Department of Neurobiology and Behavior, State University of New York at Stony Brook, Stony Brook, New York 11794
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Li, Weiyan, Christopher Thaler, and Paul Brehm. Calcium Channels in Xenopus Spinal Neurons Differ in Somas and Presynaptic Terminals. J. Neurophysiol. 86: 269-279, 2001. Calcium channels play dual roles in cell signaling by promoting membrane depolarization and allowing entry of calcium ions. Patch-clamp recordings of calcium and calcium-dependent currents from the soma of Xenopus spinal neurons indicate key functional differences from those of presynaptic terminals. Both terminals and somas exhibit prominent high-voltage-activated (HVA) calcium current, but only the soma expresses additional low-voltage-activated (LVA) T-type current. Further differences are reflected in the HVA current; N- and R-type channels are predominant in the soma while the terminal calcium current is composed principally of N type with smaller contribution by L- and R-type channels. Potential physiological significance for these different distributions of channel types may lie in the differential channel kinetics. Activation of somatic HVA calcium current occurs more slowly than HVA currents in terminals. Additionally, somatic LVA calcium current activates and deactivates much more slowly than any HVA calcium current. Fast-activating and -deactivating calcium current may be critical to processing the rapid exocytotic response in terminals, whereas slow LVA and HVA calcium currents may play a central role in shaping the somatic firing pattern. In support of different kinetic behavior between these two compartments, we find that somatic calcium current activates a prominent slow chloride current not observed in terminal recordings. This current activates in response to calcium entering through either LVA or HVA channels and likely functions as a modulator of excitability or synaptic input. The restriction of this channel type to the soma lends further support to the idea that differential expression of fast and slow channel types in these neurons is dictated by differences in signaling requirements for somatic and terminal compartments.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Individual classes of neurons
can often be distinguished on the basis of expression of distinct
receptor and voltage-activated ion channel types. In each case, it is
likely that the overall composition of channel types is dictated by the
specific functional requirements of each cell type. Furthermore this
principle appears to apply to the functionally differentiated
compartments within the same neuron. In motorneurons for example, the
functional requirements are thought to match the specific needs of each
cellular compartment, differing in dendritic, somatic, axonal, or
terminal regions (Mallart and Brigant 1982; Yuste
and Tank 1996
). The somatic compartment plays a central role in
integrating synaptic inputs while the terminal compartment is
principally responsible for release of transmitter in response to
individual spikes. Of particular interest is the distribution of
calcium channel isoforms within the motorneurons because these channels
are the central players in both of these processes (Barish
1991
; Katz and Miledi 1967
). In the soma,
calcium channels participate in shaping the depolarization as well as providing calcium signals for second-messenger signaling and gene activation. By contrast, calcium-triggered release of transmitter appears to be the principal responsibility of calcium channels at
terminals (Catterall 1998
). Functional segregation of
calcium channel isoforms within cells was suggested by several studies indicating a differential distribution of channel subtypes between soma
and dendrites (Christie et al. 1995
; Mouginot et
al. 1997
; Westenbroek et al. 1990
). However,
electrophysiological comparisons of somatic and terminal calcium
currents are difficult due to the small size of the terminals. In fact,
direct electrophysiological recordings of presynaptic calcium currents
have been made in only a handful of cell types (Borst et al.
1995
; Llinas et al. 1981
; Stanley and
Goping 1991
; Yawo and Momiyama 1993
).
Recent studies have shown that it is possible to voltage clamp nerve
terminals of Xenopus spinal neurons (Thaler et al.
2001; Yazejian et al. 1997
), permitting direct
comparison of somatic and terminal calcium channels. The results of
such comparisons indicate that large differences in calcium channel
isoforms exist between terminal and soma. Additionally, those isoforms
that are shared by the two compartments exhibit differences in
activation kinetics. We also identify a pronounced slow
calcium-activated chloride current that is restricted to the soma. Our
findings suggest that functional distinctions, principally involving
differences in kinetics, dictate the types of channels expressed in
somas and terminals.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture
Xenopus nerve and muscle co-cultures were prepared
using the methods as previously described (Thaler et al.
