PDP-11/73 computer. Experiments were performed at room temperature (21-25°C).
Linear leak and capacitative currents were digitally subtracted
from all records used for analysis. Current amplitude histograms were obtained from the data directly, with bin width equal to our
maximal resolution (323.6 points/pA). For display, each histogram was normalized to the value of the zero current peak. Open
probability, Po, was computed by measuring the average current
in a given single channel current record and dividing it by the
unitary single channel current. Open-channel current amplitudes were measured by manually fitting cursors to well-resolved
channel openings. A channel opening or closure was detected
when more than one sampling point crossed a discriminator line
at 50% of the elementary current. Histograms of open and
closed times were fitted with sums of decaying exponentials. The
best fit was determined by maximum likelihood maximization (Colquhoun and Sigworth, 1983) and the best minimum number of exponential components was determined by the maximum likelihood ratio test (Rao, 1973
). Log binning and fitting of
the binned distributions were done as described by McManus et
al. (1987)
and Sigworth and Sine (1987)
. Openings occurring
with a delay of more than one sampling point after repolarization of the membrane at
80 or
60 mV were considered as reopenings. In the measurement of the fraction of traces with reopenings, reopenings were detected using a discriminator line at
33% of the elementary current, to decrease the number of
missed short reopenings. To calculate the fraction of traces with
long and short reopenings, we used a discriminating open time
value, calculated from the double exponential open time histogram as the open time that equalized the number of openings of
the fast exponential component falsely assigned as long openings and the number of openings of the slow exponential component
falsely assigned as short openings (Demo and Yellen, 1991
). Reopenings of duration longer than this value were considered as
long reopenings and those of shorter duration as short reopenings. The pipette solution contained (mM) 90 BaCl2, 10 TEACl,
15 CsCl, 10 HEPES, pH 7.4 with TEAOH. The bath solution was
(mM) 140 K-gluconate, 5 EGTA, 35 L-glucose, 10 HEPES, pH 7.4 with KOH. The high-potassium bath solution was used to zero
the membrane potential outside the patch. The dihydropyridine
agonist (+)-(S)-202-791 (gift from Dr. Hof, Sandoz Co., Basel,
Switzerland) was added (1 µM) to the bath solution in most recordings. Liquid junction potential at the pipette tip was +12 mV (pipette positive), and this value should be subtracted to all voltages
to obtain the correct values of membrane potentials in cell attached recordings (Neher, 1992
).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Figs. 1 and 2 show that embryonic rat spinal motoneurons express L-type channels which reopen after repolarization of the membrane and display anomalous gating properties similar to those of anomalous L-type
channels of cerebellar granule cells (Forti and Pietrobon, 1993). The single channel current recordings in Figures 1 and 2 were obtained from cell-attached membrane patches of rat spinal motoneurons in primary
culture, which contained only one channel. The membrane was held at
80 mV and depolarized to four different voltages for 724 ms, every 4 seconds, in the presence of the dihydropyridine (DHP)1 agonist (+)-(S)-
202-791 in the bath in Figure 1 and in its absence in
Figure 2. The representative current traces and the
normalized current amplitude histograms from all
traces with activity display the main unusual voltage-
dependent properties of anomalous L-type channels.
|
|
The first unusual property is represented by the reopenings occurring with some delay after repolarization
of the membrane at 80 mV, a voltage well below the
threshold for channel activation. Some of these reopenings are quite long, much longer than the openings of
the same channel during the preceding depolarization (compare Fig. 1, +30- and +40-mV traces). The second
unusual property is represented by the voltage dependence of the open probability and of the open and
closed times. The open probability (Po) does not increase
with voltage in the usual sigmoidal manner, but reaches a maximum at +20 mV, and then decreases with increasing voltage, remaining low in the entire voltage range
(Fig. 3 C). In three single-channel patches, the maximal
Po at +20 mV in the presence of DHP agonist was 0.12 ± 0.02. In each patch, average open probabilities were obtained from the traces with activity, without including nulls, which were a minority at each voltage (0-7% at
+10 mV and 10-20% at +40 mV). In the absence of
DHP agonist, the activity of anomalous L-type channels
is characterized by brief, mostly unresolved and infrequent openings and by an extremely low open probability at all voltages: the maximal value of Po was 0.024 in
the single channel patch of Fig. 2. Kinetic analysis of the open and closed time histograms reveals that the low
open probability and its anomalous voltage dependence
are due to the anomalous voltage dependence of both
open and closed time constants. As shown in Fig. 3, the
time constants of the two exponential components best
fitting open time histograms both decrease with increasing voltage, and the two larger time constants of the
three exponential components best fitting closed time
histograms decrease with voltage up to +20 mV, and
then start to increase (see also Fig. 10 B). Interestingly,
the contribution of the slow exponential component in
the open time histograms increases with increasing voltage, with a symmetrical decrease of the fast component.
|
|
Thanks to these anomalous gating properties, anomalous L-type channels in the presence of DHP agonist
could be easily distinguished from the other L- and
non-L-type channels of rat spinal motoneurons. We
have found that rat spinal motoneurons coexpress, together with the anomalous L-type channels characterized in this study, two additional DHP-sensitive channels, one similar to cardiac L-type channels and the
other inactivating quite rapidly (Hivert and Pietrobon,
1995, 1997
; and our unpublished observations). They
can be distinguished from L-type channels with anomalous gating on the basis of their larger unitary current
and conductance (24 vs. 20 pS), their larger mean
open time and open probability at V > +20 mV (not
decreasing with increasing voltage), and the complete
absence of reopenings. Moreover, rat spinal motoneurons express several different DHP-insensitive calcium
channels, including two channels sharing the same
conductance of 20 pS but differing in inactivation and
pharmacological properties (Hivert and Pietrobon, 1995
; and our unpublished observations).
