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Volume 17, Number 8,
Issue of April 15, 1997
pp. 2658-2668
Copyright ©1997 Society for Neuroscience
Identification of the Single Channels that Underlie the N-Type
and L-Type Calcium Currents in Bullfrog Sympathetic Neurons
Keith S. Elmslie
Department of Physiology, Tulane University Medical School, New
Orleans, Louisiana 70112
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Most of the whole-cell calcium current of frog sympathetic neurons
is an N-type current, blocked by  -conotoxin GVIA (CGVIA). Thus,
these cells should be an excellent system to study the properties of
single N-type channels. However, a channel that is active near  10 mV
in isotonic Ba2+, originally identified as "N-type,"
corresponds more closely to a CGVIA-resistant component of the
whole-cell current observed in 100 mM Ba2+.
That conclusion would imply that the true single-channel correlate of
the macroscopic N-current remains to be identified in frog sympathetic
neurons. I report here recordings from cell-attached patches of a
calcium channel that activates in the appropriate voltage range (>0
mV, in isotonic Ba2+) and is blocked by CGVIA. This
channel has a slope conductance of 20 pS (range, 17-25 pS) and a
single-channel current of 1.3 pA at 0 mV. Other channels active in
the same voltage range (24 pS, 1.3 pA at 0 mV) were identified as
L-type channels because they exhibited long openings after
repolarization in the presence of 1 µM Bay K 8644 and
were resistant to CGVIA. A third channel type (13-19 pS) was
distinguished by current amplitude (0.6 pA at 0 mV) and strong
inactivation at 40 mV. The similarity in slope conductance among
these channels demonstrates that distinguishing them requires the
consideration of additional properties. The CGVIA-sensitive channel
can be identified as an N-type calcium channel.
Key words:
N-type calcium channels;
L-type calcium channels;
-conotoxin GVIA;
BayK 8644;
sympathetic neurons;
cell-attached patch
clamp
INTRODUCTION
N-type calcium current is important for many
neuronal functions such as neurotransmitter release and controlling
neuronal excitability. In frog sympathetic neurons, the calcium current recorded in 2 mM Ba2+ is primarily N-type
because ~90% of the current is blocked by -conotoxin GVIA
(CGVIA) (Jones and Jacobs, 1990 ; Elmslie et al., 1992). Therefore,
these neurons should be an ideal preparation to study single N-channels
and have been used in previous studies (Lipscombe et al., 1989; Delcour
and Tsien, 1993; Delcour et al., 1993). In these early studies, a
channel was identified as "N-type," which was dihydropyridine
(DHP)-resistant and had properties that superficially match those of
the whole-cell N-current (e.g., activation near 30 mV and
inactivation at a holding potential of 40 mV). However, DHP
resistance is a property shared by many calcium channel types. In
addition, the single-channel and whole-cell data were not directly
comparable because the recordings were made under different conditions,
110 mM Ba2+ versus 2 mM
Ba2+, respectively. More recent experiments have shown that
changing the external Ba2+ concentration from 2 to 90 mM shifts the voltage-dependent activation of N-current
~40 mV to the right (Elmslie et al., 1994). Thus, N-channels activate
near +10 mV in 100 mM Ba2+. Voltage-dependent
inactivation was also shifted because N-current was available from a
holding potential of 40 mV, which corresponds to a holding potential
of 80 mV in 2 mM Ba2+. Unlike
low-Ba2+ recordings, CGVIA block of N-current was
completely reversible in 100 mM Ba2+ (Boland et
al., 1994; Elmslie et al., 1994).
The experiments in isotonic Ba2+ also revealed a previously
unrecognized "novel" current, which was DHP- and CGVIA-resistant (Elmslie et al., 1994; Zhou and Jones, 1995). Recently, single "novel" channels were shown to be similar to channels expressed in
HEK 293 cells injected with E-class mRNA (Lewis et al., 1995). In
addition, E-class mRNA has been identified in frog sympathetic neurons.
Primarily to facilitate discussion, I have tentatively termed the
"novel"-channel Ef for frog E-class channel.
Surprisingly, Ef-channels showed characteristics
similar to the putative N-channel previously identified by Tsien and
colleagues (Lipscombe et al., 1989; Delcour et al., 1993). Both
channels activate near 30 mV, are strongly inactivated at a holding
potential of 40 mV, have a slope conductance of ~19 pS, and have a
current amplitude at 0 mV of approximately 0.6 pA. These similarities lead us to conclude that the two channels were the same (Elmslie et
al., 1994). If this were true, the CGVIA-sensitive N-channel in frog
sympathetic neurons remained to be identified. I report here a calcium
channel that activates near +10 mV and is reversibly blocked by
CGVIA. This channel resembles some putative N-channels (Plummer et
al., 1989; Mathie et al., 1992; Rittenhouse and Hess, 1994; Carabelli
et al., 1996) but differs from others, including the first putative
N-channels (Nowycky et al., 1985; Fox et al., 1987) and those
previously reported from frog sympathetic neurons (Lipscombe et al.,
1989; Delcour and Tsien, 1993; Delcour et al., 1993).
MATERIALS AND METHODS
Cells. Neurons were dissociated from caudal
paravertebral sympathetic ganglia of adult bullfrogs (Rana
catesbeiana) by a collagenase/dispase digestion and trituration
(Kuffler and Sejnowski, 1983; Jones, 1987; Elmslie, 1992). Cells were
maintained in L-15 culture medium, supplemented with 10% fetal bovine
serum and penicillin/streptomycin, at 4°C until use (usually 1-14
d).