2001). Briefly, myotomal muscle and spinal cord were removed
from stage 20 to 22 Xenopus laevis embryos (Nieuwkoop
and Faber 1956
) and dissociated in Ca2+-
and Mg2+-free solution. The cell suspension was
placed on basement membrane matrix (Collaborative Biomedical
Products)-coated glass coverslips in modified 60% Leibovitz's L-15
medium with the addition of 10 mM Na-HEPES, 0.5% horse serum (Life
Technologies, Gaithersburg, MD), 100 U/ml penicillin/streptomycin, 20 nM Neurotrophic Factor-3 (Regeneron, Tarrytown, NJ), and 1 µM
testosterone propionate (Sigma, St. Louis, MO). Cultures were kept in a
dark environment at room temperature.
Cells were visualized with a Zeiss 63× power LD Phase Achroplan objective and identified primarily on the basis of morphology. Muscles were easily distinguished by their prominent striations. Spinal neurons were rounded cells with diameters of 10-15 µm. The neurons projected long processes that occasionally exhibited swellings at locations where they contacted muscle cells. For the aim of side-by-side comparisons, most of the somatic and terminal recordings were performed during the third and the fourth day after plating when the accessibility to both the soma and terminal were optimal.
Electrophysiology
All voltage-clamp recordings of calcium current utilized the
perforated-patch method (Horn and Marty 1988). Electrode
tip (~2 µm OD after polished) was dipped in recording solution for 10 s. The electrode was then backfilled with a recording solution containing 200 µg/ml Amphotericin B and used immediately. Typically within 10 min after the gigaseal formation, the series resistance dropped below 20 M
, which was deemed acceptable for voltage-clamp recordings. The series resistance was routinely compensated by 50%
with a 10-µs lag time.
Patch electrodes were made from borosilicate glass (Garner glass type:
7052) with Sutter Instrument P-97 micropipette puller and fire-polished
before use to a pipette resistance of ~5 M. The internal solution
used to fill the electrode contained (in mM) 52 CsCH3SO4, 38 CsCl, 1 EGTA,
5 HEPES, and 50 glucose taken to a pH of 7.2 with CsOH.
To reduce the space-clamp artifacts resulting from the presence of
prominent neurites, we used the puff-suck system to restrict the region
of activation of calcium current (Thaler et al. 2001). As a variation of the method first introduced by Katz and Miledi (1967)
, we used a low-Ca2+ bath solution
containing (in mM) 80 TEACl, 10 NaCl, 2 KCl, 5 MgCl2, 5 HEPES, 0.4 CaCl2,
3 glucose, 1 MnCl2, and 1 µM tetrodotoxin (Alomone Labs) taken to a pH of 7.2 with
N-methyl-D-glucamine. A puffer pipette was used
to deliver a 5 mM calcium bath solution (without
Mg2+ and Mn2+) to the cell
body or terminal under recording. A second sucking pipette was used to
actively remove the high-calcium solution to avoid accumulation in the
bath (for recording configuration see Fig. 1 in Thaler et al.
2001
). By careful manipulations of the position and pressure of
both puffing and sucking pipettes, a steady laminar flow of
"high"-calcium solution could be created covering most part of the
soma or terminal while avoiding the neurites. Calcium channels outside
of the laminar flow were blocked by the manganese in the bath while
those channels in the path of the laminar flow were relieved from
block. Moving the calcium puffer a few microns off either the soma or
the terminal led to a complete loss of calcium current (Thaler
et al. 2001
). With the combination of aforementioned pipette
solution and puffing solution, voltage errors due to liquid junction
potentials corresponded to 10 mV on the bases of estimated values
(Barry and Lynch 1991
) and direct measurements
(Neher 1992
) and all data were corrected for this error.
Drug delivery was achieved via a quartz filament placed within 2 mm of
the tip of the puffing pipette. Full exchange of puffing solution could
be completed within 2 min via injection of solution containing drugs.
All drugs including -Ctx GVIA (Alomone Labs),
-Ctx MVIIC
(Peptides International),
-Aga IVA (Peptides International) nitrendipine (Biomol), and niflumic acid were diluted from a stock solution to the working concentration just prior to use to ensure viability. Dihydropyridines were kept as stock solutions in DMSO.