In this study, we have investigated the mechanism giving rise to the unusual voltage-dependent properties of
anomalous L-type channels of rat spinal motoneurons.
To discriminate between different mechanisms, it was
essential to be able to study the activity of a single
anomalous channel during both the depolarization
and repolarization periods. Since anomalous L-type
channels represent only a small fraction of the different types of calcium channels with similar conductance
expressed in motoneurons and patches with only one
anomalous L-type channel were very rare, and since, in
addition, the open probability of single anomalous
L-type channels in the absence of DHP agonist is extremely low (Figs. 2 and 3 C), a property that they share
with the more abundant inactivating L-type channel of
24 pS, it was necessary to prolong the openings of
L-type channels with a DHP agonist to be sure that only
one anomalous L-type channel was present in the
patch. The comparison between Figs. 1 and 2 shows
that the peculiar voltage-dependent properties of
anomalous L-type channels above described are essentially similar with or without agonist, as previously
shown in cerebellar granule cells (Forti and Pietrobon,
1993).
It has been proposed that reopenings of anomalous
L-type channels reflect recovery from voltage- and/or
current-dependent inactivation (Slesinger and Lansman, 1991, 1996
; Thibault et al., 1993
). If this interpretation is correct, then, in an experiment in which the
membrane is depolarized at increasingly positive voltages, one should find a correlation between the extent
of inactivation of single anomalous L-type channels
during the depolarization and the fraction of traces
with reopenings upon repolarization. Fig. 4 shows that
such a correlation is absent. The ensemble average currents from a patch containing a single anomalous
L-type channel in Fig. 4 A shows a lack of inactivation
during long depolarizations at positive voltages (+10 to
+40 mV) elicited from quite negative holding potentials (
80 mV). In the same voltage range, the fraction of traces with reopenings at
80 mV of the same channel increased as shown in Fig. 4 B (from 0 to 62%).
Similar results were obtained for single anomalous
L-type channels in cerebellar granule cells (Forti and
Pietrobon, unpublished observations). Thus, the previously reported absence of inactivation of cerebellar
anomalous L-type channels during depolarizations effective in inducing reopenings (Forti and Pietrobon,
1993
) cannot be ascribed to the relatively depolarized
holding potentials, as recently suggested (Slesinger and
Lansman, 1996
), but appears as a general distinctive
property of anomalous L-type channels.
|
As already pointed out, after long depolarizations,
both short and long reopenings at 80 mV could be
observed. The existence of two clearly different open
states, one short and the other long lasting, is even
more evident if one analyzes the reopenings at
60 mV
after a 400-ms long depolarization to +40 mV (Fig. 5, left). The recordings in Fig. 5 were obtained from a cell-attached patch containing only one anomalous L-type
channel. The open time histogram of reopenings at
60 mV required two exponential components with
time constants of 1.8 and 29 ms for best fit according to the maximum likelihood criterion. Strikingly, both
open time constants were larger (1.6 ± 0.1 and 26 ± 3 ms,
n = 5) than those measured for the anomalous L-type
channel during the predepolarization at +40 mV (0.54 ± 0.19 and 1.4 ± 0.2 ms, n = 3; see Fig. 3).
|
A common feature of the different single L-type
channels described so far is their voltage-dependent
modal gating, whereby increasing voltage progressively
drives the channels from a short-opening mode of activity (mode 1), prevailing at low voltages, to a long-opening mode (mode 2) prevailing at high positive
voltages (Pietrobon and Hess, 1990; Forti and Pietrobon, 1993
; Kavalali and Plummer, 1996
). Forti and Pietrobon (1993)
proposed that the anomalous gating arises
from the presence of a nonadsorbing closed state outside the activation pathway connected to the open state
through a voltage-dependent transition within each
mode, and used the simplified kinetic scheme of Fig. 6
to explain, at least qualitatively, the peculiar properties
of cerebellar anomalous L-type channels. In this kinetic
model, the individual open and closed states underlying the two modes within the two boxes are lumped together and connected by single voltage-dependent forward (kf) and backward (kb) rate constants. To account
for the anomalous voltage dependence of both open
and closed times (see Fig. 3), the open states within each mode (O, O*) are connected to a closed state outside the activation pathway (Cb, C*b), and the rate constants
,
* for entry into the closed states Cb in mode 1 and C*b in mode 2 are assumed to increase with voltage, while the rate constants
,
* for exit from these
closed states are assumed to decrease with voltage. Although the kinetic scheme in Fig. 6 is likely an oversimplification, we will use it here as a useful conceptual
framework for the analysis and discussion of our data
on anomalous L-type channels of motoneurons.
|
A possible interpretation of the presence of both short and long reopenings at negative voltages is that they represent reopenings of the channel in either mode 1 (from Cb) or mode 2 (from C*b), respectively. If this interpretation is correct, then any intervention that changes the probability of finding the channel in mode 2 at the end of the depolarization should change the relative proportion of long with respect to short reopenings, owing to the change in the relative probability of finding the channel in C*b with respect to Cb when the membrane is repolarized. The probability of finding the channel in the long-opening mode at the end of the depolarization can be changed by either changing the length or the amplitude of the depolarization. One expects that if the depolarization is shortened the relative proportion of long with respect to short reopenings should decrease. Indeed, Fig. 5 shows that when the depolarization was shortened from 400 to 50 ms, most of the reopenings of the single anomalous channel in the patch became short. The long reopenings were too few to define the second slower component in the open time histogram of the reopenings. The histogram was best fitted by a single exponential with a time constant of 2 ms, quite similar to the time constant of the fast component best fitting the histogram of reopenings of the same channel after 400 ms.
