Department of Physiology, Tulane University Health Science Center, New Orleans, Louisiana 70112
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ABSTRACT |
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Liang, Haoya and
Keith S. Elmslie.
Ef-Current Contributes to Whole-Cell Calcium Current
in Low Calcium in Frog Sympathetic Neurons.
J. Neurophysiol. 86: 1156-1163, 2001.
Because
Ca2+ plays diverse roles in intracellular signaling in
neurons, several types of calcium channels are employed to control Ca2+ influx in these cells. Our experiments focus on
resolving the paradox of why whole-cell current has not been observed
under typical recording conditions for one type of calcium channel that is highly expressed in frog sympathetic neurons. These channels, referred to as Ef-channels, are present in the
membrane at a density greater than the channels that carry ~90% of
whole-cell current in low Ba2+; but,
Ef-current has not been detected in low
Ba2+. Using Ca2+ instead of Ba2+ as
the charge carrier, we recorded a possible E-type current in frog
sympathetic neurons. The current was resistant to specific blockers of
N-, L-, and P/Q-type calcium channels but was more sensitive to
Ni2+ block than was N- or L-current. Current
amplitude in Ca2+ is slightly greater than that in
Ba2+. In 3 mM Ca2+, the current contributed
~12% of total current at peak voltage and increased at voltages more
hyperpolarized to the peak, reaching ~40% at 30 mV, where
whole-cell current starts to activate. The presence of
Ef-current in 3 mM Ca2+ suggests a
potential role for Ef-channels in regulating calcium influx into sympathetic neurons.
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INTRODUCTION |
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Calcium current resistant to
blockers of N-, L-, and P/Q-type channels are widely distributed in the
nervous system, where they have been implicated in dentritic
integration (Delmas et al. 2000) and synaptic release of
neurotransmitters and hormones (Wang et al. 1999
;
Wu et al. 1998
). These currents have been receiving increased attention since the cloning of
1E
channels (CaV 2.3). Resistant current (R-current) has been
detected in frog sympathetic neurons in 2 mM Ba2+
(Elmslie et al. 1992
) as well as in 100 mM
Ba2+ (Elmslie et al. 1994
). In 2 mM Ba2+, it has been shown that 5% of whole-cell
current is resistant to both
-conotoxin GVIA (
CGVIA) and
dihydropyridines (DHPs). Except for its insensitivity to
CGVIA, the
resistant current in low Ba2+ shares several
biophysical properties with N-current, including a similar
current-voltage relationship (I-V),
voltage-dependent inhibition by norepinephrine (NE), and increased
inactivation by phosphorylation (Elmslie et al. 1992
;
Werz et al. 1993
). We therefore refer to it as
"N-like" current.
Surprisingly, the properties of the current resistant to CGVIA and
DHPs change in high Ba2+. In 100 mM
Ba2+, the current contributes 30-50% of the
whole-cell current and activates and inactivates at voltages more
hyperpolarized than those of N-current (Elmslie et al.
1994
). The current is not inhibited by NE but can be
preferentially blocked by Ni2+. Pharmacological
characteristics of the resistant current resemble those of current
through channels expressed from E-class mRNA (
1E channels) (Bourinet et al.
1996
). Therefore, the resistant current in high
Ba2+ was termed Ef-current
(Elmslie 1997
), which is the abbreviation for E-type
current in frog neurons.
Single-channel studies in isotonic Ba2+ have
identified Ef-channels and possible N-like
channels (Elmslie 1997). Both channel types are
insensitive to
CGVIA and DHPs. Voltage-dependent properties of
Ef-channels match those of whole-cell
Ef-current in 100 mM Ba2+. In contrast, the potential N-like channels
were indistinguishable from N-channels in their activation voltage
range (>0 mV), single-channel conductance (~20 pS), or
unitary current amplitude (~1.4 pA at 0 mV). Single-channel
evidence also shows that the density of putative
Ef-channels is equal to or greater than
that of
CGVIA-sensitive N-channels (Elmslie 1997
).
N-channels are responsible for the majority of calcium current in
frog sympathetic neurons. The objective of our experiments is to
search for a current that may be attributed to channels coded by E-type
genes under more physiological recording conditions.
