Voltage-Dependent Sodium and Calcium Currents in Acutely Isolated
Adult Rat Trigeminal Root Ganglion Neurons
Hyung-Chan
Kim1,2 and
Man-Kyo
Chung1
1Department of Oral Physiology and
Institute of Oral Biology, School of Dentistry, and
2East-West Medical Research Institute,
Kyung Hee University, Seoul 130-701, Korea
 |
ABSTRACT |
Kim, Hyung-Chan and
Man-Kyo Chung.
Voltage-dependent sodium and calcium currents in acutely isolated
adult rat trigeminal root ganglion neurons. Voltage-dependent sodium (INa) and calcium
(ICa) currents in small (<30 µM) neurons from
adult rat trigeminal root ganglia were characterized with a standard
whole cell patch-clamp technique. Two types of
INa showing different sensitivity to
tetrodotoxin (TTX) were recorded, which showed marked differences in
their activating and inactivating time courses. The activation and the
steady-state inactivation kinetics of TTX-resistant
INa were more depolarized by about +20 and +30
mV, respectively, than those of TTX-sensitive
INa. Voltage-dependent ICa was recorded under the condition that
suppressed sodium and potassium currents with 10 mM Ca2+ as
a charge carrier. Depolarizing step pulses from a holding potential of
80 mV evoked two distinct inward ICa,
low-voltage activated (LVA) and high-voltage activated (HVA)
ICa. LVA ICa was first
observed at
60 to
50 mV and reached a peak at about
30 mV.
Amiloride (0.5 mM) suppressed ~60% of the LVA
ICa, whereas ~10% of HVA
ICa was inhibited by the same concentration of
the amiloride. LVA ICa was far less affected by
the presence of external Cd2+ or the replacement of
Ca2+ by 10 Ba2+ than HVA
ICa. The
-conotoxin GVIA (
-CgTx), an
N-type ICa blocker, suppressed ~65% of the
whole cell HVA ICa at the concentration of 1 µM. The
-CgTx-resistant HVA ICa was
sensitive to nifedipine (10 µM), a dihydropyridine (DHP) calcium
channel antagonist, which produced an additional blockade by ~25% of
the drug-free control (~70% of the
-CgTx-resistant
ICa). The combination of 10 µM nifedipine and
1 µM
-CgTx left ~13% of the drug-free control
ICa unblocked. The DHP agonist S(
)-BayK8644 (5 µM) shifted the activation of the HVA ICa to
more negative potentials and increased its maximal amplitude.
Additionally, S(
)-BayK8644 caused the appearance of a slowed
component of the tail current. These results clearly demonstrate that
the presence of two types of sodium channels, TTX sensitive and
resistant, and three types of calcium channels, T, L, and N type, in
the small-sized adult rat trigeminal ganglion neurons.
 |
INTRODUCTION |
Dorsal root ganglia (DRG) neurons transmit various sensory
information such as touch, pressure, pain, and temperature from the
peripheral region to the CNS. Similarly, a pair of trigeminal root
ganglia (TRG) is responsible for the sensory inputs from the
oromaxillofacial region to which the trigeminal nerve is innervated.
Intracellular recordings in the mammalian TRG neurons have shown that
three types of action potential exist: 1) fast spikes, most
of which were completely blocked by the external application of
tetrodotoxin (TTX), 2) Co2+- and TTX-resistant
humped spikes (Puil et al. 1986
), and 3)
slowly decaying TTX-resistant and Cd2+-sensitive action
potentials (Galdzicki et al. 1990
). These reports suggest that voltage-dependent sodium current
(INa) and calcium current
(ICa) may be involved in the electrical
activities of the TRG neurons. However, high-voltage activated
ICa only was reported in the rat (Nah and
McCleskey 1994
) and chicken TRG neurons (Galdzicki et
al. 1990
). Recently, TRG neurons were used in the study to
elucidate the mechanisms of a chemosensory transduction (Liu and
Simon 1996
) and a nociception evoked by protons
(Pidoplichko 1992
). However, there was little effort to
investigate further electrophysiological characteristics of TRG
neurons, which may be due to the assumption that the
electrophysiological characteristics of TRG neurons would be identical
to those described for DRG neurons.
The electrophysiological properties of the primary afferent neurons of
DRG in various species were investigated in detail, not only because
they are simple and accessible model neurons but also because there are
some similarities between the properties of the somata and those of
their central and peripheral terminals (Bellmonte and Gallego
1983
; Jeftinija 1994
; Scroggs and Fox
1992
). The sensory neurons of mammalian DRG are known to
express various types of voltage-dependent ion channels: 1)
at least two types of INa distinguished by their
sensitivity to TTX (Bossu and Feltz 1984
; Caffrey
et al. 1992
; Elliott and Elliott 1993
;
Fedulova et al. 1991
; Kostyuk et al.
1981
; McLean et al. 1988
; Ogata and Tatebayashi 1993
; Ogata and Tatebayashi
1992a
,b
;
Roy and Narahashi 1992
; Schwartz et al.