In three single channel patches, long reopenings were on average 40 ± 1% of the total number of reopenings after a 400-ms long depolarization, and decreased to 7 ± 4% of the total number of reopenings when the depolarization was shortened to 50 ms (Fig. 7 A). In one single channel patch, after shortening the depolarization at +40 mV from 400 to 50 ms, the voltage was increased to +150 mV keeping the duration constant at 50 ms. The long reopenings decreased from 38 to 12% of the total number of reopenings when the moderate depolarization was shortened, and increased again to 36% of the total number of reopenings when the amplitude of the short depolarization was increased. The relative increase of long with respect to short reopenings with increasing length and amplitude of the previous depolarization is consistent with the interpretation that short and long reopenings are associated with two different gating modes of the channel (mode 1 and mode 2) and with the existence of a voltage-dependent equilibrium between the gating modes whereby the probability of the long-opening mode (mode 2) increases with increasing voltage.
|
Consistent with this interpretation is also the finding that the probability of (long + short) reopenings, which depends on the probability of finding the channel in either Cb or C*b at the end of the depolarization, was not much affected by changing the length of the depolarization. In three single-channel patches, the probability of reopenings changed from 63 ± 7% after 400 ms at +40 mV to 50 ± 5% after 50 ms at the same voltage (Fig. 7 B), but the change was not statistically significant (P < 0.2). The model in Fig. 6 predicts that changing the length of the depolarization should produce mirror changes in the probabilities of long and short reopenings, with a consequent unchanged probability of (long + short) reopenings, if the intrinsic probabilities of Cb and C*b within the two individual modes were identical and as long as the rate constants of the transitions to Cb and C*b were fast with respect to the duration of the depolarization.
As a consequence of the relative increase of long with
respect to short reopenings with increasing length of
the depolarization, the peak ensemble average current
at 60 mV was larger and decayed more slowly after
the longer depolarization (Fig. 5). In three single channel patches, the average time constant of decay of the
ensemble current at
60 mV changed from 48 ± 1 ms
after the long depolarization to 9.9 ± 5 ms after the
short depolarization. On average, the ratio of the peak
average currents at
60 mV after short and long predepolarizations was 0.49 ± 0.07. The ensemble averages
at
60 mV after both long and short depolarizations
showed a clear rising phase, which originates from the
delay with which most reopenings occurred after the
repolarization. The time constant of the rising phase after short depolarizations (1.5 ± 0.3 ms) was slightly
smaller than that after long depolarizations (2.6 ± 0.5 ms), but the difference did not reach statistical significance (P < 0.1).
Fig. 8 shows directly that long moderate depolarizations are able to drive anomalous L-type channels from
a short- into a long-opening mode. The unitary current
recordings in Fig. 8 A were obtained from a patch containing a single anomalous L-type channel, in an experiment in which after 400 ms at +30 mV the membrane
was repolarized at 10 mV, a voltage just above the
threshold for channel activation. Control depolarizations to
10 mV for 800 ms were alternated with the
prepulse protocol. In the large majority of control depolarizations at
10 mV (in 98 of 102 traces with openings), the unitary activity was characterized by relatively short openings and long closings and a low open probability, as shown by the first five representative traces in
Fig. 8 A (right). The last trace represents a small minority of depolarizations (4 of 102 active traces) in which
the channel shifted to a different mode of activity, characterized by long-lasting bursts with longer openings and
shorter closings and by a much larger open probability.
|
The representative traces and the ensemble average
current of Fig. 8 A (left) show that a preceding depolarization to +30 mV for 400 ms increased the probability
of observing the long opening mode, with a consequent potentiation of the average current at 10 mV.
The fraction of active traces with the long opening mode increased from 4% in control depolarizations to
35% after 400 ms at +30 mV. These fractions were calculated using a discriminating open probability value
of 0.1 to separate the traces with the long opening
mode from those with activity similar to that in control.
Fig. 8, B-D, shows that, while the open time histogram of all traces at
10 mV after the prepulse required two
exponential components with time constants of 1.7 and
9.1 ms for best fit, the open time histogram of the
traces with Po < 0.1 could be best fitted by a single exponential with a time constant of 1.6 ms, similar to that
of the fast component in the overall histogram and to
the time constant obtained from the best fit of the
open time histogram of control traces (excluding the
four traces with the mode shift having Po > 0.1).
The average current at 10 mV after the predepolarization shows a clear rising phase, and after reaching a
maximum value, slowly decays towards the control
value. On average, the time constant of decay was 177 ± 28 ms (n = 4). The decay of the potentiated current
should mainly reflect the kinetics of return of the channel from the long-opening mode to the short-opening
mode prevailing in control. The slower decay of the average current at
10 mV with respect to that at
60 mV
after the depolarization (46 ± 2 ms, n = 6, see Fig. 5) is
consistent with the voltage dependence of kb, whereby
kb decreases with increasing voltage (Pietrobon and
Hess, 1990
; Forti and Pietrobon, 1993
). The rising
phase of the current at
10 mV is clearly slower than
the rising phase of the current at
60 mV after a similar prepulse (see Fig. 5), indicating that the rate of reopening from the closed states accessed during the depolarization (Cb and C*b) increases with more negative repolarization voltages. On average, the time constant
of the rising phase increased from 2.6 ± 0.3 ms (n = 6)
at
60 mV to 11 ± 2 ms (n = 4) at
10 mV. This finding is consistent with and supports the voltage dependence of the rate constants of exit from the closed
states Cb and C*b in the model in Fig. 6, whereby
and
* decrease with increasing voltage. Consistent with
this voltage dependence is also the peculiar lengthening of the closed times with increasing voltage of the
depolarization (see Figs. 3 B and 10 B).