If channels expressed from E-class mRNA are present in frog sympathetic
neurons, they should pass larger current in Ca2+
than they do in Ba2+, according to the relative
permeability of the two divalent cations in 1E
channels (Bourinet et al. 1996
). L- and N-channels,
which have been identified in sympathetic neurons (Elmslie et
al. 1992
), carry smaller current in Ca2+
than they do in Ba2+ (Bourinet et al.
1996
; Hess et al. 1986
; Wakamori et al.
1998
). These channel properties suggest that using
Ca2+ as the charge carrier will facilitate
detection of potential E-type current. The criteria used to identify
E-type current were based on three prominent characteristics of
currents through
1E recombinant channels,
i.e., resistance to specific blockers of N-, L-, and P/Q-type calcium
channels; sensitivity to being blocked by Ni2+;
and current amplitude in Ca2+ that is the
same as or larger than that in Ba2+.
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Methods |
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Cells
Adult bullfrogs (Rana catesbeiana) were chilled to
4°C, brain pithed, decapitated, and spine pithed before their
paravertebral sympathetic ganglia were removed. The method of sacrifice
was approved by the Institution Animal Care and Usage Committee.
Neurons were dissociated with collagenase/dispase digestion and
trituration (Elmslie et al. 1992; Jones
1987
; Kuffler and Sejnowski 1983
). Cells were
maintained for 1-14 days at 4°C in L-15 medium, which was
supplemented with 10% fetal bovine serum and penicillin/streptomycin.
Electrophysiology
Neurons were voltage-clamped in the whole-cell configuration.
Series resistances ranging from 0.3 to 1.5 M were compensated at
80%. Currents were recorded using an Axopatch 200A amplifier (Axon
Instruments, Foster City, CA). Experiments were controlled with 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 hard disk. Leak current was subtracted using a P/4 protocol.
Voltage steps were 10 ms in length unless otherwise noted. Step and
tail currents were sampled at 50 kHz and were typically filtered at 5 kHz. In the ramp voltage protocol, the membrane was depolarized from
80 to +80 mV during 160 ms. Ramp currents were sampled at 5 kHz and
filtered at 1 kHz. Ramp I-Vs were produced by
plotting ramp current against ramp voltage after subtracting leak
current, which was estimated by fitting the current between
80 and
60 mV to linear function. All recordings were carried out at 25°C.
Solutions
To isolate calcium currents, Na+ and K+ were replaced in the internal and external solutions with the impermeant cation N-methyl-D-glucamine (NMG+). The internal solution contained (in mM) 51.6 NMG-Cl, 6.0 MgCl2, 14 creatine-PO4, 2.5 NMG-HEPES, 5 Tris2-ATP, 20 NMG-bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), and 0.3 Li2-guanosine 5'-triphosphate (GTP). External solutions contained either Ba2+ or Ca2+ at 3 or 100 mM as the charge carrier. Other components in external solutions included (in mM) 10 NMG-HEPES and 10 tetraethylammonium chloride (TEA-Cl). Finally, NMG-Cl was added to maintain iso-osmolarity between internal and external solutions. The osmolarity of the internal solution was within the range of 260 to 290 mOs and that of the extenal solutions was from 260 to 320 mOs. All solutions were titrated to pH 7.2 with NMG base.
Data analysis
Data were analyzed using Igor Pro (WaveMetrics, Lake Oswego, OR)
running on a Quadra 630 Macintosh computer. Step current was measured
as the average of 10 points on the current trace at the end of the
voltage step. Fractional block = 1 (ICa during block/ICa control). For the
Ni2+ block experiments, rundown was corrected by
averaging control before and after each Ni2+
concentration was applied, i.e., ICa
control = (ICa before + ICa after)/2. Half-maximum block
(IC50) was estimated from a least-square regression fit of the data points according to the mass action equation
B/Bmax = 1/(1 + Kd/[Ni2+]o).
Group data were calculated as mean ± SD throughout the study.
