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The Journal of Neuroscience, December 1, 1998, 18(23):9607-9619
Slow Closed-State Inactivation: A Novel Mechanism Underlying Ramp
Currents in Cells Expressing the hNE/PN1 Sodium Channel
Theodore R.
Cummins1, 3,
James R.
Howe2, and
Stephen
G.
Waxman1, 2, 3
Departments of 1 Neurology and
2 Pharmacology, Yale University School of Medicine, New
Haven, Connecticut 06510, and 3 Neuroscience Research
Center, Veterans Administration Medical Center, West Haven, Connecticut
06516
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ABSTRACT |
To better understand why sensory neurons express voltage-gated
Na+ channel isoforms that are different from those
expressed in other types of excitable cells, we compared the properties
of the hNE sodium channel [a human homolog of PN1, which
is selectively expressed in dorsal root ganglion (DRG) neurons] with
that of the skeletal muscle Na+ channel (hSkM1)
[both expressed in human embryonic kidney (HEK293) cells]. Although
the voltage dependence of activation was similar, the inactivation
properties were different. The V1/2 for steady-state inactivation was slightly more negative, and the rate of open-state inactivation was ~50% slower for hNE. However, the greatest
difference was that closed-state inactivation and recovery from
inactivation were up to fivefold slower for hNE than for hSkM1
channels. TTX-sensitive (TTX-S) currents in small DRG neurons also have
slow closed-state inactivation, suggesting that hNE/PN1 contributes to
this TTX-S current. Slow ramp depolarizations (0.25 mV/msec) elicited
TTX-S persistent currents in cells expressing hNE channels, and in DRG neurons, but not in cells expressing hSkM1 channels. We propose that
slow closed-state inactivation underlies these ramp currents. This
conclusion is supported by data showing that divalent cations such as
Cd2+ and Zn2+ (50-200
µM) slowed closed-state inactivation and also
dramatically increased the ramp currents for DRG TTX-S currents and hNE
channels but not for hSkM1 channels. The hNE and DRG TTX-S ramp
currents activated near 
65 mV and therefore could play an important
role in boosting stimulus depolarizations in sensory neurons. These results suggest that differences in the kinetics of closed-state inactivation may confer distinct integrative properties on different Na+ channel isoforms.
Key words:
sodium channel; persistent current; dorsal root ganglion; excitability; tetrodotoxin; expression
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INTRODUCTION |
One of the hallmarks of most
excitable cells is the presence of voltage-gated sodium currents, which
underlie the rapid action potentials characteristic of neurons and
muscle cells. Nearly a dozen distinct voltage-gated sodium channels
have been cloned from mammals (Black and Waxman, 1996
). Many of these
channels have specific developmental, tissue, or cellular
distributions. Rat brain type III neuronal channels are primarily
expressed early during development (Felts et al., 1997).
Immunocytochemical experiments indicate that although rat brain type I
(rbI) channels are concentrated in cell bodies, rat brain type II
(rbII) channels may be preferentially targeted to neurites (Westenbroek
et al., 1989). Other neuronal isoforms are predominantly expressed in
peripheral tissues (Akopian et al., 1996; Felts et al., 1997;
Toledo-Aral et al., 1997; Dib-Hajj et al., 1998).
It is becoming apparent that the different isoforms may also have
distinct functional properties. For example, Smith and Goldin (1998)
have shown that although rbI and rbII channels both encode fast sodium
currents, the voltage dependence of activation and steady-state
inactivation is significantly more positive for the rbI channels.
Recent evidence indicates that the Na6 isoform underlies resurgent and
subthreshold persistent sodium currents in cerebellar Purkinje cells
(Raman and Bean, 1997). One of the isoforms primarily expressed in
peripheral neurons such as dorsal root ganglion (DRG) neurons,
SNS or PN3, encodes a TTX-resistant channel that has slow
inactivation kinetics when expressed in Xenopus oocytes
(Akopian et al., 1996; Sangameswaran et al., 1996). Another isoform
that is also highly expressed in DRG neurons (hNE, NaS, or PN1)
has been cloned from human (Klugbauer et al., 1995), rabbit (Belcher et
al., 1995) and rat (Sangameswaran et al., 1997; Toledo-Aral et al.,
1997) and encodes a TTX-sensitive (TTX-S) channel (Klugbauer et al.,
1995).