The two open time constants, obtained from the
biexponential open time histogram at 10 mV were
both smaller than those obtained for the reopenings at
60 mV after a similar depolarization (compare Figs. 8
and 5). On the other hand, the open time constants at both
10 and
60 mV were larger than those obtained
from the histogram of open times during the preceding depolarization (Fig. 3). In the voltage range
60 to
+40 mV, the open time constants decreased as shown
in Fig. 9 A. This anomalous voltage dependence of the open times supports the existence, within each mode,
of a closed state outside the activation pathway to which
the open state is connected through a transition whose
rate constant increases with increasing voltage. Accordingly, in Fig. 6, the open states within each mode (O,
O*) are connected to a closed state outside the activation pathway (Cb, C*b), and the rate constants
,
* for entry into these closed states increase with increasing
voltage. Our interpretation that the fast and slow components in the open-time histograms reflect sojourns in
the open states of modes 1 and 2, respectively, is further
supported by the finding that, as already pointed out,
the contribution of the slow exponential component in
the open time histograms increases with increasing depolarization voltage, with a symmetrical decrease of the
fast component (Fig. 3 A, right).
|
At voltages higher than +20 mV, the two open time
constants become similar (Figs. 3 A and 9 A). At these
positive voltages, the anomalous L-type channel in
mode 2 opens only for brief times, thus explaining the
absence of bursts of activity with long openings during
depolarizations effective in inducing the change to the long-opening mode as seen on repolarization (Fig. 8 A).
One predicts that the potentiation of the anomalous
L-type current by positive depolarizations should decrease with increasing repolarization voltage and there
should be no potentiation of the current at repolarization voltages higher than +20 mV. Fig. 9 B shows that, in a single channel patch, the unitary activity of an
anomalous L-type channel at +20 mV after a predepolarization to +40 mV for 400 ms was hardly distinguishable from that in control depolarizations at +20 mV. As
a result, the same depolarization that produced a robust potentiation of the average current at 10 mV
(Fig. 8), hardly potentiated the current at +20 mV.
Although Forti and Pietrobon (1993) did not speculate on the nature of Cb and C*b, these states might correspond to either particular conformations of the channel or to open-pore blocked states, as proposed by
Slesinger and Lansman (1996)
. In the latter case,
,
and
*,
* would be the rate constants of voltage-dependent block and unblock of the channel in the short-
and long-opening modes, respectively, and their voltage dependence would be consistent with block by a
positively charged cytoplasmic particle (Slesinger and
Lansman, 1996
). However, the result shown in Fig. 10
excludes any diffusible ion as the blocking particle. After excision in the K-gluconate/EGTA solution without
divalents, the anomalous gating can persist unaltered
for 40 min. Indeed, the data shown in Figs. 4 and 8
were derived after excision of the patch. The voltage-dependent induction of the long-opening mode in the
inside-out patch in Fig. 8 shows that a diffusible cytosolic factor is not necessary for voltage-dependent potentiation of anomalous L-type channels.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we have shown that embryonic rat spinal
motoneurons express anomalous L-type calcium channels, which reopen upon repolarization to resting potentials, displaying both short and long reopenings.
The probability of reopening increases with increasing
voltage of the preceding depolarization without any apparent correlation with inactivation during the depolarization. The probability of long with respect to short
reopenings increases with increasing length of the depolarization, with little change in the total number of
reopenings and in their delay. With less negative repolarization voltages, the delay increases, while the mean
duration of both short and long reopenings decreases,
remaining longer than that of the openings during the
preceding depolarization. Open times decrease with increasing voltage in the range 60 to +40 mV, while
closed times tend to increase with voltage at V > 20 mV.
The open probability during depolarizing pulses is low at all voltages and has an anomalous bell-shaped voltage dependence.
We have provided evidence that the two open states,
leading to short and long reopenings, correspond to
two gating modes of the channel, whose relative probability depends on voltage. Since the sojourn of the
channel in both open states decreases with increasing
voltage, the two open states must be connected to a closed state outside the activation pathway with a voltage-dependent transition whose rate constant increases
with increasing voltage. The anomalous voltage dependence of the closed times suggests that the rate constant of reopening from this closed state decreases with
increasing voltage. This voltage dependence leads to
reopening of the channel upon repolarization and predicts a faster rate of reopening at more negative repolarization voltages, as found. According to our data,
positive voltages favor both the transition from a short-opening gating mode (mode 1) to a long-opening
mode (mode 2), and the occupancy of a closed state
within each mode from which the channel reopens on
repolarization, displaying short reopenings when it reopens from the closed state of mode 1 and long reopenings when it reopens from the closed state of mode 2 (Fig. 6, Cb and C*b) (Forti and Pietrobon, 1993). The
voltage dependence of the probability of reopenings
reflects the voltage dependence of the occupancy of
the closed states from which the channel reopens,
while the relative probability of long with respect to
short reopenings reflects the voltage dependence of
the equilibrium between modes.
The properties of the first latency distribution of reopenings of anomalous L-type channels of mouse cerebellar granule cells, measured by Slesinger and Lansman (1996) as a function of repolarization voltage, are
consistent with our conclusions. To explain their data,
Slesinger and Lansman (1996)
assumed the existence
of a positively charged cytoplasmic blocking particle
that may reversibly block the pore during the depolarization and be released upon repolarization at negative
membrane potentials. According to this interpretation,
Cb and C*b would correspond to open-pore blocked states. Our finding that the anomalous gating persists
after excision of the patch in divalent-free solution
rules out block by a diffusible ion. It does not rule out
block by a membrane-bound particle. The abrupt
switch from the anomalous gating to the cardiac-type gating, observed by Forti and Pietrobon (1993)
in one
single channel patch, tends to exclude part of the channel as the blocking particle. An alternative interpretation, consistent with all the available data, is that Cb and
C*b represent conformational states of the channel and
that voltage-dependent pore block is not involved in
anomalous gating.