Chemicals
CGVIA and
-conotoxin MVIIC (
CMVIIC) were obtained from
Bachem Bioscience (King of Prussia, PA). (±) Bay K 8644 and nimodipine were obtained from Research Biochemicals (Woburn, MA).
Li2-GTP was obtained from Boehringer Mannheim
(Indianapolis, IN). All other chemicals were obtained from Sigma (St.
Louis, MO).
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RESULTS |
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Whole-cell current in Ba2+ versus Ca2+
Previous reports suggest that, in frog sympathetic neurons,
current resistant to CGVIA and DHPs results from at least two types
of current, Ef and N-like. The former was
observed in isotonic Ba2+ but not in 2 mM
Ba2+ whereas the latter was the only detectable
CGVIA- and DHPs-resistant current in 2 mM Ba2+
(Elmslie et al. 1992
, 1994
). If E-type current
contributes to resistant current in low Ca2+ and
Ba2+, we postulated that the current would be
more prominent with Ca2+ as the charge carrier.
Because Ef-current was detected in high but not in low Ba2+, we first compared ramp
currents in 100 mM Ba2+ with those in 100 mM
Ca2+. Ef-current was
observed as a prominent "shoulder" on I-V in 100 mM Ba2+ when compared with 3 mM
Ba2+ (Fig.
1A). The difference in the
shape of the ramp I-V was highlighted after the peak
currents were normalized to unity and the I-V was shifted
along the voltage axis to superimpose at the peaks (Fig. 1B). These shifts were required to compensate for the
surface charge effects of different concentrations and/or types of
divalent cations (Elmslie et al. 1994
; Zhou and
Jones 1995
). In 100 mM Ca2+, a
shoulder on the I-V was also observed that was
even more pronounced than that in Ba2+. At 30 mV
negative to the peak, the relative amplitude of the shoulder was
25 ± 10% of peak current in Ca2+, which
was significantly larger than the 14 ± 7% of peak in
Ba2+ (n = 5, P < 0.01 from one-tail paired t-test). This is consistent with
the idea that the relative permeation ratio of
Ca2+ to Ba2+ is higher in
Ef-channels than in N- and L-channels.
Interestingly, when the current in 3 mM Ba2+ was
compared with that in 3 mM Ca2+, there was also a
larger shoulder component in Ca2+ (Fig. 1,
C and D). Subsequent experiments were designed to
determine the identity of the current generating the shoulder in low
Ca2+.
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Fraction of resistant current in 3 mM Ba2+ versus in 3 mM Ca2+
If Ef-current underlies the larger
shoulder in 3 mM Ca2+, a larger fraction of the
whole-cell current should be resistant to CGVIA and DHPs in
Ca2+ than in Ba2+.
Contribution from each current component was evaluated
pharmacologically with 3 µM each of
CGVIA, nimodipine, and
CMVIIC sequentially applied to the external solutions containing
either 3 mM Ba2+ or 3 mM
Ca2+ (Fig. 2).
CMVIIC was used to test if the N-like current was caused by
P/Q-current. The amplitude of peak current decreased with time after
blockers were applied in Ba2+ (Fig.
2A) and in Ca2+ (Fig. 2D).
In 3 mM Ba2+, 85 ± 3, 10 ± 2, and
2 ± 1% of total current at peak voltage (0 mV) was blocked by
CGVIA, nimodipine, and
CMVIIC, respectively; the remaining 3 ± 1% was resistant to all three blockers (n = 7). In
3 mM Ca2+, the percentages of peak current (10 mV) sensitive to
CGVIA, nimodipine, and
CMVIIC were 70 ± 6, 14 ± 4, and 5 ± 2%, respectively. On average, 12 ± 5% persisted in the presence of all blockers (n = 7). Hereafter, we refer to the current that is resistant to N-, L-
and P/Q-channel blockers as resistant current.
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The increased effect of CMVIIC in Ca2+
relative to Ba2+ was surprising. However, we
noticed that the
CGVIA block in Ca2+ was
slower than that in Ba2+ (Fig. 2). Thus it seemed
likely that the increased
CMVIIC block resulted from residual
N-current. To test this idea, we examined the effect of
CMVIIC after
~9 min application of 3 µM
CGVIA (range 8-10 min). Under this
condition, the
CGVIA block was complete and
CMVIIC had no
additional effect (1 ± 1%, n = 7, not shown). Thus the block by
CMVIIC in Ca2+ (Fig.