1990
), 2) T, L, N, and P types of
ICa showing distinct pharmacological and kinetic
properties (Mintz and Bean 1992
; Scroggs and Fox
1991
, 1992
), and
3) a number of potassium currents
(IK) (Gold et al. 1996
). In
addition, the distribution of these numerous ion channels in DRG
neurons, according to the size of cells and developmental stages, was
widely studied to elucidate the developmental and functional role of
each type of ion channels in the modulation of specific sensory
modalities (Roy and Narahashi 1992
; Schwartz et
al. 1990
; Scroggs and Fox 1991
,
1992
).
The main goals of this study were to survey the ranges of
voltage-dependent INa and
ICa in adult rat TRG neurons and to compare them
with those of DRG neurons as a part of an attempt to understand their
role in the processing of sensory information, especially pain, from
oromaxillofacial region. The results show that two types of
INa distinguished by their TTX sensitivity and
T-, L-, and N-type ICa are present in the adult
rat TRG neurons.
 |
METHODS |
Preparation of rat TRG neurons
Rat TRG neurons were prepared by the method described by
Liu and Simon (1996)
as follows. After the decapitation
of the ketamine-anesthesized (100-125 mg/kg im) adult Sprague-Dawley
rat (100-150 g), a pair of trigeminal ganglia were dissected and
washed several times in cold (4°C) modified Hanks' balanced salt
solution (HBSS) containing (in mM) 130 NaCl, 5 KCl, 0.3 KH2PO4, 4 NaHCO3, 0.3 Na2HPO4, 5.6 D-glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.3. They were incubated for 40 min at 37°C in HBSS
containing 1 mg/ml of type XI collagenase, triturated with flamed
Pasteur pipettes, and finally incubated at 37°C for 6 min with 1 mg/ml of type IV DNase I. Then they were retriturated and washed/centrifuged three times in Dulbecco's modified Eagle medium (DMEM) and maintained in room temperature. Cells were used in electrophysiological recording within 6 h.
Recording solutions and drugs
The composition of internal solution used for the isolation of
INa was (in mM) 110 CsF, 10 NaCl, 0.1 CaCl2, 10 HEPES, and 11 ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), pH 7.3 adjusted with CsOH. The external solution used for
INa contained (in mM) 60 choline-Cl, 60 NaCl, 20 tetraethylammonium (TEA)-Cl, 5 KCl, 5 MgCl2, 2 CaCl2, 10 HEPES, and 20 glucose, pH 7.4 adjusted with NaOH.
0.1 mM CdCl2 was added to this solution to suppress
ICa. The internal solution used for
ICa consisted of (in mM) 100 Cs-methanesulfonate
(Cs-MeSO3), 0.1 CaCl2, 20 TEA-Cl, 10 HEPES, 11 EGTA, and 2 Mg-ATP, pH 7.3 adjusted with CsOH. The external solution
used for ICa consisted of (in mM) 140 choline-Cl, 2 MgCl2, 10 CaCl2, 10 HEPES, and 20 glucose, pH 7.4 adjusted with CsOH. All the enzymes and chemicals were
purchased from Sigma, except DMEM and methanesulfonic acid, which was
purchased from Gibco Laboratory and Fluka, respectively. TTX (Research
Biochemical International) was dissolved first in ethyl alcohol and
then diluted in the external solution before use. A stock solution of
amiloride-HCl, nifedipine (Sigma), and S(
)BayK8644 (Research
Biochemical International) was made by dissolving them in dimethyl
sulfoxide. The
-conotoxin GVIA (
-CgTx, Research Biochemical
International) was stored as a stock solution of 1 mM in distilled
water. All experiments with dihydropyridine (DHP) drugs were carried
out in dim light.
Electrophysiology
The isolated cells were plated onto a polyethyleneimine-coated
glass coverslip in a recording chamber (100-µl volume) for superfusion (3 ml/min) with control and drug-containing solutions. Patch-clamp pipettes were manufactured from soda lime glass capillaries with a two-stage vertical pipette puller (L/M-3P-A, List Electronics) and fire-polished with a microforge (MF-83, Narishige). Because we had
large currents, low-resistance pipettes (~0.7-1.5 M
) were employed to reduce the voltage drop across the series resistance, which
was compensated 70-75% usually. We gave up further recordings when
the resultant series resistance was >3 M
after compensations. Whole
cell patch-clamp recordings were performed with an Axopatch 200A
patch-clamp amplifier (Axon Instruments). Stimulus application and data
acquisition were controlled by an IBM-compatible personal computer in
conjunction with a Labmaster (PP-50) DMA interface (Warner Instrument).
The data were low-pass filtered at 2 kHz (
3 dB) with the amplifier's
Bessel filter and digitized every 20 µs (INa)
or 50 µs (ICa). All data collection and
analysis were carried out with pCLAMP 6.0 software (Axon Instruments).
Neuron diameter was measured with an eyepiece micrometer under phase
contrast illumination and categorized into three groups: small (20-27
µm), medium (33-38 µm), and large (45-51 µm) cells, according
to the classification of Scroggs and Fox (1992a)
. We recorded voltage-dependent ion currents primarily in the small cells of
15-30 µm in diameters without or with short processes because
1) they are known to be related to pain sensation
(Harper and Lawson 1985a
), 2) the amplitude
of current recorded from the medium or large cells was so great that a
serious series resistance error and the saturation of an amplifier
could not be avoided under the ionic condition in this experiment
(i.e., 10 Ca2+ or 60 Na+ in external bathing
solution), and 3) the complete compensation of cell membrane
capacitances was difficult frequently and the signs of poor space-clamp
condition such as a slow deactivating current used to be detected in
some medium and large cells or cells with processes.