Our data, both in motoneurons and cerebellar granule cells, do not show any apparent inactivation of single anomalous L-type channels during depolarizations effective in inducing reopenings, even though the kinetic scheme in Fig. 6 is clearly compatible with inactivation and actually might seem to be inconsistent with lack of inactivation. Simulations performed using the model in Fig. 6 show that, depending on the rate constants of the transitions between the states within each mode, the model can generate both noninactivating and inactivating currents during depolarizations effective in inducing reopenings (not shown). The extent of inactivation depends crucially on the ratio between forward and backward rate constants, and increases with increasing ratios above a certain value. Thus, the model predicts that macroscopic inactivation should become apparent at sufficiently high voltages. The fact that it was not apparent from our single channel ensemble averages in the range from +10 to +40 mV may signify that these voltages were not sufficiently high. Alternatively, a small extent of inactivation at high voltages might have been missed, due to the stochastic behavior in the records.
Voltage-dependent potentiation is an interesting
property shared by the different L-type channels described so far. This name refers to the ability of earlier
depolarization to transiently increase macroscopic L-type
current. In different cells, different voltage dependence,
different time course, and different duration of voltage-dependent potentiation of L-type channels have
been reported, reflecting different L-type channels
and/or different modulatory mechanisms (Fenwick et
al., 1982; Hoshi et al., 1984
; Lee, 1987
; Pietrobon and
Hess, 1990
; Artalejo et al., 1991
, 1992
; Forti and Pietrobon, 1993
; Nakayama and Brading, 1993
; Sculptoreanu et al., 1993a
,b, 1995; Bourinet et al., 1994
; Johnson et
al., 1994
; Kleppisch et al., 1994
; Fleig and Penner, 1996
;
Kavalali and Plummer, 1996
; Parri and Lansman, 1996
;
Cloues et al., 1997
). The progressive shift towards a
long-opening mode induced by increasing voltage can
explain the voltage-dependent potentiation of L-type channels of cardiac (Pietrobon and Hess, 1990
) and
smooth muscle cells (Kleppisch et al., 1994
), as well as
that of cardiac-type neuronal L-type channels (Bourinet et al., 1994
; Kavalali and Plummer, 1996
, and our
unpublished observations). We have shown here and in
our previous work (Forti and Pietrobon, 1993
) that a
voltage-dependent change in gating mode is also at the
basis of voltage-dependent potentiation of anomalous
L-type channels.
The closed states giving rise to the anomalous gating
confer to the voltage-dependent potentiation of anomalous L-type channels two specific properties. The transient increase of anomalous L-type current following a
depolarization is delayed, and falls with increasing repolarization voltage to become almost zero at +20 mV,
where the open probability in the two modes becomes
similar. In similar experimental conditions, potentiation of cardiac L-type channels (Pietrobon and Hess,
1990) and brain
1C (Bourinet et al., 1994
) is still observed at +20 mV, where the open probability of the
two modes is still quite different. Anomalous L-type
channels differ from cardiac-type channels also in the
voltage range controlling the mode change, which is
shifted to lower voltages for anomalous L-type channels
(Pietrobon and Hess, 1990
; Kavalali and Plummer, 1996
). Furthermore, the potentiation lasts longer for
anomalous L-type channels with respect to cardiac
channels (see Figure 4 of Forti and Pietrobon, 1993
).
An important specific property of anomalous L-type
channels is that even very short or small depolarizations, insufficient to significantly shift the channels towards the long-opening mode, can induce short reopenings and thus transiently increase, for a brief time,
the current upon repolarization. By delaying channel opening at resting potential, where the driving force is
larger, the closed/blocked state from which anomalous
L-type channels reopen provides a mechanism to maximize calcium influx after a transient membrane depolarization such as an action potential. With increasing
duration and amplitude of the depolarization, the relative contribution of long reopenings, due to the mode shift, increases, leading to a potentiated and longer
lasting transient increase of the current upon repolarization. The anomalous gating warrants maximal potentiation at resting potentials. One can predict that a
presynaptic train of action potentials at high frequency
may lead to a progressive increase of calcium influx through presynaptic anomalous L-type channels during
the train, and/or may generate a large surge of calcium
influx through postsynaptic anomalous L-type channels
at the end of the train. Reopenings of L-type channels after single action potentials, with increasing open times at
more negative repolarization voltages, and a progressive shift of the same channels to a long-opening gating
mode during trains of action potentials have been observed in sensory neurons (Ferroni et al., 1996). Reopenings of calcium channels induced by single backpropagating action potentials have been observed also
in dendrites of hippocampal cells (Magee and Johnston,
1995
). Most likely, anomalous L-type channels were involved in both cases. Kavalali and Plummer (1994
, 1996
)
and Kavalali et al. (1997)
have characterized L-type
channels in hippocampal neurons with biophysical properties quite similar to those of anomalous L-type
channels of cerebellar granule cells and motoneurons.
The similarities include single channel current and conductance, low open probability, mean open times at +20
mV shorter than at
30 mV, potentiation after modest
depolarizations (LVP) and lack of potentiation at repolarization voltages higher than +20 mV, slower decay of
the potentiated current than that of cardiac L-type channels, and persistent activity in excised patches.