3) appears to result from an incomplete
block of N-current by
CGVIA. Therefore our estimation of each
component in 3 mM Ca2+ is 75% N-current
(
CGVIA +
CMVIIC sensitive components), l4% L-current, and 12%
resistant current.
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Comparing each component in Ba2+ with that in
Ca2+ (Fig. 3) showed that the largest changes
were in the reduced fraction of CGVIA-sensitive current (N-current)
and the increased fraction of resistant current. The fraction of
resistant current increased at voltages more hyperpolarized to the peak
both in Ca2+ and Ba2+, but
the percentage of resistant current in Ca2+ was
significantly higher (P < 0.05) than that in
Ba2+. Although the percentage of L-current also
increased with hyperpolarization both in Ca2+ and
Ba2+, there was no significant difference
(P > 0.05) in its percentage at 30 mV hyperpolarized
to the peak when external solution was switched from
Ca2+ to Ba2+. These
findings provide quantitative support for the hypothesis that the more
prominent shoulder on the I-V in 3 mM
Ca2+ results from resistant current. The
hyperpolarized activation voltage of resistant current is consistent
with a substantial contribution from
Ef-current.
Amplitude of resistant current in 3 mM Ba2+ versus 3 mM Ca2+
Because of the slow washout of peptide toxins, the comparison of
component currents (Figs. 2 and 3) was carried out on data collected
from two groups of cells, one group in Ba2+ and
the other group in Ca2+. To determine the
relative permeability of Ca2+ versus
Ba2+ through channels underlying resistant
current, we further examined the effect of switching between
Ba2+ and Ca2+ on the
amplitude of resistant current in the same cell. With 3 µM each of
CGVIA, nimodipine, and
CMVIIC in the external solutions, I-V of resistant current in 3 mM Ba2+
was compared with that in 3 mM Ca2+ (Fig.
4). Peak current occurred at more
hyperpolarized voltages in 3 mM Ba2+ than it did
in 3 mM Ca2+, as expected from more effective
screening of surface charge by Ca2+. The
amplitudes of the peak current in Ca2+ were
consistently larger than those in Ba2+ regardless
of the order in which Ca2+ and
Ba2+ were applied. On average, the ratio of peak
current in 3 mM Ca2+ to that in 3 mM
Ba2+ was 1.14 ± 0.07 (n = 5). Because
1E recombinant channels carry larger Ca2+ current than
Ba2+ current (Bourinet et al.
1996
), the results support the idea that the activity of
Ef-channels contributes to resistant current.
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Ni2+ sensitivity of N-, L-, and resistant current in 3 mM Ca2+
The data presented so far are consistent with the hypothesis that
the larger shoulder in 3 mM Ca2+ results from
Ef-current. Another characteristic of
Ef-current in high
Ba2+ was its higher sensitivity to block by
Ni2+ (Elmslie et al. 1994).
Therefore the Ni2+ sensitivity of resistant
current in 3 mM Ca2+ was examined and compared
with that of N- and L-current. Estimation of the
IC50 of Ni2+ for blocking
N-current was carried out in the presence of 3 µM nimodipine to block
L-current. By eliminating L-current, the Ni2+
sensitivity of the remaining current would be weighed toward that of
N-current. In the example cell (Fig.
5A), step current to +10 mV
decreased with increasing
[Ni2+]o and the
fractional block was best fit with a single-site mass action equation.
The IC50 of Ni2+ was
calculated from the fit to be 245 µM in the cell illustrated (Fig.
5C). On average, Ni2+ blocked
N-current with IC50 of 322 ± 73 µM
(n = 7). Our experiment probably underestimated the
value of IC50 of Ni2+ for
blocking N-current because 1) in the presence of 3 µM
nimodipine, whole-cell current is the mixture of N-current and
resistant current and the latter will be shown to be more sensitive to
Ni2+ block and 2) we had evidence
that, at concentrations >1 mM, Ni2+
right-shifted the N-channel I-V because the brief
outward current on stepping to +10 mV was decreased. The outward
current is thought to result from the movement of gating particles
(Jones and Marks 1989
) and its reduction
suggests that Ni2+ is screening surface charge,
leading to a shift in channel activation to more depolarized voltages.