Cell-attached patch recording. The pipette solution
consisted of (in mM) 100 BaCl2, 10 tetraethylammonium (TEA) chloride, 5, 4-aminopyridine (4-AP), and 10 N-methyl-D-glucamine (NMG)-HEPES, pH 7.2. The
TEA and 4-AP were required to suppress K+ currents because
intracellular K+ was not replaced. Gigaseals were formed in
an extracellular solution designed to zero the cell's membrane
potential. This zeroing solution contained (in mM) 100 KCl,
10 K-HEPES, and 5 NMG-EGTA, pH 7.2. In a few experiments
(n = 5), cell-attached macropatches were excised to
demonstrate that the cells do not maintain a membrane potential in the
high-K+ solution. Voltage ramps (1 mV/msec) were used to
monitor the macroscopic I-V before and after
excision. Although the calcium channel current rundown was rapid after
excision, peak current did not move along the voltage axis, indicating
that the membrane potential and bath potential were equal (data not
shown). Unless noted otherwise, the zeroing solution contained the
L-channel agonist (±)Bay K 8644 (1 µM) to reveal the
presence of L-channels in the patch (Plummer et al., 1989).
Electrodes for single-channel experiments were fabricated from Corning
7052 glass (OD 1.5 mm, ID 0.86 mm; A-M Systems, Everett, WA) and had
resistances of 3-15 M . The electrodes were coated with the General
Electric equivalent of SYLGARD (GE Silicones RTV615, General Electric
Company, Waterford, NY). The small tip size made filling the electrode
difficult. Therefore, recordings were done using fiber-filled glass to
facilitate filling the electrode without compromising seal formation.
Non-fiber-filled glass was used for the CGVIA experiments, in which
the tip-filling and back-filling solutions were different (see
below).
Data acquisition. Calcium-channel currents were recorded
using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA).
The experiment was controlled by a Macintosh II computer (Apple
Computer, Cupertino, CA) running S3 data acquisition software written
by Dr. Stephen Ikeda (Guthrie Research Institute, Sayre, PA). Currents
were digitized with a MacAdios II analog-digital converter (GW
Instruments, Somerville, MA) and stored on a 170 MB hard disk.
Currents were filtered at 2 kHz with the 4-pole Bessel filter on the
Axopatch 200A and amplified an additional 10× with a Frequency Devices
900 Bessel filter (Frequency Devices, Haverhill, MA) before being
digitized at 10 kHz (5 times the filter cutoff frequency). Data were
analyzed using IgorPro (WaveMetrics, Lake Oswego, OR) and Excel
(Microsoft, Redmond, WA) run on a Quadra 800 microcomputer (Apple
Computer).
Data were obtained in sets of 100 voltage steps of 100 msec duration
delivered at a 2 sec interval (~3.5 min/data set). The test voltage
was varied randomly across data sets to compensate for drifts in open
probability (Po) during the recording. During the 100 sweep data sets, the noise level would sometimes briefly increase. These brief noise events tended to occur more frequently as
the recording time increased. Sweeps recorded during these noise events
were excluded from analysis.
Analysis of single-channel records. Uncorrected capacitative
current and voltage-independent leakage current were removed from the
records by averaging sweeps without channel openings (nulls) and
subtracting this from active sweeps. This procedure was done within a
single data set (typically 100 sweeps) to decrease the possible effect
of changes in leakage currents or capacitative currents over time. If
null sweeps were rare or absent, a null record was made by fitting the
null regions of low activity sweeps with the sum of exponentials to
obtain a line that closely approximated the capacitative and leakage
currents. When generating pseudomacroscopic currents for comparison
within a patch, active sweeps for each current were subtracted using a
leakage record averaged from an equal number of null sweeps. This was
done to ensure similar noise levels in each of the pseudomacroscopic
currents (see Figs. 1, 2, 3, 5, 6, 7).
Fig. 1.
Putative N-type calcium channel. A,
Eight consecutive single-channel records are shown for three voltages,
along with the pseudomacroscopic current for each voltage. This patch
contained a single putative N-channel and an
Ef-channel, which was occasionally active during
steps from the holding potential of 40 mV. The number of sweeps
averaged for the pseudomacroscopic currents were 34, 70, and 100 for 0 mV, +20 mV, and +40 mV, respectively. The interval between sweeps was 2 sec, and the illustrated records were Gaussian-filtered at 1 kHz. This
patch was not exposed to Bay K 8644 until after these data were taken.
Note the apparent substate visible in the second sweep
from the top in the +20 mV steps and in the low-variance histogram for
+20 mV (peak ~ 0.6 pA). This was the only patch where such a
sublevel was observed. B, Low-variance-mean current
histograms are shown for each of the three voltage steps in
A. The window size was 3 points for 0 mV and 10 points
for both +20 and +40 mV. The binwidth was 0.05 pA. The smooth
lines are single Gaussian fits. Note that the
Ef-channel is evident as a shoulder on the zero peak
in the +20 mV histogram. C, Current-voltage
(I-V) relation averaged
from five sweeps where voltage was ramped from 80 to +80 mV over 160 msec (1 mV/msec). D, The single-channel current
amplitude taken from the low-variance-mean histograms was plotted
against the step potential. A linear regression fit to all of the
points yields a slope conductance of 21 pS and a 0 mV intercept of
1.38 pA. All data are from the same patch.
[View Larger Version of this Image (42K GIF file)]
Fig. 2.