Given their presence in cerebellar, hippocampal, sensory, and motor neurons, L-type channels with anomalous gating are probably widely expressed in the nervous system, while they are absent from cardiac and
pituitary endocrine cells (our unpublished observations). The predicted capability of anomalous L-type
channels to produce a delayed influx of calcium at resting potentials after previous neuronal electrical activity,
whose extent depends on the duration and strength of
such activity, makes them particularly suited to play a
critical role in coupling transient neuronal activity with
long-term changes in nervous system development and
function. Indeed, several of these changes have been
found to be specifically inhibited by dihydropyridine
drugs (see INTRODUCTION). Calcium entry through
anomalous L-type channels might play an important role also in neurotoxic pathogenesis, since these channels are probably still active under conditions of metabolic stress such as hypoxia, which would favor the voltage-dependent change in gating mode leading to
potentiation of calcium influx. In addition, anomalous
L-type channels may play a role in neuronal deterioration during aging (Thibault and Landfield, 1996).
The molecular basis for the anomalous L-type channel is unknown. Its absence in pituitary endocrine cells,
which express the 1D subunit in large amounts (Fomina
et al., 1996
), and, on the other hand, its abundance
in rat cerebellar granule cells in primary culture, where
the
1D subunit is either not expressed or expressed in
small amounts (Schramm et al., 1999
), suggest that,
most likely, the pore-forming subunit of anomalous
L-type channels is not
1D. Since the
1C subunit was detected as a major transcript in rat cerebellar granule
cells in primary culture using degenerated oligonucleotide primer pairs under highly stringent conditions, and no new
1-related sequences were amplified under
the same conditions (Schramm et al., 1999
), it appears
likely that the anomalous L-type channel is related to
the
1C subunit. It remains to be established whether
the anomalous behavior arises from an unknown splice
variant of the
1C subunit, from a particular subunit composition, or from an unknown type of modulation.
![]() |
FOOTNOTES |
---|
Original version received 19 January 1999 and accepted version received 25 March 1999.
The financial support of Telethon-Italy to D. Pietrobon (grant 720) and to B. Hivert is gratefully acknowledged. This work was also partially supported by a grant from the Regione del Veneto (Giunta Regionale Ricerca Sanitaria Finalizzata-Venezia-Italia).We thank Dr. C.E. Henderson for providing the IgG-192 hybridoma and Dr. M. Cantini for providing the muscle-conditioned medium.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Aniksztejn, L., and Y. Ben-Ari. 1991. Novel form of long-term potentiation produced by a K+ channel blocker in the hippocampus. Nature. 349: 67-69 [Medline]. |
2. | Artalejo, C.R., D.J. Mogul, R.L. Perlman, and A.P. Fox. 1991. Three types of bovine chromaffin cell Ca2+ channels: facilitation increases the opening probability of a 27 pS channel. J. Physiol. 444: 213-240 [Abstract]. |
3. | Artalejo, C.R., S. Rossie, R.L. Perlman, and A.P. Fox. 1992. Voltage-dependent phosphorylation may recruit Ca2+ current facilitation in chromaffin cells. Nature. 358: 63-66 [Medline]. |
4. | Bading, H., D.D. Ginty, and M.E. Greenberg. 1993. Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways. Science. 260: 181-186 [Medline]. |
5. | Bolshakov, V.Y., and S.A. Siegelbaum. 1994. Postsynaptic induction and presynaptic expression of hippocampal long-term depression. Science. 264: 1148-1152 [Medline]. |
6. |
Bourinet, E.,
P. Charnet,
W.J. Tomlinson,
A. Stea,
T.P. Snutch, and
J. Nargeot.
1994.
Voltage-dependent facilitation of a neuronal
![]() |
7. |
Brosenitsch, T.A.,
D. Salgado-Commissariat,
D.L. Kunze, and
D.M. Katz.
1998.
A role for L-type calcium channels in developmental
regulation of transmitter phenotype in primary sensory neurons.
J. Neurosci.
18:
1047-1055
|
8. | Camu, W., E. Bloch-Gallego, and C.E. Henderson. 1993. Purification of spinal motoneurons from chicken and rat embryos by immunopanning. Neuroprotocols 2: 191-199 . |
9. |
Cloues, R.K.,
S.J. Tavalin, and
N.V. Marrion.
1997.
b-Adrenergic
stimulation selectively inhibits long-lasting L-type calcium channel facilitation in hippocampal pyramidal neurons.
J. Neurosci.
17:
6493-6503
|
10. | Collins, F., M.F. Schmidt, P.B. Guthrie, and S.B. Kater. 1991. Sustained increase in intracellular calcium promotes neuronal survival. J. Neurosci. 11: 2582-2587 [Abstract]. |
11. | Colquhoun, D., and F.J. Sigworth. 1983. Fitting and statistical analysis of single-channel records. In Single Channel Recordings. B. Sakmann and E. Neher, editors. Plenum Publishing Corp., New York. 191-264. |
12. | Deisseroth, K., K.E. Heist, and R.W. Tsien. 1998. Translocation of calmodulin to the nucleus supports CREB phosphorylation in hippocampal neurons. Nature. 392: 198-202 [Medline]. |
13. | Demo, S.D., and G. Yellen. 1991. The inactivation gate of the Shaker K+ channel behaves like an open-channel blocker. Neuron. 7: 743-753 [Medline]. |
14. | Fenwick, E.M., A. Marty, and E. Neher. 1982. Sodium and calcium channels in bovine chromaffin cells. J. Physiol. 331: 599-635 [Medline]. |
15. | Ferroni, A., A. Galli, and M. Mazzanti. 1996. Functional role of low-voltage activated dihydropyridine-sensitive Ca channels during the action potential in adult rat sensory neurons. Pflügers Arch. 431: 954-963 [Medline]. |
16. | Finkbeiner, S., and M.E. Greenberg. 1996. Ca2+-dependent routes to Ras: mechanisms for neuronal survival, differentiation, and plasticity? Neuron. 16: 233-236 [Medline]. |
17. |
Fisher, R.E.,
R. Gray, and
D. Johnston.
1990.