Therefore the fractional block may be overestimated when
[Ni2+]o >1 mM because
the decrease in current size was the cumulative effect of the block and
a shift in the I-V caused by Ni2+.
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The Ni2+ sensitivity of L-current was determined
by measuring Ni2+ block of the slow tail current
induced by 1 µM Bay K 8644 (Fig. 6).
Under these conditions, tail current deactivation at 40 mV could be
fitted with the sum of two exponential functions, yielding
fast
(
f = 0.3 ms) and
slow
(
s = 2.3 ms). The fast deactivating current
resulted from non-L-current because
f was
similar to the deactivation time constant at the same tail voltage in
the presence of nimodipine (Fig. 6, inset). The slow
deactivating component has been shown to result exclusively from
L-current (Elmslie et al. 1992
; Plummer et al.
1989
). L-current amplitude was measured at 4 ms after
repolarization, when the deactivation of the fast component was
complete, and plotted against
[Ni2+]o (Fig.
6C). Ni2+ block was best fit with a
single-site mass action equation yielding an IC50
of 261 µM in the example cell and 244 ± 39 µM in all cells examined (n = 6).
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Ni2+ sensitivity of resistant current was
determined after other currents were blocked with 3 µM each of
CGVIA, nimodipine, and
CMVIIC (Fig.
7). IC50 of
Ni2+ block was 76 µM according to a single-site
mass action equation in the example cell and 83 ± 21 µM for the
group (n = 10). Given the possibility that
Ef- and N-like current both contribute to resistant current, we tried to further dissect resistant current based
on Ni2+ sensitivity. To this end, fractional
block of resistant current was also fitted with the sum of two mass
action equations, which gave a slightly closer fit than assuming a
single site. From two-site fit, 79% of the current was blocked by
Ni2+ with IC50 of 49 µM
and 21% of the current was blocked with IC50 of
491 µM for the example cell. In all cells tested, the
IC50 of Ni2+ at the high
affinity site was 39 ± 9 µM and that at the low affinity site
was 436 ± 109 µM (n = 10). In Fig.
8, fractional block versus [Ni2+]o relationship was
compared among N-, L-, and resistant current. Resistant current was
more sensitive to Ni2+ block than either N- or
L-current. The IC50 of Ni2+
for blocking resistant current in Ca2+ is close
to that required for blocking current through expressed
1E channels (IC50 = 25 µM) (Jouvenceau et al. 2000
). The results support the
idea that resistant current in low Ca2+
predominantly derives from Ef-current. The
Ni2+-insensitive component of resistant current,
which was ~20% of resistant current according to the two-site fit,
could arise from the activity of the N-like channels.
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Ni2+-sensitive component of whole-cell current in 3 mM Ca2+
Finally, we examined the effect of 30 µM Ni2+ on whole-cell calcium current to test our initial hypothesis that the larger shoulder on the I-V in Ca2+ (Fig. 1D) resulted from Ef-current. Based on the IC50 for Ni2+ blocking different current components, 30 µM Ni2+ should block 40% of the Ni2+-sensitive component in resistant current and 10% of N- and L-current. Thus this concentration of Ni2+ was expected to suppress the shoulder but have less effect on the peak current. In the presence of 30 µM Ni2+ (Fig. 9), the shoulder in 3 mM Ca2+ was substantially decreased. On average, 30 µM Ni2+ blocked a significantly larger fraction of the total current at the shoulder (measured at 30 mV hyperpolarized to the peak) than at the peak (28 ± 9% vs. 10 ± 3%, n = 11, P < 0.01, paired t-test). The results support the hypothesis that the larger shoulder on the I-V that is observed in low Ca2+ results from Ef-current.