L-channel recorded in the presence of 1 µM BayK 8644. A, Eight consecutive sweeps
are shown for each of three voltages. The pseudomacroscopic currents
are averages of 59, 100, and 100 sweeps for 20, 0, and +20 mV,
respectively. Note the long tail openings at 40 mV and
the slow tail current in the pseudomacroscopic currents. The illustrated single-channel sweeps were Gaussian-filtered at 1 kHz,
and two L-channels were present in the patch. B,
Low-variance-mean histograms were generated with a window size of 5 points for 20 mV and 10 points for both 0 and +20 mV. The
smooth lines are single Gaussian fits. C,
The ramp I-V was averaged from 15 voltage ramps (1 mV/msec) given with an interval of 2 sec. The holding
potential for the ramps was 40 mV. D, The
single-channel current amplitude taken from the low-variance-mean
histograms was plotted against the step potential. A linear regression
fit to all of the points yields a slope conductance of 25 pS and a 0 mV
intercept of 1.31 pA. All data are from the same patch.
[View Larger Version of this Image (34K GIF file)]
Fig. 3.
Ef-channel activity recorded in
the presence of CGVIA. A, Eight consecutive records
are shown for each of three voltages. Note that the holding potential
was 80 mV. The pipette was front-filled with the standard pipette
solution, but back-filled with that solution containing 100 µM CGVIA (see CGVIA application in Materials and
Methods). The pseudomacroscopic currents are averages of 83, 90, and 51 sweeps for 20, 0, and +20 mV, respectively. This patch contained at
least three Ef-channels. B,
Low-variance-mean histograms were generated using windows sizes of 10 points for 20 and 0 mV and 15 points for +20 mV. C,
Pseudomacroscopic current was averaged from 70 voltage ramps (1 mV/msec) given with an interval of 2 sec. D,
Single-channel current amplitude was plotted against the step
potential. A linear regression to all of the points from 40 to 0 mV
gave a slope conductance of 18 pS and a current amplitude at 0 mV of
0.6 pA. All data are from the same patch.
[View Larger Version of this Image (35K GIF file)]
Fig. 5.
Diffusion of 100 µM CGVIA to the
patch blocks single N-channels. A, Single-channel sweeps
and pseudomacroscopic currents are shown for three time points. Time
was measured from the point that the toxin-free tip solution and the
CGVIA back-filling solution are allowed to mix. The
pseudomacroscopic currents are averages of 46 sweeps for 5 min, 90 sweeps for 29 min, and 39 sweeps for 57 min. This patch contained at
least three N-channels and an Ef-channel.
B, The time course of CGVIA block measured from
pseudomacroscopic currents. The current amplitude was measured as the
average between 10 and 100 msec of the +20 mV step. Each patch is
represented by a separate symbol, and the asterisk
indicates the patch illustrated in A. C,
Pseudomacroscopic ramp current from the patch illustrated in
A is shown for comparison with Figure 1C
and whole-cell N-current (Elmslie et al., 1994). The current is an
average of 15 sweeps.
[View Larger Version of this Image (34K GIF file)]
Fig. 6.
Single N-channels recover from CGVIA block.
A, Single-channel sweeps and pseudomacroscopic currents
are shown for three time points. The clock started when the CGVIA
solution in the electrode tip and the toxin-free back-filling solution
were allowed to mix. The pseudomacroscopic currents are averages of 100 sweeps for 12 min, 100 sweeps for 25 min, and 22 sweeps for 54 min.
Note the channel with a smaller single-channel current
in the sweeps recorded at 12 min. This was a
Ef-channel that briefly recovered from inactivation.
This patch contained at least two N-channels. B, The
time course of the recovery from CGVIA block measured from
pseudomacroscopic currents. The currents were measured as the average
between 10 and 100 msec of the +20 mV step. Each patch is represented
by a separate symbol, and the asterisk indicates the
patch illustrated in A. C, The
single-channel I-V relation for
CGVIA-sensitive N-channels. This plot includes channels that were
blocked during the diffusion of CGVIA to the patch (Fig. 5;
n = 5) and channels that recovered during diffusion
of CGVIA from the patch (n = 4). The
line is a linear regression fit to all of the points
from 0 to +40 mV. The slope conductance was 20 pS, and the 0 mV
intercept was 1.3 pA.
[View Larger Version of this Image (37K GIF file)]
Fig. 7.
L-channels were not blocked by CGVIA.
A, L-channel activity is shown for three time points
after allowing the front- and CGVIA-containing back-filling
solutions to mix. This patch appeared to contain a single L-channel.
B, Current amplitudes were measured from
pseudomacroscopic currents as an average from 10 to 100 msec during the
voltage step. For each L-channel patch, 100 µM CGVIA was in the back-filling solution while the tip was filled with toxin-free pipette solution. Each patch (n = 4) is
represented by a different symbol, and the asterisk
indicates the patch shown in A. C,
Pseudomacroscopic ramp current is shown from the patch illustrated in
A as an average of 15 sweeps.
[View Larger Version of this Image (34K GIF file)]
Low-variance histograms. Because of the brief open times
exhibited by the calcium channels at some voltages, the use of
all-points histograms has been avoided. Instead, low-variance analysis
has been used to estimate single-channel current levels because it excludes points from regions of high variance (e.g., channel opening or
closing transitions) (Patlak, 1988, 1993; Elmslie et al., 1994). Briefly, the mean current amplitude and variance were calculated for a
window (generally 5-20 points; 0.5-2 msec) advanced across the record
in 1 point increments. If the variance in the window was  baseline
variance, the mean current amplitude was kept for the histogram. The
histogram binwidth was 0.05 pA. The y-axis of the histogram
was log-transformed to truncate large peaks relative to small
peaks.