The human homolog of this isoform, hNE, has been expressed in the
mammalian human embryonic kidney cell line HEK293, but the initial characterization did not demonstrate any exceptional
differences between hNE and other TTX-S isoforms (Klugbauer et al.,
1995). This isoform is particularly interesting because it is expressed in a majority of small DRG neurons (Black et al., 1996) and may be the
predominant TTX-S channel in these sensory neurons. We (Cummins and
Waxman, 1997) and others (Elliott and Elliott, 1993) have shown that
the predominant TTX-S current in small DRG neurons from adult rats has
slow repriming (recovery from inactivation) kinetics, much slower than
those observed in adult CNS neurons (Costa, 1996) and axotomized DRG
neurons (Cummins and Waxman, 1997). Therefore we were interested in
determining whether the hNE sodium channel had slow repriming kinetics
or other unique properties.
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MATERIALS AND METHODS |
Transfection and preparation of stably transfected cell
lines. Transfections were performed using the calcium phosphate
precipitation technique. HEK293 cells are grown under standard
tissue culture conditions (5% CO2; 37°C) in
DMEM supplemented with 10% fetal bovine serum. The calcium
phosphate-DNA mixture was added to the cell culture medium and left
for 15-20 hr, after which time the cells were washed with fresh
medium. After 48 hr, antibiotic (G418, Geneticin; Life Technologies,
Gaithersburg, MD) was added to select for neomycin-resistant cells.
After 2-3 weeks in G418, colonies were picked, split, and subsequently
tested for channel expression using whole-cell patch-clamp recording techniques.
Culture of dorsal root ganglion neurons. DRG cells were
studied after short-term culture (12-24 hr). The culture was performed as previously described (Caffrey et al., 1992). Briefly, the L4 and L5
DRG ganglia were harvested from adult female Sprague Dawley rats. The
DRG were treated with collagenase and papain, dissociated in DMEM and
Ham's F12 medium supplemented with 10% fetal bovine serum, and plated
on glass coverslips. Recordings were made within 24 hr of dissociation.
Whole-cell patch-clamp recordings. Whole-cell patch-clamp
recordings were conducted at room temperature (~21°C) using an
EPC-9 amplifier. Data were acquired on a Macintosh Quadra 950 computer using the Pulse program (version 7.89; HEKA
Electronic). Fire-polished electrodes (0.8-1.5 M
) were
fabricated from 1.65 mm Corning 7052 capillary glass using a Sutter
P-97 puller (Novato, CA). To minimize space-clamp problems, we selected
for recording only isolated cells with a soma diameter of <25 µm.
Cells were not considered for analysis if the initial seal resistance
was <5 G or if they had high leakage currents (holding current > 0.1 nA at 80 mV), membrane blebs, or an access resistance >4
M. The average access resistance was 2.3 ± 0.6 M (mean ± SD; n = 116) for cells expressing hNE channels and
2.3 ± 0.6 M (n = 52) for cells expressing
hSkM1 channels. Voltage errors were minimized using 80% series
resistance compensation, and the capacitance artifact was canceled
using the computer-controlled circuitry of the patch-clamp amplifier. Linear leak subtraction, based on resistance estimates from four to
five hyperpolarizing pulses applied before the depolarizing test
potential, was used for all voltage-clamp recordings. Membrane currents
were usually filtered at 2.5 kHz and sampled at 10 kHz. The pipette
solution contained (in mM): 140 CsF, 1 EGTA, 10 NaCl, and
10 HEPES, pH 7.3. The standard bathing solution was (in
mM): 140 NaCl, 3 KCl, 1 MgCl2, 1 CaCl2, and 10 HEPES, pH 7.3. The liquid junction
potential for these solutions was <8 mV; data were not corrected to
account for this offset. The osmolarity of all solutions was adjusted
to 310 mOsm (Wescor 5500 osmometer, Logan, UT). The offset potential
was zeroed before patching the cells and checked after each recording
for drift; if the drift was >10 mV per hour, the recording was discarded.