All experiments were carried out at room temperature (22-25°C).
Data analysis
The peak conductance (G) of
INa or ICa at each
potential was calculated from the corresponding peak current by using
the equation
where ERev is the reversal potential of
INa or ICa, I
is the peak current amplitude of INa or
ICa, and E is the membrane potential.
Normalized peak conductance (G/Gmax)
and the data describing the fractional decrease in the peak current
during the steady-state inactivation
(I/Imax) were fitted with a Boltzmann
function
where V1/2 is the membrane potential at
which 50% inactivation of the current is observed,
Vm is the prepulse membrane potential, and
k is the slope of the function. All data are expressed as means ± SE (n = number of observations) for at
least three measurements, unless otherwise stated.
 |
RESULTS |
TTX-s and TTX-r INa
We tried to isolate INa from the
other kinds of voltage-dependent ionic currents in whole cell
patch-clamp recordings by 1) suppressing
IK with the use of the equimolar replacement of
K+ with Cs+ in the internal solution and by the
addition of 20 mM TEA in the external solution, 2)
suppressing ICa by 0.1 mM Cd2+ in
the external solution and 5 mM Mg2+ in the internal and
external solutions, and 3) decreasing the concentration of
Na+ in the external solution to 60 mM by the isomolar
substitution of choline to prevent the saturation of the amplifier by
large INa. Inward current recorded under this
ionic condition was reduced as expected by the partial replacement of
the external Na+ by choline (Fig.
1A). The observed potential
for zero current flow was 46.7 ± 0.5 mV (n = 30),
as expected for a sodium-selective channel, and moved 11.3 ± 0.3 mV (n = 3) in the hyperpolarizing direction when the
external Na+ concentration was reduced from 60 to 40 mM
(Fig. 1B), which is identical to the predicted value by the
Nernst equation for the sodium-selective channel. In addition, when the
Na+ in the bathing solution was totally removed, the
current traces became much smaller and the direction of the current
became outward (n = 3, not shown). These observations
indicate that the inward current is the voltage dependent
INa.

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Fig. 1.
Inward current component carried by Na+. A:
current traces evoked by the depolarizing pulse to 10 mV from a
Vh of 70 mV. External solution contained (in
mM) 60 choline-Cl, 60 NaCl (or 80 choline-Cl and 40 NaCl), 20 tetraethylammonium (TEA)-Cl, 5 KCl, 5 MgCl2, 2 CaCl2, 0.1 CdCl2, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES), and 20 glucose, pH 7.4. The composition of internal
solution was (in mM) 10 CsF, 10 NaCl, 0.1 CaCl2, 10 HEPES,
and 11 bis( -aminoethyl
ether)-N,N,N',N'-tetraacetic
acid (EGTA), pH 7.3. B: peak I-V relationships
obtained from the current traces in A. Note the significant
reduction in the amplitude of the inward current and the shift of
Erev in a negative direction in the external
solution with 40 Na+.
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Figure 2A shows typical
INa recorded from the acutely isolated rat TRG
neuron under the ionic condition described above.
INa was first observed at the potential of
40
to
30 mV and peaked at
20 to +10 mV (Fig. 2A). The
current appeared to be composed of two components of the
voltage-dependent INa distinguished by their
sensitivity to TTX. In most cases, 0.1 µM TTX failed to inhibit
INa completely (Fig. 2Ab). The
component that was resistant to 0.1 µM TTX was not also significantly
inhibited by 20 µM TTX (n = 2, not shown). TTX-r
INa was observed in the >90% of the small TRG
neurons <30 µm in diameter in the presence of 0.1 µM TTX. However,
external application of 0.1 µM TTX suppressed most of the current in
some preparations (Fig. 2B). This suggests that most of the
small TRG neurons from adult rat may express TTX-r INa with or without TTX-s
INa, which resulted in difficulties in direct
observation of isolated TTX-s INa in small
neurons.

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Fig. 2.
Two types of INa; tetrodotoxin (TTX)-s and TTX-r
INa. A: typical
INa recorded from a rat TRG neuron before
(a) and after (b) the application of 0.1 µM
TTX. Current traces in a and b were evoked by the
depolarizing step pulses from a Vh of 70 mV.
TTX-sensitive component in c was obtained from the digital
subtraction of the TTX-resistant component in b from the
control current traces in a. Peak amplitudes of the control
current, TTX-resistant component, and the subtracted TTX-sensitive
component were plotted against the membrane potentials in d.
Ba: superimposed current traces recorded from another rat
TRG neuron. INa was evoked by the step pulse to
20 mV before and after the application of 0.1 µM TTX, which
inhibited most of the inward current in this cell. Bb:
magnitudes of currents shown in Ba were plotted against the
membrane potentials.
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|
TTX-s INa started to activate at the voltage of
near
40 mV, which was less depolarized than those for TTX-r
INa. The peak voltage for TTX-s
INa was also slightly hyperpolarized to that for
TTX-r INa. The maximal current density of the
TTX-r INa was quite various from cell to cell.