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DISCUSSION |
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Our data show that, in frog sympathetic neurons, a notable
fraction (~10%) of the whole-cell current in physiological
Ca2+ exhibits characteristics distinct from those
of N- and L-current. Several lines of evidence support the
identification of this current as
Ef-current. First, this current shares a
pharmacological profile with 1E recombinant
channels, including resistance to specific blockers of N-, L-, and
P/Q-channels, and sensitivity to Ni2+ block.
Second, the current was larger with Ca2+ as the
charge carrier than it was with Ba2+, which has
been shown to be a salient property of
1E
recombinant channels. Finally, the current activates at voltages
negative to N-current, as has been shown for
Ef-current in high Ba2+
(Elmslie et al. 1994
). As a result of the hyperpolarized
activation voltage, the current forms a shoulder on the I-V
relationship that is highlighted when the I-V in
Ca2+ is superimposed on that in
Ba2+.
Composition of whole-cell current in Ba2+ versus Ca2+
N-, L-, Ef-, and N-like current have
been identified at the whole-cell and single-channel levels in frog
sympathetic neurons (Elmslie 1997; Elmslie et al.
1992
; Jones and Marks 1989
). In low
Ba2+, the percentage of N-current from our data
(85%) is consistent with earlier findings. The development of
N-current block by
CGVIA followed a single exponential function with
a time constant of 20.9 ± 2.8 s (n = 11),
which agrees well with reported data (Boland et al.
1994
). In low Ca2+, the percentage of
N-current was ~75%, which was significantly lower than that in
Ba2+.
CGVIA blocked N-current with a time
constant of 67.3 ± 8.9 s (n = 7). Previous
reports demonstrated the effect of divalent cations on
CGVIA block
(Boland et al. 1994
). The effect has been interpreted
either as a charge screening effect or as divalent competition for
binding (Boland et al. 1994
; McDonough et al. 1996
). Regardless of the mechanism, Ca2+
more strongly slows
CGVIA binding than does
Ba2+.
The fraction of L-current increased slightly when the switch is made
from Ba2+ (10%) to Ca2+
(14%). This increase was unexpected because it has been shown that
Ba2+ current is greater than
Ca2+ current in L- and N-channels
(Bourinet et al. 1996; Hess et al. 1986
;
Wakamori et al. 1998
). One reason may be that L-channels pass Ca2+ better than do N-channels. Within-cell
comparisons showed that peak current (primarily N-current) in 3 mM
Ba2+ is 2.0 ± 0.2 (n = 16)
times greater than that in 3 mM Ca2+.
Single-channel data from cardiac myocytes provide an estimate for
relative unitary L-current amplitude in Ba2+
versus Ca2+ (Hess et al. 1986
).
From the current-concentration relationship, unitary current in 3 mM
Ba2+ is 1.4 to 1.6 times greater than that in 3 mM Ca2+. This suggests that switching from
Ba2+ to Ca2+ may have less
effect on the current size through L-channels than that through
N-channels. Therefore the percentage of L-current in whole-cell current
increased after 3 mM Ba2+ was replaced with 3 mM
Ca2+ whereas that of N-current decreased. The
relative amount of L-current in low Ba2+ was
higher than reported previously (~5%, Elmslie et al.
1992
). The reason for the larger fraction of L-current in our
experiments is not clear. One possible explanation is that the creatine
phosphate added to the internal solution in our experiments slowed the
rate of L-current rundown.
Our experiments suggest that there is no detectable P/Q-current in frog
sympathetic neurons. In 3 mM Ba2+, application of
CMVIIC had a negligible effect on peak current size (2 ± 1%,
n = 7), which could not be differentiated from current rundown. In 3 mM Ca2+, the reduction in peak
current size in response to
CMVIIC was caused by block of residual
N-current because prolonged
CGVIA application completely eliminated
CMVIIC effect.
Although Ef-current is more sensitive to Ni2+ block than are the other current components in frog sympathetic neurons, there is no Ni2+ concentration that would selectively block only Ef-current in low Ca2+. 30 µM Ni2+ was used to examine the potential contribution of Ef-current to whole-cell current in 3 mM Ca2+ because, at this concentration, Ni2+ should produce a substantial block of the Ef-current (~40%) with minimum block (~10%) of N- and L-current. The observation that 30 µM Ni2+ reduced the shoulder in low Ca2+ (Fig. 9) supports the idea that Ef-current is a major component of whole-cell current at voltages hyperpolarized to the peak.