Data analysis. Data were analyzed using the Pulsefit (HEKA
Electronic) and Origin (Microcal Software, Northampton, MA) software programs. Unless otherwise noted, statistical significance was determined by p < 0.05 using an unpaired t
test. Results are presented as mean ± SEM, and error bars in the
figures represent SEs. The curves in the figures are drawn to
guide the eye unless otherwise noted. Time course data were fitted with
single-exponential functions. Although in some instances fitting to a
dual exponential would improve the overall fit, the second component
was typically small (<10%), and the predominant component was not
much different from that estimated with the single-exponential fits.
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RESULTS |
Sodium current activation
To compare the properties of the hNE (Klugbauer et al., 1995) and
hSkM1 (George et al., 1992) sodium channels, we created HEK293 cell
lines that stably expressed the hNE and hSkM1 channels. Fast-inactivating TTX-S sodium currents were observed in cells transfected with hNE and hSkM1 channels (Fig.
1A). Figure
1B shows that the current-voltage
(I-V) curve for the peak sodium current was similar
for hNE and hSkM1 channels. The midpoint of activation was 25.8 ± 0.8 mV (mean ± SE; n = 45) for hNE currents
and 27.0 ± 0.8 mV (n = 31) for hSkM1
currents.
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Figure 1.
hNE and hSkM1 channels have similar activation
properties. A, Family of traces from
representative HEK293 cells expressing either hNE channels
(left) or hSkM1 channels (right). The
currents were elicited by 40 msec test pulses to various potentials
from 60 to 30 mV. Cells were held at 100 mV. B,
Normalized peak current-voltage relationship for hNE
(filled squares; n = 14) and
hSkM1 (open circles; n = 12).
C, The deactivation time course of hNE and hSkM1
channels examined at potentials ranging from 120 to 50 mV after a
0.5 msec activation prepulse to 0 mV. Deactivation time constants were
obtained from single-exponential fits to the tail currents for hNE
(filled squares; n = 16) or
hSkM1 (open circles; n = 12)
channels. Inset. Traces showing
representative hNE (solid line) and hSkM1 (dotted
line) deactivation tail currents at 70 mV.
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Other parameters relating to activation were also examined. The time
course of activation, estimated using a Hodgkin and Huxley fit of
currents elicited with a step depolarization to 30 mV, was similar
for hNE channels (
m = 303 ± 17 µsec;
n = 25) and hSkM1 channels (m = 289 ± 23 µsec; n = 20). Deactivation kinetics
was examined at potentials ranging from 120 to 50 mV after a
short (0.5 msec) activating pulse (see Fig. 1C,
current traces). Figure 1C shows that the
time constants for deactivation were also similar for hNE and hSkM1
channels. Resurgent currents such as those reported by Raman et al.
(1997) in Purkinje neurons were not observed in HEK293 cells expressing either hNE or hSkM1 channels. Noninactivating currents, defined as the
residual current measured at 100 msec during a step depolarization to 0 mV, were small for both hNE channels (0.1 ± 0.1% of peak; n = 23) and hSkM1 channels (0.1 ± 0.1% of peak;
n = 17).
Steady-state inactivation and inactivation kinetics
Although the activation kinetics is similar for hNE and hSkM1
currents, Figure 2A
shows that the decay phase is slower for the hNE current. The rate of
inactivation was quantified by fitting the decay phase of the
macroscopic current with a single-exponential function. The time
constants estimated from these fits are plotted as a function of the
test potentials in Figure 2B. The time constants were
greater for hNE currents than for hSkM1 channels over the entire
voltage range from 50 to +30 mV. At 0 mV, for example, hNE currents
inactivated with a time constant of 0.77 ± 0.03 msec (n = 10), and hSkM1 channels inactivated with a time
constant of 0.51 ± 0.05 msec (n = 10). This
difference was statistically significant (p < 0.01).