The maximal TTX-r INa was elicited at a test
potential of 0 or +10 mV (Fig. 2Ad), and the current density
of TTX-r INa elicited by voltage steps to +10 mV
from a holding potential (Vh) of
70 mV was
268.5 ± 20.6 pA/pF (n = 20). The peak current
density of the TTX-s INa was
356.5 ± 56.0 pA/pF (n = 7). Figure
3 illustrates that TTX-resistant
component of INa activated and decayed more
slowly than TTX-s INa by ~10- and 7-fold,
respectively, at the voltage of +10 mV. These results indicate that at
least two types of INa, i.e., TTX-s and TTX-r INa showing different sensitivity to TTX,
activation range, and distinct activating and inactivating time
courses, are expressed in the small neurons from rat TRG.

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Fig. 3.
Rise and decay time for TTX-s and TTX-r INa. An
example of the TTX-s INa in A and
TTX-r INa in B. TTX-r
INa was recorded in the presence of 0.1 µM
TTX. Note the distinct differences in the onset and the decay of
current; 10-90% rise time and 10-90% decay time were averaged and
plotted against the membrane potentials in C and
D, respectively. Data were obtained from 6 and 3 cells for
TTX-r and TTX-s INa, respectively.
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Voltage dependence of INa
We investigated the voltage dependence of the activation and the
steady-state inactivation of INa in the presence
of 2 mM Ca2+, 5 mM Mg2+, and 0.1 mM
Cd2+ in the external bathing solution. For the evaluation
of the steady-state inactivation properties, 1-s conditioning pulses to
various potentials ranging from
100 mV to 0 mV were followed by a
constant step pulse to +10 mV. Current traces were not leak-subtracted,
and the current amplitude was measured relative to the plateau phase near the end of the test pulse. Figure
4A shows the steady-state inactivation curves of a cell expressing TTX-s and TTX-r
INa. The availability curve (Fig. 4D,
) of the INa from the cell had an inflection
because of the presence of both TTX-s and TTX-r sodium channels.
Isolated inactivation curves of TTX-r INa (Fig. 4, B and D,
) and TTX-s
INa (Fig. 4, C and D,
)
were obtained with 0.1 µM TTX. TTX-r and TTX-s
INa showed marked differences in their
inactivating properties. A half-maximal potential
(V1/2) for the inactivation of TTX-s
INa was more hyperpolarized than that of TTX-r
INa by approximately
30 mV (Fig.
4D). We evaluated the steady-state inactivation properties
of the two types of INa with this protocol.
Figure 5 shows that the mean value of
V1/2 was
60.5 ± 1.6 mV for the TTX-s
INa (Fig. 5,
) and
29.3 ± 1.1 mV for
the TTX-r INa (Fig. 5,
), giving a mean
difference in V1/2 of 31.1 ± 1.4 mV
(n = 5). The slope of the steady-state inactivation curve was much steeper for the TTX-r INa
(k = 4.9 ± 0.5 mV for the TTX-r and 7.2 ± 0.3 mV for TTX-s INa, n = 5),
and the mean ratio of the k was 1.5:1 (n = 5). The peak sodium conductance for TTX-r INa
was calculated as described in METHODS. The conductance curve for TTX-r INa (Fig. 5,
) was obtained
in the presence of 0.1 µM TTX, and a curve for TTX-s
INa (Fig. 5,
) was derived from the cells
showing virtually complete inhibition (i.e., >95%) of the
INa by the 0.1 µM TTX. The
V1/2 for the activation of TTX-s INa was approximately
20 mV more
hyperpolarized than that of the TTX-r INa. The
averaged V1/2 was
19.7 ± 2.2 mV for
TTX-s INa and 0.1 ± 2.0 mV for the TTX-r
INa. The slope of the peak conductance curve was
slightly steeper for the TTX-s INa
(k = 5.6 ± 0.5 mV for TTX-r
INa and 4.1 ± 0.5 mV for TTX-s
INa).

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Fig. 4.
Inactivation properties of two types of INa. The
availability of INa was evaluated in a partially
TTX-sensitive cell. A: current traces evoked by the constant
step pulses to +10 mV preceded by 1-s prepotentials from 100 to 0 mV
with 10-mV increment. B: steady-state inactivation of TTX-r
INa obtained from the same cell in A
after the application of 0.1 µM TTX. Leaks were not subtracted in
A and B. C: TTX-s
INa isolated by the subtraction of TTX-r
INa from the control current traces.
D: plots of normalized current amplitudes measured at the
peak of each current trace shown in A-C.  in
and were derived from the curve fit to
Boltzmann function, which yielded V1/2 and
k of 29.9 mV and 5.7 mV in TTX-r
INa and 61.7 mV and 7.9 mV in TTX-s
INa, respectively.
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Fig. 5.
Voltage-dependent kinetics of INa. Midpoints of
maximal activation and inactivation for TTX-s
INa were less depolarized by about 30 and 10
mV, respectively, than those of TTX-r INa.
Steady-state inactivation curves were derived from the experiment in
Fig. 4, and normalized peak conductances were calculated as described
in METHODS.