Molecular basis of resistant current
In rat cerebellar granule cells, resistant current could also be
dissected into Ni2+-sensitive and -insensitive
components (Tottene et al. 1996, 2000
). Single-channel
conductance of the Ni2+-sensitive component
matches that of Ef-channels in frog
neurons whereas that of the Ni2+-insensitive
component is larger than that of
Ef-channels. Interestingly, both
Ni2+-sensitive and -insensitive components were
diminished after the neurons were injected with
1E antisense mRNA, which suggests that they
are coded by the same gene (Tottene et al. 2000
).
Other results support the idea that multiple genes can give rise to
resistant currents. In one case, expressed 1D
channels in HEK 293 cells were shown to be incompletely blocked by DHPs but sensitive to Ni2+ block (Xu and
Lipscombe 2000
). In a second example, substantial resistant
current was observed in neurons isolated from
1E knockout mice. The identity of this channel
was speculated to be
1A (Wilson et al.
2000
). These results demonstrated that the type of channel generating resistant currents cannot be determined by pharmacology alone. We used pharmacology to detect and isolate the resistant current
in frog sympathetic neurons, but it was the permeation properties of
the current that helped us to further identify the likely gene.
The molecular basis of resistant current in frog sympathetic neurons
has not been examined in our experiments, but our data are consistent
with the notion that 1E gene product gives
rise to the Ni2+-sensitive component of resistant
current, which we have tentatively identified as
Ef-current. The
Ni2+-insensitive component of resistant current
could arise from a distinct gene. Alternatively, it is possible that,
despite their difference in Ni2+ sensitivity,
both components of resistant current derive from the activity of
channels coded by the E-class gene, mirroring findings in rat
cerebellar granule cells.
Functional significance of Ef-channels
Single-channel experiments in 100 mM Ba2+
indicated that the density of Ef-channels
is as high as that of N-channels in frog sympathetic neurons but, in 3 mM Ca2+, Ef-current
makes up only ~10% of the total current at peak. This inconsistency
may be attributed to three major differences in
Ef- and N-channels. First,
Ef-channels tend to gate in a low Po mode with brief open times (Elmslie
1997) whereas N-channels normally gate in a mode characterized
by high Po (0.8-0.9) and long open times
(Lee and Elmslie 1999
). Second, single-channel conductance as well as unitary current are smaller for
Ef-channels than they are for N-channels
(Elmslie 1997
). Last, steady-state inactivation of
Ef-channels at resting membrane potential
(
80 mV) may result in fewer channels being available for opening on depolarization, as compared with N-channels (Elmslie
1997
; Elmslie et al. 1994
).
Although Ef-current accounts for only ~10% of whole-cell current at peak, the fraction of the current increases at hyperpolarized voltages. This negative activation voltage suggests that Ef-channels can open with moderate membrane depolarization, such as that induced by large excitatory postsynaptic potentials. Given the density of Ef-channels, influx of Ca2+ through these channels is likely to have widespread effects by increasing local [Ca2+]i and initiating cellular processes that are highly sensitive to Ca2+, such as the opening of Ca2+-activated channels and the assembly of machinery that leads to neurotransmitter release. Therefore the unique physiological role of Ef-channels may lie in the fine-tuning of neuronal firing patterns and neurotransmitter release.
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ACKNOWLEDGMENTS |
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We thank Drs. Geoffrey G. Schofield and Norman R. Kreisman for valuable comments on the manuscript.
This study was supported by National Institute of Neurological Disorders and Stroke Grant NS-33671.
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FOOTNOTES |
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Address for reprint requests: K. S. Elmslie, Dept. of Physiology SL-39, Tulane University Health Science Center, 1430 Tulane Ave., New Orleans, LA 70112 (E-mail: kelmslie{at}tulane.edu).
Received 21 November 2000; accepted in final form 10 May 2001.
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