CGVIA application. Single-channel data were recorded
while the concentration of CGVIA at the patch was altered with a
technique analogous to that used for introducing nystatin to the
membrane in perforated-patch recordings (Horn and Marty, 1988). The
electrode tip was filled with one solution (e.g., toxin-free), and the
electrode was back-filled with another (e.g., toxin-containing pipette
solution). The patch concentration of a test substance could be
increased or decreased by switching tip-filling and back-filling
solutions. Time was zeroed when the bubble separating the two solutions
was tapped out to allow the solutions to mix. The amount of solution in
the tip was measured before the electrode was back-filled. On average,
1.3 mm of the tip was filled (range, 0.93-2.1 mm) and strong positive
pressure was maintained on the pipette until cell contact was made. In
general, less solution was used when 100 µM CGVIA was
in the tip solution because CGVIA dissociation is slow in 100 mM Ba2+ (Boland et al., 1994; Elmslie et al.,
1994). Filling the tip with 100 µM CGVIA did not
compromise seal formation. These experiments used only nonfilament
electrode glass so that the two solutions would not mix until the
bubble was tapped out.
Drugs. (±)-Bay K 8644 was obtained from Research
Biochemicals (Natick, MA). CGVIA was obtained from either LC
laboratories (Woburn, MA) or Bachem Bioscience (King of Prussia, PA).
TEA, 4-AP, EGTA, and HEPES were obtained from Sigma (St. Louis, MO). All other chemicals were reagent grade.
RESULTS
The goal of this work was to identify the single calcium channel
that generates macroscopic N-current. Therefore, a majority of
recordings were done from a holding potential of 40 mV, where Ef-channels are primarily inactivated (Elmslie et
al., 1994). N-channels in 100 mM Ba2+ are
active from a holding potential of 40 mV, which is equivalent to 80
mV in 2 mM Ba2+ (Elmslie et al., 1994). Bay K
8644 (1 µM) was bath-applied to all patches to help
separate N-channels from L-channels. Bay K 8644 increases the open time
of L-channel gating both during voltage steps and on repolarization to
the holding potential (tail openings). The presence of these tail
openings was used to tentatively identify L-channels (Plummer et al.,
1989). As will be seen, the presence of these tail openings correlates
very well with other properties expected for L-channels such as
activation at voltages more than 30 mV and resistance to block by
CGVIA.
General description of recorded channels
Putative N-channels
Our whole-cell recordings in 100 mM Ba2+
showed that N-channels should activate at voltages > 0 mV, and
peak macroscopic current should occur between +25 and +40 mV (Elmslie
et al., 1994). In addition, N-channels should deactivate rapidly at
40 mV in the presence of Bay K 8644 (i.e., no long tail openings).
One type of calcium channel available from a holding potential of 40
mV matched these properties (Fig. 1). Activity of these
channels was measured in isolation from other channel activity in nine patches (not exposed to CGVIA). Channel gating was observed during voltage steps ranging from 0 to +50 mV with the open probability (Po) increasing with voltage. For the example
illustrated in Figure 1, Po increased from 0.02 at 0 mV to 0.54 at +30 mV. (The highest voltage at which
Po could be accurately measured in this patch.) The slope conductance was calculated from 0 to +40 mV by linear regression and was found to be 21 pS with a single-channel current at 0 mV of 1.3 pA (9 patches not exposed to CGVIA; Figs.
1D, 4A). Consistent with whole-cell
data, ramp currents showed measurable inward current at voltages
greater than or equal to +10 mV, and the mean voltage generating peak
current was +35 mV for the five patches examined in this data set
(range +28 to +41 mV; Fig. 1C). Voltage ramps were not
recorded for the other four patches (not exposed to CGVIA). Channel
activity showed no inactivation during 100 msec steps to +20 mV (Figs.
1A, 5A, 6A). However, a
small inactivating component could be observed in the pseudomacroscopic records for steps to +40 mV (Figs. 1A). This
incomplete inactivation is consistent with whole-cell N-current data
(Jones and Marks, 1989; Werz et al., 1993). The density of these
channels was high because only two of nine patches (not exposed to
CGVIA) appeared to contain a single N-channel.
Fig. 4.
Single-channel I-V
relation for N-channels, Ef-channels, and
L-channels. A, N- and Ef-channel
current amplitudes are superimposed to facilitate comparisons. Activity
was measured using low-variance-mean histograms during voltage steps
ranging from 0 to +50 mV for N-channels (n = 9) and
40 to +20 mV for Ef-channels
(n = 12). None of the N-channels were exposed to
CGVIA. The lines are linear regression fits to all
points from 0 to +40 mV for N-channels and 40 to 0 mV for
Ef-channels. The fit to N-channel data gave a slope
conductance of 21 pS and 0 mV intercept of 1.3 pA. The
Ef-channel data fit gave a 16 pS conductance and a 0 mV intercept of 0.6 pA. The dashed line is the
regression fit to single L-channel currents from B. It
is replotted here to facilitate comparison to N- and Ef-channels. B, Single L-channel
current amplitudes were measured during voltage steps ranging from 30
to +50 mV using low-variance-mean histograms (n = 10). The line is a linear regression fit to all points
from 20 to +20 mV. The slope conductance was 24 pS, and the 0 mV
intercept was 1.3 pA.