Calcium currents were recorded using an EPC-9/2 dual patch-clamp amplifier (List Electronics, Darmstadt, Germany) and sampled at 10-µs intervals. Data were acquired and analyzed using HEKA Pulse + PulseFit software. Activation and inactivation time constants were measured by Hodgkin and Huxley fitting of calcium current traces. Prior to analysis the currents were leak-corrected by use of a P/10 protocol and refiltered with a 4-pole Bessel filter at 2 kHz. Statistical analysis, plotting and curve fitting were performed through the use of IGOR pro software. All statistical values represent means ± SD unless specified otherwise.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In dissociated cell cultures of Xenopus spinal neuron
and muscle, both the somas and presynaptic terminals of neurons were accessible for voltage-clamp analysis. Dissociated spinal neurons represented a mixture of neuronal types, but we restricted our recordings to cholinergic neurons capable of eliciting muscle contractions when stimulated. The somatic diameter averaged 15 µm,
whereas the terminals were usually <1 µm in diameter. However, occasional swellings in the terminals were observed in regions where
the neurites contacted muscle. These regions were as large as 4 µm in
diameter permitting whole cell voltage-clamp recording. These swellings
were proved to be functional presynaptic terminals in previous study
where direct stimulation produced excitatory postsynaptic currents
(Thaler et al. 2001). Both terminal and somatic calcium
currents were difficult to properly voltage clamp due to the large size
of the calcium current and the presence of associated neuritic
processes. These technical problems were ameliorated by focal
application of calcium to the recording site. This technique restricts
the physical boundaries of channel activation to a certain region under
voltage clamp. For this purpose, a puffing pipette containing 5 mM
calcium was repositioned until optimal recording of inward calcium
current was achieved. The applied calcium was restricted to a local
region of the cell through active removal by means of a second sucking
pipette. Once the puffing and sucking pipettes were optimally
positioned, they were not moved during the course of the experiment.
Low-voltage-activated calcium current
All 232 somatic recordings revealed the presence of
voltage-activated inward current. Removal of the calcium puffing
pipette just prior to the termination of the experiment completely
abolished the inward current, indicating that it was carried by
calcium. In 79% of those somas tested, two components of inward
current could be resolved on the basis of the differences in threshold and kinetics (Fig. 1A).
Activation of a low-voltage-activated (LVA) calcium current occurred at
an average potential corresponding to 63 ± 6 mV and was fully
activated at
33 ± 7 mV (n = 183). A second
high-voltage-activated (HVA) calcium current activated at
23 ± 7 mV, and this current peaked near 0 ± 6 mV (n = 232). These two currents were evident in the raw traces and in the
current-voltage relations for peak current (Fig. 1A).
Frequently, the HVA calcium current exceeded 3 nA, rendering adequate
control over the voltage impossible and these data were discarded. All
of the 49 terminal recordings were of HVA type; no evidence of any LVA
current was obtained in any terminals tested (Fig. 1B).
|
Inspection of the raw current records indicates that the LVA current
inactivated more rapidly than the HVA calcium current (Fig.
1A). In all somatic recordings, kinetic analysis of LVA calcium current was complicated by the activation of HVA current at
potentials above 23 mV. Addition of 1 µM
-conotoxin (Ctx) GVIA
irreversibly blocked much of the HVA current, leaving the LVA component
untouched (Fig. 2). In most cells an
incomplete block of HVA calcium current was observed, but in two cells,
1 µM
-Ctx GVIA blocked 100% of the HVA component, permitting
analysis of the pure LVA calcium current (Fig. 2). The activation and
inactivation kinetics of LVA currents were both voltage dependent. The
time constants of activation ranged from 8 ± 4 ms near threshold
(
60 mV) to 3 ± 1 ms at potentials corresponding to maximal
activation (Fig. 3, inset A;
n = 10). Inactivation time constants were strongly voltage dependent, averaging 23 ± 6 ms at
60 mV and 12 ± 2 ms at
30 mV (Fig. 3, inset B; n = 10).