Properties and distribution of single voltage-gated calcium channels in adult hippocampal neurons.
J. Neurophysiol.
64:
91-104
|
18. | Fleig, A., and R. Penner. 1996. Silent calcium channels generate excessive tail currents and facilitation of calcium currents in rat skeletal myoballs. J. Physiol. 494: 141-153 [Abstract]. |
19. | Fomina, A.F., E.S. Levitan, and K. Takimoto. 1996. Dexamethasone rapidly increases calcium channel subunit messenger RNA expression and high voltage-activated calcium current in clonal pituitary cells. Neuroscience. 72: 857-862 [Medline]. |
20. | Forti, L., and D. Pietrobon. 1993. Functional diversity of L-type calcium channels in rat cerebellar neurons. Neuron. 10: 437-450 [Medline]. |
21. | Galli, C., O. Meucci, A. Scorziello, T.M. Werge, P. Calissano, and G. Schettini. 1995. Apoptosis in cerebellar granule cells is blocked by high KCl, forskolin, and IGF-1 through distinct mechanisms of action: the involvement of intracellular calcium and RNA synthesis. J. Neurosci 15: 1172-1179 [Abstract]. |
22. | Ghosh, A., J. Carnahan, and M.E. Greenberg. 1994. Requirement for BDNF in activity-dependent survival of cortical neurons. Science. 263: 1618-1623 [Medline]. |
23. | Grover, L.M., and T.J. Teyler. 1990. Two components of long-term potentiation induced by different patterns of afferent activation. Nature. 347: 477-479 [Medline]. |
24. | Hamill, O.P., A. Marty, E. Neher, B. Sakmann, and F.J. Sigworth. 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch. 391: 85-100 [Medline]. |
25. | Hivert, B., S. Bouhanna, S. Dochot, W. Camu, G. Dayanithi, C.E. Henderson, and J. Valmer. 1995. Embryonic rat motoneurons express a functional P-type voltage-dependent calcium channel. Int. J. Dev. Neurosci. 13: 429-436 [Medline]. |
26. | Hivert, B., and D. Pietrobon. 1995. Calcium channels of embryonic rat motoneurons. Soc. Neurosci. 21: 1578 . (Abstr.) . |
27. | Hivert, B., and D. Pietrobon. 1997. Anomalous L-type calcium channels of rat spinal motoneurons. Biophys. J 72: A22 . (Abstr.) . |
28. | Hoshi, T., J. Rothlein, and R.J. Smith. 1984. Facilitation of Ca2+ channel currents in bovine adrenal chromaffin cells. Proc. Natl. Acad. Sci. USA. 81: 5871-5875 [Abstract]. |
29. |
Johnson, B.D.,
T. Scheuer, and
W.A. Catterall.
1994.
Voltage-dependent potentiation of L-type Ca2+ channels in skeletal muscle cells
requires anchored cAMP-dependent protein kinase.
Proc. Natl.
Acad. Sci. USA.
91:
11492-11496
|
30. | Johnston, D., S. Williams, D. Jaffe, and R. Gray. 1992. NMDA-receptor independent long-term potentiation. Annu. Rev. Physiol. 54: 489-505 [Medline]. |
31. |
Kavalali, E.T.,
K.S. Hwang, and
M.R. Plummer.
1997.
cAMP-dependent enhancement of dihydropyridine-sensitive calcium channel
availability in hippocampal neurons.
J. Neurosci.
17:
5334-5348
|
32. | Kavalali, E.T., and M.R. Plummer. 1994. Selective potentiation of a novel calcium channel in rat hippocampal neurones. J. Physiol. 480: 475-484 [Abstract]. |
33. | Kavalali, E.T., and M.R. Plummer. 1996. Multiple voltage-dependent mechanisms potentiate calcium channel activity in hippocampal neurons. J. Neurosci. 16: 1072-1082 [Abstract]. |
34. | Kirsch, J., and H. Betz. 1998. Glycine-receptor activation is required for receptor clustering in spinal neurons. Nature. 392: 717-720 [Medline]. |
35. | Kleppisch, T., K. Pedersen, C. Strübing, E. Bosse-Doenecke, V. Flockerzi, F. Hofmann, and J. Hescheler. 1994. Double-pulse facilitation of smooth muscle a1 subunit Ca2+ channels expressed in CHO cells. EMBO (Eur. Mol. Biol. Organ.) J. 13: 2502-2507 [Abstract]. |
36. | Kullmann, D.M., D.J. Perkel, T. Manabe, and R.A. Nicoll. 1992. Ca2+ entry via postsynaptic voltage-sensitive Ca2+ channels can transiently potentiate excitatory synaptic transmission in the hippocampus. Neuron. 9: 1175-1183 [Medline]. |
37. | Lee, K.. 1987. Potentiation of calcium channel currents of internally perfused mammalian heart cells by repetitive depolarization. Proc. Natl. Acad. Sci. USA. 84: 3941-3945 [Abstract]. |
38. | Magee, J.C., and D. Johnston. 1995. Synaptic activation of voltage-gated channels in the dendrites of hippocampal pyramidal neurons. Science. 268: 301-307 [Medline]. |
39. | Magnelli, V., P. Baldelli, and E. Carbone. 1998. Antagonists-resistant calcium currents in rat embryo motoneurons. Eur. J. Neurosci. 10: 1810-1825 [Medline]. |
40. | McManus, O.B., A.L. Blatz, and K.L. Magleby. 1987. Sampling, log binning, fitting and plotting durations of open and shut intervals from single channels and the effect of noise. Pflügers Arch. 410: 530-553 [Medline]. |
41. | Murphy, T.H., P.F. Worley, and J.M. Baraban. 1991. L-type voltage-sensitive calcium channels mediate synaptic activation of immediate early genes. Neuron. 7: 625-635 [Medline]. |
42. |
Mynlieff, M., and
K.G. Beam.