[View Larger Version of this Image (18K GIF file)]
The current level of one channel in this group [shown as (double
triangle) in Fig. 4A] was larger than the
others. The slope conductance of this channel was 20 pS, like the other
channels, but the current amplitude at +20 mV was 1.16 pA compared
wiith a mean of 0.85 pA (n = 8; range 0.74 to
0.95 pA) for the others. It is possible that this is a different
channel type. Alternatively, it could be the same channel type gating
in a mode with a larger single-channel current as described for the
N-channel in rat sympathetic neurons (Plummer et al., 1989; Rittenhouse
and Hess, 1994). Mode switching was not observed. However, it could
have been missed because I recorded from only two single-N-channel
patches.
L-channels
A separate population of channels could be distinguished by long
tail openings in the presence of the DHP agonist Bay K 8644 and
activation near 20 mV (compared with +10 mV for the putative N-channel; Fig. 2). The mean voltage for peak of the
pseudomacroscopic ramp current was +23 mV (range +12 to +38 mV,
n = 9; Fig. 2C). Single L-channel patches
(4/10) were more frequently encountered compared with N-channels (2/9)
and Ef-channels (0/5; see below). Although N- and
L-channels were easily distinguished by activation voltage, the
single-channel current and slope conductance were found to be similar
between these channels (Fig. 4). The L-channel current at +20 mV ranged
from 0.62 to 0.98 pA (mean 0.81 pA for 8 patches), which overlaps
with the putative N-channel (mean 0.87 pA; see above). The
single-channel conductance measured from 40 to +20 mV was 24 pS (Fig.
4B). L-channels show a large variability in both the
single-channel conductance (range 19-33 pS) and the current amplitude
at 0 mV (range 1.0-1.4 pA). One explanation for this variability is
that frog sympathetic neurons express multiple L-channel types as shown
previously for rat cerebellar granule cells and rat hypocampal
pyramidal cells (Forti and Pietrobon, 1993; Kavalali and Plummer,
1994).
Ef-channels
Ef-channel activity was observed during many of the N-
and L-channel recordings (12/28), although the holding potential was 40 mV. The pattern of gating was brief periods of activity separated by long periods of inactivation. This is expected because whole-cell Ef-current is ~80% inactivated at 40 mV
(Elmslie et al., 1994). N- or L-channel activity was absent in five
additional patches, allowing the holding potential to be set to 80 mV
(total patches with Ef-channels 17/43; Table
1, rows 1 and 2). Each of these five patches contained
at least two Ef-channels.
Activity of Ef-channels could be recorded during
steps to 40 mV (holding potential 80 mV). However, measurable
inward current during voltage ramps was not apparent until
approximately 20 mV (Fig.
3A,C). The voltage
generating peak inward current was measured from voltage ramps to be
+20 mV (range 13-27 mV; n = 3), which is similar to
whole-cell Ef-current (Elmslie et al., 1994). The
slope conductance measured by regression fit ranged from 13 to 20 pS in
12 patches (with and without CGVIA in the pipette; mean 16 pS) with
a mean current amplitude at 0 mV of 0.6 pA (range 0.4 to 0.7 pA;
Fig. 4A). The conductance values are
in the range previously reported for these channels (15-16 pS:
Lipscombe et al., 1988, 1989; 18 pS: Delcour et al., 1993; 19 pS:
Elmslie et al., 1994). The variability in slope conductance may reflect
multiple channel types, subconductance states, and/or different gating
modes. The absence of single-Ef-channel patches in
the present study prevents distinguishing among these
possibilities.
Frequency of channel observation
Putative N-channels were observed in 18 of 43 patches (Table 1,
rows 1 and 2; excluding patches continuously exposed to CGVIA; see
below). This frequency is consistent with the whole-cell calcium current recorded in 100 mM Ba2+, which is
50-70% N-current (Elmslie et al., 1994). L-channels were observed
less frequently than N-channels (10/43 patches; Table 1, rows 1 and 2)
but more frequently than expected from whole-cell recording, in which
L-current comprises ~5% of the total calcium current. The frequency
of observing Ef-channels was similar to that of
N-channels (17/43; Table 1, rows 1 and 2). The large number of patches
with Ef-channels was expected because
Ef-current comprises 30-50% of the whole-cell
current recorded in isotonic Ba2+ (Elmslie et al.,
1994).
Identification of N-channels by sensitivity to CGVIA
The 20 pS calcium channel is the best candidate to be the N-type
calcium channel because it is active over the voltage range expected
for N-current in 100 mM Ba2+. To test this
hypothesis, CGVIA was applied during cell-attached patch recordings
of single calcium channels. This was accomplished by filling the
electrode tip with toxin-free solution and back-filling with the
pipette solution containing 100 µM CGVIA. The high
concentration of CGVIA was required to ensure block in isotonic
Ba2+ (Boland et al., 1994). Figure
5A shows the blocking effect of CGVIA on
putative N-channels in one experiment. This channel had a slope
conductance of 22 pS, and the single-channel current is plotted as a
filled square in Figure 6C. The
pseudomacroscopic current averaged from 15 voltage ramps showed
activation near +10 mV and peak at +44 mV (Fig. 5C). The
time course of the block was measured from pseudomacroscopic step
currents. Figure 5B shows the time course of the CGVIA
effect in six patches that contained putative N-channels. In five of
six patches, channel activity decreased with time, as expected for
increasing concentration of CGVIA at the patch.
Ef-channel activity was noticed in four of the five
N-channel patches. However, the activity periods were short (holding
potential 40 mV) and the small current amplitude made identification
easy (see Fig. 6). One of the N-channel patches contained an L-channel
in addition to an Ef-channel. The single L-channel
current was smaller (0.7 vs 1.0 pA at +20 mV) and activated at more
hyperpolarized voltages than the N-channel. The block of N-channel
activity was reflected in the reduction of the pseudomacroscopic
current at +20 mV (Fig. 5B, filled diamonds). Over this time, the activity of the single L-channel increased, as
shown by the pseudomacroscopic current at 0 mV (Fig.