Deactivation time constants at
90 mV were measured by exponential
fitting of the tail currents associated with repolarizations from brief
steps to
30 mV, prior to the inactivation of the current. The tails
were well fit with a single exponential curve, yielding time constants
of 2.5 ± 0.5 ms (n = 10).
|
|
Inactivation of LVA calcium current was determined by use of 500-ms
conditioning depolarizations prior to application of the test pulse.
The midpoint of steady state availability was 67 mV (Fig.
4A), a potential that does not
inactivate HVA calcium current. Comparisons of steady-state
availability and normalized I-V relations from the same cell
indicate a small region of overlap near threshold of the LVA current
(Fig. 4A). At potentials corresponding to this region of
overlap, persistent inward LVA calcium currents can be observed (Fig.
3). In cells with both LVA and HVA calcium current, it was possible to
isolate the HVA component by means of long depolarizations to
50 mV,
which inactivated the LVA current (Fig. 4B) as predicted by
the steady-state inactivation curve in Fig. 4A. The
resultant HVA current showed a reduction in inactivation reflecting the
loss of the inactivating LVA component. However, the HVA current still
showed modest inactivation (Fig. 4B, inset).
|
HVA calcium current
An incomplete block of somatic HVA current by -Ctx GVIA in the
majority of cells tested suggested the existence of additional calcium
channel isoforms (Fig. 5). Overall, 1 µM
-Ctx GVIA blocked an average 62 ± 16% of HVA current
(n = 25). Two lines of evidence indicate that the
residual HVA current represents the presence of a
-Ctx
GVIA-insensitive calcium current rather than a sub-saturating block by
the toxin. First, the time course of inhibition of the late
steady-state inward current by 1 µM
-Ctx GVIA indicated that
maximal block was achieved within 2 min (Fig. 5A), and this block was irreversible (data not shown). Because this toxin blocks irreversibly over the time course of our experiment, the presence of a
plateau block in Fig. 5A reflects a toxin-resistant
component. As a second line of evidence, 6 µM
-Ctx GVIA was tested
on 13 cells, resulting in an average block of 66 ± 15% of the
HVA current, a value not significantly different from the effects of 1 µM
-Ctx GVIA (Student's t-test, P > 0.05).
|
Similar measurements from terminal calcium currents indicate that
-Ctx GVIA blocks dihydropyridine (DHP)-sensitive channels in
addition to N-type channels (Thaler et al. 2001
). To
further determine that the
-Ctx GVIA sensitivity reflects N- and not L-type current in the soma, we tested the effects of another known N-type calcium channel antagonist,
-Ctx MVIIC. This toxin also blocks P/Q type channels so it was necessary to first test for the
presence of these latter channel types in the soma. For this purpose
-agatoxin (Aga) IVA, a specific blocker of P/Q-type channel, was
applied at a concentration of 500 nM. In five cells tested,
-Aga IVA
exerted no effects on calcium current (Fig.
6), consistent with the negative findings
on terminal calcium current. Therefore any effects of
-Ctx MVIIC
were considered to be mediated through block of N-type calcium current.
In 12 cells tested, 10 µM
-Ctx MVIIC inhibited 51 ± 25% of
the HVA calcium current (Fig. 7). Previously published dose dependence of inhibition of calcium current
by
-Ctx MVIIC in Xenopus spinal neurons showed that this concentration produces a steady-state block of 80% of the
-Ctx MVIIC-sensitive current (Thaler et al. 2001
). Therefore
following correction, the estimated block by
-Ctx MVIIC corresponded
to 64%, which was not significantly different from the average block by 1 µM
-Ctx GVIA. Taken together, these data indicate that
~60-65% of the somatic current is N-type and the balance of current
is resistant to both toxins.
|
|
Curiously, the proportion of toxin-resistant current appears to vary
widely among somatic recordings. In 8 of 50 cells tested with either
-Ctx GVIA or
-Ctx MVIIC, more than 90% (95 ± 4%) of the
HVA current was inhibited indicating almost complete contribution by
N-type channels. However, in the other 42 cells tested with either
toxin, an average 57 ± 14% of HVA current was inhibited, signaling a considerable contribution by calcium channels other than
N-type (Fig. 8A). We
considered that the balance of channel types in these cells might be
DHP-sensitive due to the existence of this channel type in terminals.