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Figure 2.
hNE and hSkM1 channels have distinct inactivation
properties. A, Representative currents from whole-cell
recordings of a cell expressing hNE channels (trace
labeled hNE) and a cell expressing hSkM1 channels
(unlabeled trace). Currents were elicited by a
step depolarization to 30 mV from a holding potential of 100 mV and
were scaled for comparison. The hNE current decays more slowly than
does the hSkM1 current. B, Inactivation kinetics as a
function of voltage. The macroscopic decay time constant is greater for
hNE currents (filled squares;
n = 10) than for hSkM1 currents (open
circles; n = 10) at each voltage. Time
constants were estimated from single-exponential fits to the decay
phase of currents elicited by 100 msec step depolarizations to the
indicated potential. C, Comparison of hNE
(filled squares; n = 13)
and hSkM1 (open circles; n = 12) steady-state inactivation. Steady-state inactivation was
estimated by measuring the peak current amplitude elicited by 20 msec
test pulses to 10 mV after 500 msec prepulses to potentials over the
range of 130 to 10 mV. Current is plotted as a fraction of the
maximum peak current.
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The voltage dependence of steady-state inactivation
(h
) was examined by holding the cells at
prepulse potentials between 130 and 10 mV for 500 msec before
stepping to the test potential (10 mV) for 20 msec. The
h curves are plotted in Figure 2C.
The midpoint of the h curve was significantly
(p < 0.001) more negative for hNE channels
(78 ± 1 mV; n = 45) than for hSkM1 channels
(72 ± 1 mV; n = 31).
Because TTX-S currents in DRG neurons recover relatively slowly from
inactivation and because hNE transcripts are detected in the majority
of DRG neurons (Black et al., 1996), we wanted to compare the time
course for recovery from inactivation for hNE and hSkM1 channels.
Recovery was examined after 20 msec inactivating pulses at 20 mV
(protocol shown in Fig. 3A).
We used 20 msec inactivating prepulses to allow complete fast
inactivation without inducing slow inactivation. Similar results were
also obtained with 5 and 100 msec inactivating prepulses (data not
shown). Figure 3A shows currents from a representative hNE
cell and hSkM1 cell illustrating recovery at 80 mV. Although only
~50% of the hNE current has recovered after 100 msec at 80 mV,
virtually all of the hSkM1 current has recovered at this time. In
general, the time course for recovery from inactivation for both hNE
and hSkM1 currents could be fitted well with a single-exponential
function. The average recovery time course at 80 mV for hNE and hSkM1
currents is shown in Figure 3B. The time constant for
recovery of hNE channels ( = 104 ± 8 msec; n = 12) was more than sixfold greater than the corresponding time constant
for hSkM1 channels ( = 16 ± 4 msec; n = 11).
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Figure 3.
Recovery from inactivation and development of
inactivation are slower for hNE channels than for hSkM1 channels.
A, Family of current traces from cells
expressing hNE or hSkM1 currents showing the rate of recovery from
inactivation at 80 mV. The standard recovery from inactivation
voltage protocol is shown above the current
traces. The cells were prepulsed to 20 mV for 20 msec
to inactivate all of the current and then brought back to 80 mV
for increasing recovery durations before the test pulse to 20 mV. The
maximum pulse rate was 0.5 Hz. B, Time course for
recovery from inactivation of peak currents at 80 mV. Recovery is
much slower for hNE (filled squares;
n = 12) than for hSkM1 (open circles; n = 11)
currents. The solid curves show single-exponential
functions fitted to the data, with time constants of 104 msec (hNE) and
16 msec (hSkM1). The data are plotted on a logarithmic time axis to
allow comparison of the disparate time courses. C,
Family of current traces from cells expressing hNE and
hSkM1 currents showing the rate of development of inactivation at 80
mV. The standard development of inactivation voltage protocol is shown
above the current traces. From a holding
potential of 100 mV, the cells were prepulsed to 80 mV for
increasing durations and then stepped to 20 mV to determine the
fraction of current inactivated during the prepulse. D,
Time course for development of inactivation for the peak current.