7B, filled diamonds).
Although the reduction in N-channel activity is consistent with channel
block by CGVIA, single channels can "drop out" of the patch for
unknown reasons. Therefore, I took advantage of the reversibility of
CGVIA block of frog N-channels recorded in ~100 mM
Ba2+ (Boland et al., 1994; Elmslie et al., 1994). For these
experiments, the electrode tip was filled with the pipette solution
containing 100 µM CGVIA and the electrode back-filled
with toxin-free solution. Figure 6A shows the
increase in N-channel activity over time for one experiment. In four
patches treated this way, channel activity increased over time and in
each case the recovered channel showed N-channel properties (Fig.
6B). Thus, the N-channel appears to recover from
block as CGVIA diffuses away from the patch. Two of these patches
also contained Ef-channel channels, but no L-channel activity was observed in any of the four patches. In three additional patches, no channel activity could be observed up to 1.75 hr after removal of the bubble separating the two solutions in the pipette.
The single-channel I-V for all
CGVIA-sensitive channels (n = 9) is plotted in
Figure 6C. The regression fit gives a slope conductance of
20 pS and a single-channel current at 0 mV of 1.29 pA. The
pseudomacroscopic ramp current was examined in six CGVIA patches and
showed activation near +10 mV with a mean peak current at +38 mV (range
+31 to +44 mV). Although simultaneous N-channel openings were rarely
observed in these CGVIA experiments, the presence of the blocker
prevented me from determining the number of N-channels present in each
patch. The properties of the CGVIA-sensitive channels match those of
the putative N-channels identified by activation voltage and the
absence of long tail openings in Bay K 8644. Table 2
shows the good agreement between the properties of the macroscopic
N-current recorded in 90-100 mM Ba2+ and the
single N-channel presented here.
L-channels
CGVIA (100 µM) was specific for N-channels
because L- channels were not blocked. L-channel activity was observed
in four patches where the tip of the pipette was filled with toxin-free pipette solution and where the pipette was back-filled with pipette solution containing 100 µM CGVIA. The example channel
shown in Figure 7A had a 23 pS conductance and a
single-channel current at 0 mV of 1.4 pA. Although the
pseudomacroscopic current showed a slight decrease over time, channel
activity was still high even after 60 min (Fig. 7A). In the
other patches, L-channel activity increased in one patch, decreased
slightly in another, and varied around a central point (approximately
1.4 pA) in the third patch (Fig. 7B).
Channels recorded in the presence of CGVIA
Ef-channel activity was observed in six
CGVIA-diffusion patches at times when N-channel activity in the
patch was blocked. However, only one patch was held at 80 mV for as
long as 60 min (channel illustrated in Fig. 3). Although this patch did
not show a decrease in activity, a single observation does not
adequately support the conclusion that Ef-channels
are not CGVIA-sensitive. Therefore, an additional set of recordings
was made with the entire pipette filled with solution containing 100 µM CGVIA. Under this condition,
Ef-channels were encountered in ~25% of the
patches, which is similar to the frequency in the other experimental
conditions (Table 1). As expected, L-channels were not also blocked
because the frequency of L-channels patches remained at 25% in
CGVIA (Table 1). However, the constant presence of CGVIA in the
pipette reduced frequency of N-channel patches to ~5% compared with
~43% in both control and CGVIA-diffusion experiments (Table 1).
Not surprisingly, the frequency of patches with no channel activity was
nearly doubled by constant exposure to CGVIA. These results are
consistent with the conclusion that CGVIA blocks the 20 pS N-channel
but that Ef-channels and L-channels are not
affected.
Activity of N-like channels was not blocked in two patches tested
with CGVIA. In one patch in which the pipette had been backed-filled
with CGVIA, channel activity was not altered during the 50 min
recording time (Fig. 5B). This channel had a low
Po that was manifested in the small amplitude of
the pseudomacroscopic currents (Fig. 5B). In a second patch,
N-like channel activity was observed in the constant presence of
CGVIA. The slope conductance for these channels calculated over four
voltages (range +10 to +40 mV) was 19 pS, and the single-channel
current at +20 mV was 0.95 pA (data not shown). The
I-V relation measured from voltage ramps showed
activation near +10 mV and peak near +40 mV. These channels may be
responsible for the small CGVIA-insensitive whole-cell calcium
current that shares several N-current properties such as
voltage-dependent inhibition by norepinephrine (Elmslie et al.,
1992).
DISCUSSION
The CGVIA-sensitive N-channel in frog sympathetic neurons
The identification of single N-channels has been
controversial (Elmslie et al., 1994). Putative N-channels have widely
variable conductances and current amplitudes (Table 3).
The strategy here is to use two fundamental criteria for identification
of true N-channels: (1) the pseudomacroscopic
I-V relation should agree with the whole-cell
CGVIA-sensitive current recorded in isotonic Ba+2, and
(2) the true N-channel should be reversibly blocked by CGVIA (for
frog in high Ba+2). N-channel activity was characterized in
complete isolation from other channels in 17 different patches. The
N-channel identified here fits the properties expected from whole-cell
N-current recorded in 100 mM Ba2+ (Table 2).