1992.
Characterization of voltage-
dependent calcium currents in mouse motoneurons.
J. Neurophysiol.
68:
85-92
|
43. | Nakayama, S., and A.F. Brading. 1993. Evidence for multiple open states of the Ca2+ channels in smooth muscle cells isolated from the guinea-pig detrusor. J. Physiol. 471: 87-105 [Abstract]. |
44. | Neher, E.. 1992. Correction for liquid junction potentials in patch clamp experiments. Methods Enzymol. 207: 123-131 [Medline]. |
45. |
Parri, H.R., and
J.B. Lansman.
1996.
Multiple components of Ca2+
channel facilitation in cerebellar granule cells: expression of facilitation during development in culture.
J. Neurosci.
16:
4890-4902
|
46. | Pietrobon, D., and P. Hess. 1990. Novel mechanism of voltage-dependent gating in L-type calcium channels. Nature. 346: 651-655 [Medline]. |
47. | Rao, C.R. 1973. Linear statistical inference and its applications. 2nd edition. John Wiley & Sons, New York. 400 pp. |
48. | Schramm, M., R. Vajna, A. Pereverzev, A. Tottene, U. Klockner, D. Pietrobon, J. Hescheler, and T. Schneider. 1999. Isoforms of alpha1E voltage-gated calcium channels in rat cerebellar granule cells. Detection of major calcium channel alpha1-transcripts by reverse transcription-polymerase chain reaction. Neuroscience. In press. |
49. | Sculptoreanu, A., A. Figourov, and W.C. De Groat. 1995. Voltage-dependent potentiation of neuronal L-type calcium channels due to state-dependent phosphorylation. Am. J. Physiol. 269: C725-C732 [Abstract]. |
50. | Sculptoreanu, A., E. Rotman, M. Takahashi, T. Scheuer, and W.A. Catterall. 1993a. Voltage-dependent potentiation of the activity of cardiac L-type calcium channel alpha 1 subunits due to phosphorylation by cAMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA. 90: 10135-10139 [Abstract]. |
51. | Sculptoreanu, A., T. Scheuer, and W.A. Catterall. 1993b. Voltage- dependent potentiation of L-type Ca2+ channels due to phosphorylation by cAMP-dependent protein kinase. Nature. 364: 240-243 [Medline]. |
52. |
Shitaka, Y.,
N. Matsuki,
H. Saito, and
H. Katsuki.
1996.
Basic fibroblast growth factor increases functional L-type Ca2+ channels in
fetal rat hippocampal neurons: implications for neurite morphogenesis in vitro.
J. Neurosci.
16:
6476-6489
|
53. | Sigworth, F.J., and S.M. Sine. 1987. Data transformation for improved display and fitting of single-channel dwell time histograms. Biophys. J. 52: 1047-1054 [Abstract]. |
54. | Slesinger, P.A., and J.B. Lansman. 1991. Reopening of Ca2+ channels in mouse cerebellar neurons at resting membrane potentials during recovery from inactivation. Neuron. 7: 755-762 [Medline]. |
55. | Slesinger, P.A., and J.B. Lansman. 1996. Reopening of single L-type Ca2+ channels in mouse cerebellar granule cells: dependence on voltage and ion concentration. J. Physiol. 491: 335-345 [Abstract]. |
56. | Snutch, T.P., W.J. Tomlinson, J.P. Leonard, and M.M. Gilbert. 1991. Distinct calcium channels are generated by alternative splicing and are differentially expressed in the mammalian CNS. Neuron. 7: 45-57 [Medline]. |
57. |
Soldatov, N.M.,
A. Bouron, and
H. Reuter.
1995.
Different voltage-dependent inhibition by dihydropyridines of human Ca2+ channel splice variants.
J. Biol. Chem.
270:
10540-10543
|
58. | Thibault, O., and P.W. Landfield. 1996. Increase in single L-type calcium channels in hippocampal neurons during aging. Science. 272: 1017-1020 [Abstract]. |
59. | Thibault, O., N.M. Porter, and P.W. Landfield. 1993. Low Ba2+ and Ca2+ induce a sustained high probability of repolarization openings of L-type Ca2+ channels in hippocampal neurons: physiological implications. Proc. Natl. Acad. Sci. USA. 90: 11792-11796 [Abstract]. |
60. | Umemiya, M., and A.J. Berger. 1994. Properties and function of low- and high-voltage-activated Ca2+ channels in hypoglossal motoneurons. J. Neurosci. 14: 5652-5660 [Abstract]. |
61. | Umemiya, M., and A.J. Berger. 1995. Single-channel properties of four calcium channel types in rat motoneurons. J. Neurosci. 15: 2218-2224 [Abstract]. |
62. |
Williams, M.E.,
D.H. Feldman,
A.F. McCue,
R. Brenner,
G. Velicelebi,
S.B. Ellis, and
M.M. Harpold.
1992.
Structure and functional expression of ![]() ![]() ![]() |