Inactivation develops more slowly at 80 mV for hNE channels
(filled squares; n = 12) than
for hSkM1 channels (open circles; n = 11). The solid curves are single-exponential functions
fitted to the data, with time constants of 144 msec (hNE) and 26 msec
(hSkM1). E, The time constants for recovery from
inactivation (squares) and development of inactivation
(circles) plotted as a function of voltage. Time
constants were estimated from single-exponential fits to time courses
measured with the protocols shown in A and
C for cells expressing hNE channels
(filled symbols; n = 15) and
hSkM1 channels (open symbols; n = 11). For comparison the inactivation time constants for the TTX-S
current in small DRG neurons are shown (dotted curve).
F, Basic multistate gating scheme for sodium channel
activation and fast inactivation. Closed states are indicated by
Cs, inactivated closed states are indicated by
ICs, the open state is indicated by O,
and the inactivated open state is indicated by IO. At
very negative potentials, channels reside in the leftmost closed state,
and with depolarization the channels progress toward the open state.
Channels can inactivate from any state.
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We also compared the time course for the development of inactivation of
hNE and hSkM1 channels. The protocol for these experiments is shown at
the top of Figure 3C. Cells were stepped
to the inactivation potential (from a holding
potential of 100 mV) for increasing durations and then stepped to the
test potential (20 mV) to measure the fraction of current remaining
available. Figure 3C shows representative currents
illustrating the development of inactivation at 80 mV for hNE and
hSkM1 channels. Although less than one-half of the hNE inactivation has
occurred after the 100 msec inactivating pulse (at 80 mV), hSkM1
inactivation is nearly complete at this time point. The average time
course for development of inactivation at 80 mV from 12 hNE and 11 hSkM1 cells is shown in Figure 3D. The data from these cells
were fitted well with a single-exponential function, and the time
constant was almost fivefold greater for hNE channels ( = 144 ± 11 msec; n = 12) than for hSkM1 channels ( = 26 ± 7 msec; n = 11).
The time course for recovery from inactivation was measured at voltages
ranging from 140 to 60 mV, and the time course for development of inactivation was measured from 90 to 40 mV for both
hNE and hSkM1. At most voltages both the rate of recovery from
inactivation and the development of inactivation were much slower for
hNE channels (Fig. 3E). The time constants for inactivation of hNE currents are similar to those measured for the TTX-S currents in
small DRG neurons (Fig. 3E, dotted curve)
(Cummins and Waxman, 1997). By contrast, the predominant time constants
measured for cloned rbII channels (Sakar et al., 1995) and the sodium
currents in adult hippocampal neurons (Costa, 1996) are similar to
those for the hSkM1 channels.
The difference between hNE and hSkM1 inactivation kinetics can be
considered in the context of a simple multistate sodium channel gating
scheme (Vandenberg and Bezanilla, 1991; Kuo and Bean, 1994) such as
that shown in Figure 3F. This model has multiple closed
states leading to the open state. Channels progress through these
closed states during depolarization. In this model, inactivation can
occur from any of the closed states as well as from the open state. The
rate of macroscopic inactivation at potentials positive to 20 mV is
thought primarily to reflect inactivation of channels in the open
state. The results shown in Figure 2B thus suggest that open-state inactivation is ~50% slower for hNE channels than it
is for hSkM1 channels. At potentials less than 60 mV, at which the
probability of channel opening is very low, inactivation would occur
primarily from the closed states. The time course for the development
of inactivation between 90 to 60 mV was much slower for hNE than
for hSkM1 channels, and therefore our data indicate that closed-state
inactivation is relatively slow for hNE channels.