N-channels activated near +10 mV and were available from a holding
potential of 40 mV. Peak pseudomacroscopic current was measured at
voltages ranging from 28 to 44 mV, which is similar to N-current in
isotonic Ba2+. The time course of activation was
monophasic, as expected for N-current in the absence of modulation by
activated G-proteins, and deactivation was rapid after repolarization
to 40 mV. N-channel inactivation was incomplete over 100 msec at
voltages that generated peak inward current (i.e., +40 mV). This fits
very well with whole-cell N-current data (Jones and Marks, 1989; Werz
et al., 1993). N-channels were reversibly blocked by 100 µM CGVIA with a time course that is consistent with
whole-cell data in isotonic Ba2+ (Boland et al., 1994;
Elmslie et al., 1994). However, L- and Ef-channels
were not affected. The evidence supports the conclusion that this 20 pS
channel underlies the CGVIA-sensitive N-type calcium current in frog
sympathetic neurons.
Table 3.
Comparison of N-channels recorded in various neuronal
preparations
| Channel
properties |
Chick DRG neurons1 |
Rat
CA3 neurons2 |
Rat motoneuron3 |
Rat sympathetic4 |
Neurohypophysis
terminals5 |
Rat sympathetic6 |
Frog sympathetic7 |
|
Slope
 |
13 pS |
12 pS |
14 pS |
11 pS |
10-15
pS |
17-22 pS |
20 pS |
| i at 0 mV |
1 pA |
0.5 pA |
0.7
pA |
0.8 pA |
NR |
1.5 pA |
1.3 pA |
| Activation
voltage |
20 mV |
30 mV |
10 mV |
30 mV |
10 mV |
10
mV |
10 mV |
| Inactivation at 40
mV |
Strong |
Strong |
Strong |
Strong |
Strong |
Moderate |
Weak |
| CGVIA
sensitivity |
NT |
NT |
NT |
NT |
NT |
YES? |
YES |
|
|
NT, not tested; NR, not reported
1Nowycky et al., 1985 and Fox et al., 1987;
2Mogul and Fox, 1991; 3Umemiya and Berger,
1995; 4Hirning et al., 1988; 5Lemos and
Nowycky, 1989; 6Plummer et al., 1989; Mathie et al., 1992,
and Rittenhouse and Hess, 1994; 7Data presented in this
paper.
YES? refers to unpublished data of N-channel block by CGVIA (see
Discussion, Plummer et al., 1989).
*
The data in Mathie et al., 1992 were corrected for a 14 mV junction
potential. With this correction removed, the N- and L-channel data of
Mathie et al., 1992 matches the data presented in Plummer et al., 1989;
Rittenhouse and Hess, 1994; and this paper.
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Two patches contained channels that fit the voltage-dependent
properties of the N-channel, but they were not blocked by CGVIA. Whole-cell recordings have demonstrated an CGVIA- and
DHP-insensitive calcium current with properties similar to those of the
N-current. These similarities include voltage-dependent inhibition by
norepinephrine (Elmslie et al., 1992) and an enhancement of
inactivation rate by the phosphatase inhibitor okadaic acid (Werz et
al., 1993). This calcium current comprises 5% of the whole-cell
calcium current in 2 mM Ba2+, so the frequency
of recording this channel type should be low.
Putative N-channels from other neuronal preparations
Calcium channels in several different preparations have
been identified as "N-type" based on DHP insensitivity and strong inactivation at a holding potential of 40 mV (Table 3, columns 1-4).
However, DHP insensitivity is a characteristic of many calcium channels, and N-current in isotonic Ba2+ does not
inactivate strongly at a holding potential of 40 mV. These putative
N-channels activated at voltages hyperpolarized to N-current in
isotonic Ba2+, and their single-channel current was smaller
than that of the frog N-channel. Thus, the properties of these channels
do not match those expected for N-current.
The putative N-channel recorded from rat neurohypophysis terminals
activated at voltages close to that of N-current in isotonic Ba2+ (Table 3) but had a smaller conductance (10-15 pS)
than the frog N-channel and inactivated strongly at 30 mV (Lemos and
Nowycky, 1989). Fujita et al. (1993) observed a 14 pS channel for
B-class mRNA (N-channel) expressed in dysgenic mouse skeletal muscle
that activated at voltages greater than or equal to 10 mV. It will be
interesting to see whether future experiments demonstrate that native
N-channels can exhibit a broad range of conductances. CGVIA block of
a 13 pS calcium channel recorded from chick sensory neurons has been
reported (Aosaki and Kasai, 1989). However, there are problems equating
this "CGVIA-sensitive" channel with the N-current. The
single-channel activity inactivated more rapidly during voltage steps
than whole-cell N-current, and the channels activated near 20 mV in
isotonic Ba2+ (Aosaki and Kasai, 1989). The loss of channel
activity observed in the CGVIA blocking experiments could have been
caused by rundown because the channels were recorded in the outside-out
configuration.
Many properties of the putative N-channel in rat sympathetic neurons
match those of the frog N-channels (Table 3). One difference is that
the rat N-channel exhibited moderate inactivation at 30 mV (~50%)
(Plummer et al., 1989). This inactivation is larger than expected for
N-current in frog sympathetic neurons but is similar to a DHP- and
CGVIA-resistant calcium current recorded in rat sympathetic neurons
exposed to isotonic Ba2+ (~35% at 40 mV) (Boland et
al., 1994). Furthermore, the calcium current recorded by Boland et al.