To test for the possible contribution by L-type channels, we applied
nitrendipine to the soma. In the 15 cells tested, including the one
shown in Fig. 8B, application of 1 µM nitrendipine for
3
min had no significant effect on peak calcium current. To ensure that
nitrendipine was without effect on these neuronal somatic calcium
currents we tested 2 µM (n = 6) and 5 µM
(n = 2) nitrendipine and neither showed evidence of inhibition. As a positive control for nitrendipine inhibition, we
applied 1 µM nitrendipine to GH3 pituitary cells expressing L-type
calcium channels and observed a large inhibition of calcium current
(data not shown). Furthermore, side-by-side recordings showed that this
concentration of nitrendipine blocked ~40% of the calcium current in
the terminals of these neurons (Thaler et al. 2001
).
|
Concern over the observed lack of inhibition by nitrendipine was raised
by previous studies attesting to the presence of verapamil-sensitive currents in the somas of these neurons (Barish 1991).
Additionally, in our recordings of calcium current performed nearly 4 yr before completion of the study, we had observed partial inhibition
of somatic calcium current by 1 µM nitrendipine. However, for reasons that are completely obscure to us, nitrendipine sensitivity was never
observed in any cells tested over the past 2-yr period, despite our
efforts to reproduce the exact conditions of those recordings.
The pharmacological criteria applied to HVA calcium current thus far
point to contributions by both N-type and DHP/toxin-resistant channel
isoforms. Each current component was further analyzed for possible
differences in channel function. The -Ctx-GVIA-sensitive (N-type)
calcium current activated at
21 ± 5 mV and peaked at
2 ± 7 mV (n = 10). The time constant of activation of
N-type current averaged 4.6 ms at
10 mV and decreased at more
depolarized potentials to a stable value of 0.8 ms at 30 mV (Fig.
9C; n = 10).
The time constant of deactivation of N-type current at
90 mV averaged
0.47 ± 0.22 ms (n = 10). DHP/toxin-resistant
(R-type) calcium current activated at
15 ± 6 mV and peaked at
5 ± 7 mV (n = 7). These values obtained for
R-type current showed a slight right shift when compared with N-type
currents, but the differences were not statistically significant
(Student's t-test, P > 0.05). Activation
time constants for R-type current were similar to N-type current,
ranging from 5.0 ms at
10 mV to 0.8 ms at 30 mV (Fig. 9D;
n = 6). Deactivation time constant for R-type calcium
current at
90 mV measured 0.43 ± 0.36 ms (n = 6), again not statistically different from that of N-type current.
|
The small size of terminal calcium current impeded the reasonable
fitting of separated -Ctx-GVIA- and DHP-sensitive currents, especially at lower membrane potentials. Therefore the activation kinetics were determined for overall terminal current to compare with
somatic calcium current. Comparisons of normalized averaged traces
revealed that terminal calcium current activated faster than somatic
N-type current at potentials near threshold (Fig. 9, A and
B). Activation time constants for terminal HVA currents averaged 2.6 ms at
10 mV and decreased at more depolarized potentials to a stable value of 0.5 ms at 30 mV (Fig. 9E;
n = 10). Averaged activation time constants for
terminal calcium current were smaller than those for somatic HVA
currents at all membrane potentials tested (Fig. 9F).
However, due to large variations, the difference was significant only
at lower voltages (i.e.,
10 and
5 mV; Student's t-test,
P < 0.05).
Calcium-activated chloride current
Recordings from somas in the presence of external calcium revealed
a current that was not observed when barium was substituted for
calcium. This pronounced current initially complicated our measurements
of calcium tail currents because of its large size and slow kinetics
(Fig. 10). The current was observed in
40% of cells tested, and it appeared as a slowly decaying tail current following repolarization from potentials positive to 50 mV. At
90
mV, this tail current decayed with two time constants corresponding to
22 ± 8 and 0.4 ± 0.2 ms (n = 10). The
fast-decaying component of the tail currents exhibited time constants
similar to N- and R-type calcium currents. In the spinal neurons
exhibiting the slow-decaying tail currents, larger somas generally
tended to have larger amplitude tail current. Additionally, the tail
current also showed a clear dependence on time in culture. The slow
component of tail current was absent in recordings performed within 2 days after plating. However, 60% of cells tested on day 5 showed slow components of tail current.
|
This tail current was carried by chloride as the estimated reversal
potential for this current (30 ± 8 mV; Fig. 10A;
n = 10) was close to the theoretical equilibrium
potential (
26 mV) for chloride in our recording solutions. Also,
niflumic acid, an inhibitor of chloride channels (White and
Aylwin 1990
), fully blocked the slow tail current at a
concentration of 200 µM while sparing both the calcium current and
the fast decaying component of the tail (Fig. 10B;
n = 4).