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Whole cell ICa from adult rat TRG neurons
ICa was recorded from rat TRG neurons under
conditions that 1) suppressed IK by
the replacement of internal K+ with Cs+ and 20 mM TEA included in external solution, 2) suppressed
INa with choline replacing external
Na+, and 3) enhanced ICa
with 10 mM Ca2+ as a charge carrier.
Figure 6 shows whole cell
ICa obtained from a small-diameter neuron evoked
by depolarizing steps to various test potentials from a
Vh of
80 mV, which is thought to be comprised
of multiple components of ICa. By applying
depolarizing steps from a Vh at
80 mV,
transient inactivating ICa was first observed at
about
60 mV. The currents reached its peak in 5-30 ms after the
onset of the step pulse and decayed rapidly in a single-exponential time course with a time constant of 15-30 ms. With further
depolarization the peak amplitude increased smoothly and reached a
plateau at about
30 mV, resulting in a distinct shoulder in
I-V relationship (Fig. 6C). At the potential of
about
30 to
20 mV, a step increase in current amplitude was
observed, and the decay of the current became progressively less
pronounced. When the cell was held at
40 mV, all of the decaying
component of ICa could no longer be evoked over
the whole range of test potentials, but sustained ICa was activated at the stronger
depolarizations. The current showed little inactivation during the
80-ms step pulse (Fig. 6B) and was maximal at near
10 mV
(Fig. 6C). These observations suggest the presence of two
current components with different activation range, i.e., low-voltage
activated (LVA) and high-voltage activated (HVA)
ICa, in small-sized rat TRG neurons.

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Fig. 6.
Three components of ICa observed in a rat
trigeminal root gangion (TRG) neuron. A: current traces
evoked by step pulses to test potentials from 80 to +40 mV from a
Vh of 80 mV. The composition of the external
solution was (in mM) 140 choline-Cl, 2 MgCl2, 10 CaCl2, 10 HEPES, and 20 glucose, pH 7.4, and the internal
solution contained (in mM) 100 Cs-MeSO3, 0.1 CaCl2, 20 TEA-Cl, 10 HEPES, 11 EGTA, and 2 Mg-ATP, pH 7.3. B: current traces elicited from a Vh
of 40 mV. Note the disappearance of decaying components shown in
A. C: I-V plots derived from the
current traces in A and B. Note the low-voltage
activated (LVA) ICa was not induced by the
Vh of 40 mV ( ).
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HVA ICa was recorded in every cell investigated,
whereas LVA ICa did not appear to be recorded
uniformly. In some preparations, low-threshold transient LVA
ICa was recorded prominently, whereas the
sustained HVA ICa component was predominant
Ca2+ entry pathway in some cells. These may indicate that
the densities of the two types of the Ca2+ channels may be
various from cell to cell.
Figure 7 shows the results obtained from
a single cell expressing LVA and HVA ICa when a
10 mM Ca2+ containing medium was changed to the solution
with equimolar Ba2+. With Ba2+ as a charge
carrier the magnitude of the sustained ICa was
enhanced almost twofold at 0 mV, and the I-V relationship
was shifted ~10 mV to a hyperpolarizing direction. However,
Ba2+ showed negligible effect on the LVA
ICa, and in some cells the current amplitude was
slightly decreased by the replacement of Ba2+. The effect
of Ba2+ on ICa was completely
reversible (not shown). These results support the notion that the whole
cell ICa recorded in adult rat TRG neurons consists of independent types of ICa.

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Fig. 7.
Enhancement of high-voltage activated (HVA) ICa
by substitution of 10 Ba2+ for 10 Ca2+.
A: examples of superimposed current traces enhanced by the
equimolar substitution of Ca2+ with Ba2+.
Traces marked by asterisk represent the current traces carried by
Ba2+. B: peak I-V relationships in
10 mM Ca2+ and in 10 mM Ba2+. The peak HVA
ICa nearly doubled in the amplitude in 10 mM
Ba2+, whereas the magnitude of LVA
ICa little changed or slightly decreased.
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Effects of calcium channel blockers on ICa
Different types of Ca2+ channels show the differential
sensitivities to some inorganic Ca2+ channel blockers
(Fox et al. 1987
). We tested the effects of several
calcium channel blockers on ICa. As illustrated
in Fig. 8 Cd2+ seemed to be
more effective in blocking the HVA ICa. The
addition of 50 µM Cd2+ to the bath virtually eliminated
the HVA ICa, whereas the LVA component was
comparatively less affected; 100 µM Cd2+ also did not
abolish LVA ICa completely but suppressed HVA
ICa by >90%. The inhibitory effects of
Cd2+ were readily reversible (not shown). These results are
similar to those reported by others regarding the differential
sensitivity of LVA and HVA ICa in DRG neurons
(Fox et al. 1987
; Gross and McDonald
1987
).

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Fig. 8.
Preferential block of HVA ICa by
Cd2+. A: superimposed current traces elicited by
80-ms step pulses to 30 mV (top traces) and 0 mV
(bottom traces) from a Vh of 80 mV
in the control medium and after superfusion with 50 and 100 µM
Cd2+. Note that the blocking effects were considerably
greater on the HVA ICa than on the LVA
ICa. B: peak I-V
relationships obtained from the complete sets of records exemplified in
A. Note the incomplete block of the LVA
ICa by 100 µM Cd2+.