(1994) activated near 10 mV, which is close to the N-channel
activation voltage. Plummer et al. (1989) reported in their Discussion
that their rat N-channel was blocked by CGVIA, but the data were not
shown. Mathie et al. (1992) reported a 17 pS N-channel in rat
sympathetic neurons that was blocked by including CGVIA in the
pipette solution (data not shown). The properties of the 17 pS
N-channel match those of the frog N-channel after allowing for a 14 mV
correction for junction potential used by Mathie et al. (1992). The
putative rat N-channel has been shown to gate in three modes that were defined by open probability (Rittenhouse and Hess, 1994). As stated above, I have not noticed different gating modes in the frog N-channel experiments. However, it is nearly impossible to discern modes in
multichannnel patches like those usually recorded here (only 2 single-channel patches).
The N-channel recorded from neuroblastoma IMR32 cells by Carabelli et
al. (1996) nicely matches the frog N-channel characterized here. The
slope conductance of the IMR32 N-channel was 19 pS, and the
single-channel current at +20 mV was 0.9 pA (compared with 0.87 pA for
the frog N-channel). The IMR32 N-channel activated near 0 mV, which is
~10 mV hyperpolarized to the frog N-channel.
L-channels
L-type calcium channels were identified by long tail openings and
activation near 20 mV with 1 µM Bay K 8644 present in
the bath. Consistent with this identification, these channels were not
blocked by 100 µM CGVIA. Thus, pharmacology provides
an excellent means by which to separate N-channels from L-channels
(Plummer et al., 1989), but slope conductance and current amplitude
were too similar to be a reliable criteria for separation (Fig. 4). In
addition, one must be careful when using Po as a
criterion because both N- and L-channels are open much of the time at
depolarized potentials (greater than or equal to +30 mV). L-channels
were found in 10 of 43 patches, which is surprisingly frequent because only ~5% of the whole-cell current is sensitive to DHP antagonists (Jones and Jacobs, 1990; Elmslie et al., 1992). However, L-channel density appeared to be lower than either N- or
Ef-channels because there was a larger fraction of
single L-channel patches (7/17).
Ef-channels
The Ef/novel calcium channel was identified in
an earlier paper (Elmslie et al., 1994). The name novel was used
because the whole-cell current had not been recognized previously.
Recent experiments demonstrate that "novel" channels are likely to
be expressed from E-class mRNA (Lewis et al., 1995). First, PCR was used to demonstrate that frog sympathetic neurons express E-class mRNA.
Second, "novel" channel properties closely match those of the
channel expressed by E-class mRNA transfected into HEK 293 cells (Lewis
et al., 1995). The "novel" channel is tentatively referred to here
as Ef to facilitate discussion, but a solid identification will require further study.
Ef-channels could be separated from N- and
L-channels by strong inactivation at 40 mV and the small
single-channel current amplitude. As seen above, slope conductance was
not a good method by which to separate Ef-channels
from the others. The slope conductance for
Ef-channels ranged from 13 to 19 pS, which overlaps
the range observed for N- and L-channels. Previous measurements of
slope conductance for channels gating in this voltage range varied from 15 to 19 pS (Lipscombe et al., 1988, 1989; Delcour et al., 1993; Elmslie et al., 1994). Some of this variability could be caused by
different modes of Ef-channel gating as described
previously (Delcour et al., 1993). I have made no effort to distinguish
modes because no single-Ef-channel patches were
recorded. As demonstrated for whole-cell Ef-current,
Ef-channels were not blocked by CGVIA. Ef-channel activity was observed in CGVIA
diffusion experiments when the N-channel activity in the same patch was
blocked (n = 6). In addition,
Ef-channel activity was observed in the constant presence of CGVIA (n = 5).
The Ef-channel is primarily inactivated at a holding
potential of 40 mV but not completely. Whole-cell recordings show
that Ef-current is reduced by ~80% when the
holding potential is depolarized from 80 to 40 mV. Single-channel
recordings demonstrate that Ef-channels could
recover from inactivation during a constant holding potential of 40
mV. Once recovered, the channel could dwell in the activatable state
for many seconds. This can be seen in Figure 6A (12 min), in which the Ef-channel was active over three
consecutive sweeps that spanned 6 sec.
Summary and conclusion
There are currently three mRNAs that can be expressed to form
DHP-insensitive calcium channels. In addition, isoforms of
channel-associated proteins have been cloned. Given this diversity, it
is imperative that single calcium channels be identified as rigorously
as possible. In this paper, I identify N-type, L-type, and
Ef-type calcium channels by comparing their
biophysical and pharmacological properties with those of the whole-cell
currents recorded in isotonic Ba+2. The frog N-channel
identified here is similar to N-channels recorded from rat sympathetic
neurons (Plummer et al., 1989; Mathie et al., 1992; Rittenhouse and
Hess, 1994) and from human neuroblastoma IMR32 cells (Carabelli et al.,
1996). We currently do not have sufficient information to determine
whether putative N-channels from different preparations are indeed
CGVIA-sensitive N-channels.
FOOTNOTES
Received Nov. 14, 1996; revised Jan. 29, 1997; accepted Jan 30, 1997.
This research was supported by National Institutes of Health Grant
NS33671, the Louisiana Education Quality Support Fund (LEQSF-RD-A-28), and Pharmaceutical Research and Manufacturers of America Foundation. I
thank Dr. Stephen W. Jones for suggesting the method for applying -conotoxin to the isolated calcium channels and for his comments on
an early version of this manuscript. In addition, I thank Dr. Geoffrey
G. Schofield, Dr. Hye Kyung Lee, and Walter Robertson for helpful
discussions and comments on this manuscript.
Correspondence should be addressed to Dr. Keith S. Elmslie, Department
of Physiology, Tulane University Medical School, 1430 Tulane Avenue,
New Orleans, LA 70112.
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