Furthermore several lines of evidence identified this chloride tail current as calcium-activated. First, removal of Ca2+ by lifting the puffer eliminated both the inward calcium current and the tail current (Fig. 11A); second, when depolarizing pulses neared ECa (e.g., +60 mV), this tail current also disappeared (Fig. 11B); third, substitution of barium for calcium eliminated the long-lasting tail while sparing the fast component of tail current (Fig. 11C). Finally, the sensitivity to niflumic acid (Fig. 10B) is consistent with previously reported effects on calcium-dependent chloride channels.
|
In cells exhibiting slowly decaying tail currents, the inward current during the test pulse further indicates contribution by calcium-activated chloride current. In Fig. 10B, treatment with 200 µM niflumic acid reduced the apparent inactivation of inward calcium current following depolarization to +10 mV. Similar reduction in inward current decay was observed following substitution of 5 mM barium for extracellular calcium. These actions reflect an inhibition of an outward chloride current during the test pulse that resulted from maintained entry of calcium.
The relationship between the amplitude of calcium current and chloride tail current was determined by comparing the current-voltage relations of these two currents. For this purpose, the peak calcium current and the amplitude of the slow component of tail current were plotted as a function of test potential. Both LVA and HVA calcium currents were capable of activating chloride tail current (Fig. 12A). However, the activation of calcium-activated chloride current also depends on the accumulated calcium entry (Fig. 12B). Test pulses of increasing durations led to cumulative increases in the amplitude of chloride tail current. Comparison of tail current amplitude with the integrated calcium charge entry indicated a nonlinear region for pulses briefer than 5 ms. However, longer pulses resulted in a linear relationship between chloride current and calcium charge entry. (Fig. 12B). Because individual short pulses were seemingly less effective in triggering calcium-dependent chloride currents, we explored the possibility that under physiological conditions most of these channels are recruited only during high-frequency stimulation. Stimulation of cells using a series of high-frequency short-duration pulses resulted in an apparent reduction in inward calcium current, while the size of the slow chloride tail current exhibited cumulative increases in amplitude during the train (Fig. 12C).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Comparisons of somatic and terminal voltage-clamp recordings have
revealed differences in the distribution of functionally distinct
calcium channels. The most obvious difference is reflected in the
contribution by LVA calcium current (Barish 1991;
Yazejian et al. 1997
). Approximately 80% of the somas
tested exhibited prominent LVA current, whereas no terminals tested
showed any sign of LVA calcium current. Analysis of the voltage
dependence of activation and the kinetics both indicate potential
reasons for the exclusion of functional LVA calcium channels from
terminals. First, the low-threshold opening by these calcium channels
results in the potential for activation near resting membrane levels
(Gu and Spitzer 1993
). Such openings, even by single
channels, would likely result in unwanted transmitter release
(Stanley 1993
). Additionally, the LVA channels activate
three times slower and deactivate six times slower than HVA channels,
consistent with previous findings on Xenopus spinal neuron
somas (Barish 1991
; Gu and Spitzer 1993
).
Because the majority of calcium enters during the repolarizing phase of
action potential (Thaler et al. 2001
; Yazejian et
al. 1997
), fast activation and deactivation of current in these
terminals is advantageous. Moreover, fast kinetics provide for
synchronized entry of calcium by ensuring that conductance is maximal
at the time of calcium entry and that entry is terminated quickly on
repolarization (Sabatini and Regehr 1999
). At
half-amplitude, the duration of the presynaptic spike measures 1.4 ± 0.2 ms (Thaler et al. 2001
), which is far too short
in duration for full activation of LVA current. Also the slow
deactivation or LVA calcium current would be expected to prolong
postrepolarization entry of calcium.