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Amiloride, a K+-sparing diuretic, was known to block LVA
ICa effectively in many preparations (Fox
et al. 1987
; Scroggs and Fox 1991
,
1992
). Figure
9 illustrates the selective inhibitory effect of amiloride on LVA ICa evoked from a TRG
neuron. LVA ICa elicited from
Vh of
80 mV with a step pulse to
30 mV was
reversibly inhibited by 0.5 mM amiloride (60.4 ± 5.4%,
n = 3). However, 0.5 mM amiloride showed the smaller
inhibitory effect on HVA ICa (12.6 ± 3.9%, n = 3). The inhibitory effect of amiloride on
HVA ICa may be due to the incomplete
inactivation of T-type ICa in our protocols (see
Scroggs and Fox 1992
).

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Fig. 9.
Effects of amiloride on the LVA and HVA ICa in a
rat TRG neuron. A: LVA and HVA ICa
were evoked by step pulses to 30 and 0 mV respectively from a
Vh of 70 mV at 0.1 Hz in a small rat TRG
neuron before and after the application of 0.5 mM amiloride. The
inhibitory effect of amiloride was far more pronounced in LVA
ICa than in HVA ICa.
Numbers represent the location in B at which the current
traces were derived from. B: peak amplitudes of LVA and HVA
ICa were plotted against time. Amiloride
inhibited LVA ICa by ~65% immediately after
superfusion at the concentration of 0.5 mM. However, HVA
ICa was far less affected by the drug. The
effects of amiloride were completely reversed after washout.
|
|
Nifedipine, a DHP calcium channel antagonist, has been shown to
preferentially block the sustained component of
ICa in many other preparations (Regan et
al. 1991
), and the synthetic toxin
-conotoxin GVIA
(
-CgTx) is a potent blocker of a component of the HVA
ICa that reflects the activation of N-type
ICa in a variety of neurons (Fox et al.
1987
; Scroggs and Fox 1991
,
1992
; Tsien et al.
1981
). We tested the effects of
-CgTx and nifedipine on the
HVA ICa from cells in which the LVA
ICa produced only a very small deflection in the
I-V relationships. The HVA ICa was
evoked by repetitive step pulses to +10 mV from a
Vh of
80 mV every 10 s. After a baseline
of five to seven traces was established in the drug-free condition,
-CgTx (1 µM) was superfused over the neuron. The
-CgTx blocked
62.3 ± 4.0% (n = 4) of the peak HVA
ICa at the concentration of 1 µM (Fig.
10Aa). Subsequent
superfusion of 10 µM nifedipine in the presence of 1 µM
-CgTx
blocked an additional 26.2 ± 1.6% (n = 4) of the
drug-free control ICa, which indicates that the
69.5 ± 1.6% (n = 4) of
-CgTx-resistant
ICa was suppressed by 10 µM nifedipine. The
combination of 1 µM
-CgTx and 10 µM nifedipine left some
ICa unblocked. On average, 13.0 ± 3.3%
(n = 4) of the control ICa was
resistant to the mixture of 10 µM nifedipine and 1 µM
-CgTx in
small rat TRG neurons (Fig. 10Ab). Figure 10B
illustrates the representative current traces evoked by 400-ms step
pulses to 10 mV and corresponding I-V curves after the
application of 1 µM
-CgTx and 10 µM nifedipine. Figure 10,
Ba and Bb, show that the inactivating transient
component of the HVA ICa was blocked by the
application of 1 µM
-CgTx. The current amplitudes of the digitally
subtracted nifedipine-sensitive ICa and
-CgTx-sensitive ICa were plotted against the
membrane potentials, which revealed that the
-CgTx-sensitive
component was activated at more hyperpolarized potential than the
nifedipine-sensitive component (Fig. 10, Bc and
Bd). These results suggest that there are at least two
distinct types of the HVA ICa showing different pharmacological properties in rat TRG neurons.

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Fig. 10.
Blocking effects of nifedipine and -conotoxin GVIA ( -CgTx) on HVA
ICa. Aa: repetitive constant step
pulses to 10 mV from a Vh of 80 mV were
delivered to evoke the HVA ICa in small-sized
rat TRG neurons. Superfusion with 1 µM -CgTx blocked ~65% of
peak ICa. Additional application of 10 µM
nifedipine inhibited the remained ICa by
~70%. Superimposed current traces in b were selected from
continuous recordings in a at the locations indicated by
corresponding numbers. Ba: from a Vh
of 80 mV the HVA ICa was evoked by the 400-ms
step pulse to 0 mV in the control solution (1), in the presence of 1 µM -CgTx (2), and after the additional application of 10 µM
nifedipine (3). Bb: examples of the nifedipine-sensitive
current component (2 and 3) and the -CgTx-sensitive current
component (1 and 2) obtained by the digital subtraction of the currents
in Ba. Bc: peak I-V relationships
obtained from the current traces in Ba. Bd:
nifedipine-sensitive and the -CgTx-sensitive
ICa were plotted against the membrane
potentials. The membrane potential for maximal -CgTx-sensitive
ICa is less depolarized than that of the
nifedipine-sensitive ICa.
|
|
Finally, the DHP calcium channel agonist S(
)-BayK8644, which
increases current amplitude through L-type calcium channels by
enhancing the probability of channel opening, was tested to confirm the
presence of L-type calcium channel in rat TRG neurons. When
S(
)-BayK8644 (5 µM) was superfused over rat TRG neurons held at
80 mV, the amplitude of HVA ICa was increased,
which was greatest at weak test depolarizations compared with strong test potentials (Fig. 11, A and
B). S(
)-BayK8644 also
greatly increased the amplitude and decreased the rate of inactivation of the tail current (Fig. 11C). In addition, the calcium
channel activation threshold and the peak of the I-V curve
was shifted in the hyperpolarizing direction (Fig. 11D).