One potential solution to the problems posed by LVA calcium current in
terminals would be to uncouple the calcium entry associated with LVA
current from the exocytotic process. While slow deactivation of LVA
current would still slow repolarization, the calcium entering would not
result in persistent transmitter release. In fact, some calcium channel
types are thought to couple more effectively to transmitter release
machinery (Butz et al. 1998; Leveque et al. 1994
; Sheng et al. 1994
). However, it appears
that all three types of calcium channels (N, L, and R type) present in
spinal motor nerve terminals couple to exocytosis (Thaler et al.
2001
). On the basis of these findings, it seems unlikely that
terminal LVA current could be present and fail to participate in
transmitter release. Therefore the solution adopted by these neurons
was to differentially target LVA channels to the somatic compartment. In agreement with our findings, LVA calcium channel is not present in
most of the characterized presynaptic terminals studied to date,
such as squid giant synapse (Charlton and Augustine
1990
), the calyx of Held (Doughty et al. 1998
),
the calyx of the chick ciliary ganglion (Stanley and Goping
1991
), and axon terminal of goldfish bipolar cells
(Tachibana et al. 1993
). One exception is in leech heart
interneurons, where LVA currents were shown to support specifically
graded synaptic transmission in which a prolonged subthreshold
neurotransmitter release is evident (Ivanov and Calabrese
2000
; Lu et al. 1997
). This further agrees with our hypothesis that LVA calcium current can couple to release, yet they
are not suited for a synchronized fast synaptic transmission.
The likely importance of fast channel kinetics to transmitter release
is underscored by our finding that HVA current activation is
significantly slower in the soma than in the terminal at membrane potentials near the threshold level. Direct comparisons of calcium current in response to rectangular command pulses indicate consistently faster rise of inward HVA calcium current in terminals at these potentials. The average activation time constants of terminal HVA
calcium current also appear smaller than somatic HVA current at more
positive potentials, yet the differences were not significant due to a
large variability. Although direct comparisons of calcium channel
kinetics between somas and terminals in other preparations are lacking,
similar differences have been suggested by studies on calyx of Held
(Borst and Sakmann 1998). As in our preparation, the
mechanisms underlying the faster activation of calcium channels in
terminals have not been identified. However, the established effects of
auxiliary subunits (Wakamori et al. 1999
) and
G-protein-mediated modulation (Delmas et al. 2000
;
Dolphin 1998
) on calcium current kinetics point to
possible regulatory mechanisms.
In contrast to the terminals, LVA calcium channels have been shown to
exist in the somas of various neurons (Tsien et al. 1991). Based on their low threshold and slow kinetics, the
proposed functional roles of somatic LVA calcium currents in different neurons have included regulation of the threshold for spike generation (Huguenard 1996
), contribution to depolarizing envelope
underlying burst firing (Llinas and Yarom 1981
), and
promotion of intrinsic oscillatory behavior (McCormick and
Huguenard 1992
). In Xenopus spinal neurons, the
function of LVA calcium currents likely lies in their ability to
regulate spike initiation and synaptic integration. For example, in
these neurons, LVA channels were shown to function as the trigger
responsible for the spontaneous elevations of intracellular calcium,
which seems essential for their development (Gu and Spitzer 1993
). In mature neurons, these channels may function to
enhance postsynaptic integration via activation by subthreshold EPSPs and therefore lower the threshold for action potential initiation.
Our study points to contributions by two calcium channel types to HVA
current in the soma. The predominant calcium channel type is N type
based on the sensitivity to both -Ctx GVIA and
-Ctx MVIIC. N-type
current contributes an average 65% of overall current, and the balance
of HVA current is contributed by channels that are insensitive to both
conotoxins. P/Q- and L-type channels are ruled out on the basis of
insensitivity to
-Aga IVA and nitrendipine. Given the unusual
pharmacological profile, we tentatively refer to these channels as
resistant or R type. An appreciable, but much smaller proportion of
R-type current was observed in terminals. However, there is no direct
evidence to suggest that the R-type currents in the soma and terminal
represent the same channel types. R-type was originally defined as
"resistant" to all the known calcium channel antagonists. It has
been generally established via expression studies that
1E subunit
forms the pore for R-type calcium channels (Hilaire et al.
1997
; Piedras-Renteria and Tsien 1998
;
Zhang et al. 1993
). However, recent pharmacological
studies have demonstrated that R-type current is not homogenous across cell types (Newcomb et al. 1998
) and multiple subtypes
of R-type calcium channels may co-exist in the same neuron
(Tottene et al. 2000
; Wilson et al.
2000
).
In the process of identifying differences in calcium current between
somatic and terminal compartments, a prominent slowly deactivating
inward tail current surfaced. This current was deemed to be
calcium-activated based on its dependence on calcium entry for
activation and the absence of activation when barium was substituted for calcium. Furthermore the current was carried by chloride on the
basis of reversal potential measurements and its sensitivity to
niflumic acid, an inhibitor of chloride channels (White and Aylwin 1990). Our results agree with previously published
studies showing that calcium-activated currents in Xenopus
spinal neurons appear over time in culture (Hussy 1991
,
1992
). We now show that these currents do not activate fully in
response to a short-duration pulse corresponding to a single action
potential. A steep nonlinear dependence between stimulus duration and
chloride charge entry exists for pulse lengths between 1 and 4 ms,
whereas a shallow linear dependence of charge entry on pulse duration
exists beyond 5 ms. We interpret the nonlinear region of the
relationship to represent the local concentration dependence of
chloride channel activation. Pulse durations in excess of 5 ms may
result in cumulative calcium concentrations sufficient to fully
activate local chloride channels. The mechanisms causal to the linear
region of this relationship are less clear and may represent diffusion
of calcium and subsequent activation of distal chloride channels in the
cell soma.
In cultured Xenopus embryonic spinal neurons, the chloride
equilibrium potential is approximately 60 mV, close to the resting potential (Bixby and Spitzer 1984
). Therefore the
calcium-activated chloride current was speculated to either promote
repolarization or to modulate excitability and/or synaptic input
(Hussy 1991
). However, our results show that amplitude
of chloride current activated by single short pulses is minimal. It is
unlikely that this current has significant physiological role during
the repolarization phase of the action potential considering the large
size of the normal repolarizing potassium current. Furthermore we
showed that high-frequency stimulation recruited more chloride currents
depending on an accumulative calcium entry. It is therefore reasonable
to hypothesize that these channels may only respond to elevation of
intracellular calcium induced by high-frequency firing, exerting an
inhibitory effect on synaptic integration and spike initiation.
The complete absence of this current in the terminal may reflect the
different functional requirements of this cellular compartment. As
discussed for calcium current, fast activation and deactivation may be
required for synchronous transmitter release. If the calcium-activated chloride current were present in terminals, it would result in persistent activation of an inhibitory current. Such a current could
potentially alter the presynaptic spike waveform thereby further
altering calcium entry. While this might be an attractive mechanism for
presynaptic modulation, it may not be suitable for a follower synapse
such as the neuromuscular junction. This was indirectly supported by
the very fast kinetics of calcium-activated potassium channel
identified in the presynaptic terminal of the same preparation
(Yazejian et al. 2000). The lack of calcium-activated chloride current in terminals may reflect differential targeting to the
somatic compartment. Alternatively, the chloride channels may be
present in terminals but fail to activate due to the strong calcium
buffering shown to occur in presynaptic terminals. In either case, it
is clear that this current plays a specific role in firing of somatic spikes.
Overall, our data have indicated that the composition and possibly modulation of calcium channels are tailored to serve the specific functions for different subcellular compartments. Between the cell body and terminal of Xenopus spinal neurons, the issue of speed seems to be the dominating principle such that slow kinetics supports synaptic input integration in the soma while fast kinetics provides synchronized release of neurotransmitter at presynaptic terminals.
![]() |
ACKNOWLEDGMENTS |
---|
The authors thank Regeneron for the generous donation of NT-3.
This research was supported by National Institute of Neurological Disorders and Stroke Grant NS-18205.
![]() |
FOOTNOTES |
---|
Address for reprint requests: W. Li (E-mail: weili{at}ic.sunysb.edu).
Received 8 January 2001; accepted in final form 20 March 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|