These effects are all characteristics of DHP agonists that were
previously described (Scroggs and Fox 1991
). Similar
results were obtained from other five cells.

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Fig. 11.
Effects of S( )-BayK8644 on HVA ICa.
A: superimposed current traces elicited by 80-ms step pulses
to 20 mV in Aa and +20 mV in Ab from a
Vh of 80 mV in the control medium and after
superfusion with 5 µM S( )-BayK8644. Note the marked increase in
current amplitude at a weak depolarization to 20 mV compared with +20
mV. B: S( )-BayK8644 increased the amplitude and decreased
the rate of inactivation of tail currents compared with control.
C: peak I-V relationships obtained from the
complete sets of records exemplified in A. Note the shift of
the potential for the activation and peak of ICa
to a hyperpolarizing direction.
|
|
 |
DISCUSSION |
Classification of primary afferent neurons of the mammalian DRG is
based on the morphological and electrophysiological characteristics of
neurons (Harper and Lawson 1985a
,b
). The cell size is known to be correlated
with the axonal conduction velocity well; large neurons with myelinated
A
,
fibers are fast conducting, whereas small cells with
myelinated A
and unmyelinated C fibers are slow conducting
(Harper and Lawson 1985a
). However, properties of the somatic action potentials are thought to be associated with the cell
types at lesser extent: long action potential with inflected repolarization that is mainly recorded from the A
and C cells and
short action potential without plateau during the falling phase
recorded in A
and A
cells (Harper and Lawson
1985b
). Patch-clamp techniques were used to investigate the
range and properties of ionic channels underlying these electrical
activities in dissociated mammalian DRG neurons, and two types of
INa were characterized; in general, TTX-s
INa is found in large-diameter cells, and TTX-r INa is expressed in small-diameter cells
(Caffrey et al. 1992
; Elliott and Elliott
1993
; Kostyuk et al. 1981
; McLean et al.
1988
; Ogata and Tatebayashi 1992a
; Puil
et al. 1986
; Schwartz et al. 1990
). Although it
is generally accepted that TTX-r INa is found in
the soma of small DRG neurons that give rise primarily to C-type afferent fibers, the frequency of recording TTX-r
INa in small neurons is in disagreement among
studies from the same type of cell. For example, Caffrey et al.
(1992)
reported that TTX-r INa was
always observed in conjunction with TTX-s INa in
small DRG neurons from adult rats, where as Elliott and Elliott
(1993)
suggested that only 45% of small cells express TTX-r
INa from the same preparation. We recorded
INa primarily from the small-sized TRG neurons
ranging from 15 to 30 µm and observed TTX-r
INa in every cell investigated in the presence
of 0.1 µM TTX, in agreement to the report of Caffrey et al.
(1992)
.
These two types of voltage-dependent INa from
adult rat TRG neurons showed additional distinct characteristics as
well as a distinct sensitivity to TTX. TTX-r INa
showed slower kinetics of activation and inactivation (Figs. 2 and 3),
more depolarized potential for the activation threshold (Fig.
2Ad, and the more depolarized voltage dependence of the
activation and the steady-state inactivation (Figs. 4 and 5). These
properties of TTX-r INa are similar to those
described in DRG neurons (Fedulova et al. 1991
; Kostyuk et al. 1981
; McLean et al. 1988
;
Ogata and Tatebayashi 1993
; Roy and Narahashi
1992
).
Although the functional contribution of TTX-r
INa to the electrophysiological activities of
sensory neurons is not clear, it is suggested that TTX-r
INa may contribute to the prolongation of the
interspike interval by extending the inactivated state of TTX-s
INa during sustained depolarization of the
membrane potential (Bossu and Feltz 1984
) or may be
responsible for the slow adaptive properties and the generation of the
long trains of action potentials because TTX-r
INa shows the extremely slow inactivation
process and the rapid repriming kinetics (Elliott and Elliott
1993
; Ogata and Tatebayashi 1992b
). Recent
studies suggested that TTX-r INa may participate
in the sensitization to noxious stimuli in small capsaicin-sensitive
primary afferent neurons based on its modulation by pain-inducing
agents (Cesare and McNaughton 1997
). In addition, TTX-resistant Na+-dependent spikes are reported to be
activated selectively in primary afferent fibers by elevated
extracellular K+ concentration (Jeftinija
1994
), which may be frequently occurred at the site of injury
and depolarize the neuronal membrane potential. The more depolarized
activation and steady-state inactivation kinetics of TTX-r
INa than those of TTX-s
INa in sensory neurons may contribute to the
selective transmission of sensory signals by TTX-r
INa under the condition depolarizing sensory
neurons. It may be valuable for the further comprehension and better
management of the oromaxillofacial pain to elucidate the association of
TTX-r INa with the transmission of noxious
signals from the maxillofacial region.
Rat DRG neurons are known to have several types of calcium
channels (Scroggs and Fox 1991
, 1992
) that are similar to those reported in chick
sensory neurons (Fox et al. 1987
): 1) a
transient (T-type) or LVA ICa, 2) a
dihydropyridine-sensitive, sustained (L-type) or HVA
ICa, and 3) N-type
ICa showing kinetic properties between the T-
and L-type ICa and a selective suppression by
-CgTx. Recently, a fourth type, known as the P-type
ICa, distinguished by its unique sensitivity to
-agatoxin, also was described in the rat DRG neurons (Mintz
and Bean 1992
). We identified both LVA and HVA
ICa from acutely isolated rat TRG neurons. LVA
ICa was identified by the low threshold for the
activation at about
60 to
50 mV with maximal current amplitude at
the voltage of
30 mV, a fast rate of inactivation, and a shoulder in
the I-V curve. LVA ICa recorded in
rat TRG neurons showed several characteristics similar to those
described for T-type ICa in other neuronal
preparations (Fox et al. 1987
; Scroggs and Fox
1991
, 1992
);
1) LVA ICa inactivated when the cell
held at depolarized potential of about
40 mV (Fig. 1), 2)
the channel for the current was found to be more permeable to
Ca2+ than Ba2+ (Fig. 7), and 3) the
current was more sensitive to amiloride and resistant to
Cd2+ than HVA ICa (Figs. 8 and 9).
HVA ICa was present in the virtually all of the
rat TRG neurons investigated and typically activated at about
30 to
20 mV, with the maximal peak current occurring between 0 and 10 mV.
HVA ICa was greatly suppressed by 0.1 mM Cd2+ (Fig. 8) and greatly enhanced by Ba2+
(Fig. 7), in agreement with other preparation (Fox et al.
1987
). Several characteristics of HVA
ICa indicate that it may have at least two
components; 1) the decays of current traces at positive test
potentials (Figs. 6A and 10B) were poorly fitted
by single exponential (not shown), 2) when HVA
ICa was elicited from a
Vh of
40 mV, the amplitudes were reduced
significantly and the ICa decayed little or very
slowly (Fig. 6B), 3) the
-CgTx-resistant current was largely inhibited by nifedipine (Fig. 10), and
4) DHP agonist S(
)-BayK8644 increased the amplitude of HVA
ICa (Fig. 11). These observations strongly
suggest the presence of at least two types of HVA
ICa in acutely dissociated rat TRG neurons,
resembling the DHP-sensitive sustained L-type
ICa and
-CgTx-sensitive N-type ICa described in other preparations (Fox
et al. 1987
; Gross and McDonald 1987
;
Regan et al. 1991
; Scroggs and Fox 1991
,
1992
). When the HVA
ICa was evoked from a Vh
of
80 mV, the proportion of nifedipine/
-CgTx-resistant
ICa was relatively constant (10-15%), which
was similar to the results from small-diameter rat DRG neurons (Scroggs and Fox 1992
). This
nifedipine/
-CgTx-resistant ICa component may
be interpreted as the incomplete block of L- or N-type
ICa at those concentration of drugs or the
presence of other types of HVA ICa components in
rat TRG neurons.
The calcium channels are known to be involved in the pacemaker
depolarization, Ca2+-dependent secretion, and the
activation of Ca2+-dependent ionic conductance in neuronal
preparations, although linking a specific type of
ICa to a particular cellular process may be
difficult (Tsien et al. 1988
). It may be certain,
however, that the subtypes of ICa contribute in
different ways to the transmission of sensory signals of different
modalities in primary afferent neurons. The subtypes of
ICa are known to show diameter-dependent variation in acutely isolated rat DRG neurons, i.e., medium-diameter neurons had a large amount of T-type ICa,
whereas significantly large proportion of the whole cell
ICa was L type in the small cells
(Scroggs and Fox 1991
, 1992
), which shaped action potentials differently and
therefore may significantly affect the sensory transmission.
In conclusion, we identified the voltage-dependent TTX-r and TTX-s
sodium currents, amiloride-sensitive T-type, DHP-sensitive L-type, and
-CgTx-sensitive N-type calcium currents from acutely dissociated TRG
neurons of adult rats. Although three types of proton-gated
INa were identified in rat TRG neurons
(Pidoplichko 1992
) and TTX-r sodium channels were
detected by in situ hybridization technique in the same preparation
(Akopian et al. 1996
), no direct observation of
voltage-dependent INa was reported from TRG
neurons in any species, as far as we know. In addition, T-type
ICa, DHP-sensitive L-type, and
-CgTx-sensitive N-type ICa were not reported
in this preparation.
 |
ACKNOWLEDGMENTS |
This work was supported in part by the Basic Medical Research Fund
(L-42) in 1996 from the Ministry of Education, Republic of Korea.
 |
FOOTNOTES |
Address for reprint requests: H.-C. Kim, Dept. of Oral Physiology,
School of Dentistry, Kyung Hee University, #1, Heoki-dong,
Dongdaemoon-ku, Seoul 130-701, Korea.
Received 17 November 1997; accepted in final form 4 August 1998.
 |
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