Recovery from inactivation was also relatively slow for hNE currents
compared with hSkM1 currents. However, although the inactivation time
constants were dramatically different for hNE and hSkM1 currents, we
observed a discrepancy between the rate for development of inactivation
and the rate for recovery from inactivation when measured at the same
potential (from 90 to 60 mV). This discrepancy was observed for
both hNE and hSkM1 currents (Fig. 3E). At 80 mV, for
example, recovery from inactivation was ~30% faster than was
development of inactivation for hNE channels. Similarly, for hSkM1
channels recovery from inactivation at 80 mV was ~40% faster than
was development of inactivation at 80 mV. This discrepancy is not
completely unexpected, because the development of inactivation and
recovery from inactivation protocols examine distinct processes. Although development of inactivation at 80 mV exclusively involves closed-state inactivation, recovery from inactivation, also measured at
80 mV, follows a 20 msec depolarizing step, during which a significant proportion of the channels inactivate from the open state.
Thus recovery involves both open-state and closed-state inactivation
(Aldrich et al., 1983). Interestingly, although the values for
development of inactivation and for recovery from inactivation differed, the values did not differ markedly, and both processes could
be reasonably well fit with a single exponential (Fig.
3B,D). Kuo and Bean (1994) reported
that most sodium channels probably must close (i.e., make the IO to
IC transition) before recovering from inactivation. Thus the
rate of recovery from inactivation also probably reflects primarily the
rate of channel transition between the closed-inactivated and closed states.
Functional significance of slow recovery from inactivation
The slow rate for development of closed-state inactivation and
recovery from inactivation in hNE channels is intriguing, especially because it is so pronounced at voltages near the typical resting potential for neurons. Slower recovery from inactivation should decrease the maximum firing frequency. Figure
4 shows that during a 50 Hz pulse train
the current amplitude remains much higher for cells expressing hSkM1
channels (Fig. 4A) than for cells expressing hNE
channels (Fig. 4B). Although 45 ± 16% of the
peak current remains available for the second pulse and 36 ± 17%
remains for the 50th pulse for hSkM1 channels (n = 8),
only 23 ± 8 and 10 ± 5% remain available for the second
and 50th pulses, respectively, for hNE channels (n = 12). The lower availability for hNE channels occurs even though
macroscopic inactivation is ~50% slower for hNE channels and more
hSkM1 channels presumably inactivate during the 2 msec step
depolarizations. Thus, because hNE channels reprime more slowly than do
hSkM1 channels, a cell expressing a pure population of hNE channels
should not be capable of the high firing frequencies that might be
expected in a cell expressing hSKM1 channels. The firing rate is
limited by the repriming rate, which probably reflects the frequency of
transitions between the closed-inactivated and closed states.
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Figure 4.
Functional consequences of slow closed-state
inactivation. A, The sodium currents elicited in a cell
expressing hSkM1 channels by a 50 Hz pulse train. The cell, held at
80 mV, was depolarized to 0 mV for 2 msec every 20 msec during the 1 sec pulse train. Inset, Currents elicited by the first
and second depolarizations on an expanded time scale (calibration bar,
2 msec). The current elicited by the second pulse is approximately
one-half the amplitude of the current elicited by the first pulse.
B, The sodium currents elicited in a cell expressing hNE
channels by a 50 Hz pulse train. Details are as described in
A. The current elicited by the second pulse is much
smaller than that elicited by the first pulse. C,
Comparison of hSkM1 current elicited by a step depolarization to 0 mV
from 100 mV (left trace) with the hSkM1 current
elicited by a step depolarization from 50 mV that was preceded by a
slow ramp depolarization from 100 to 50 mV (right
traces). The voltage protocol is shown below the
current traces. Two different ramp speeds were used: 1 mV/msec (50 msec total duration) and 0.2 mV/msec (250 msec total
duration). After the 50 msec ramp, less than one-half of the current
remains available for activation, and after the 250 msec ramp, only
~10% of the current is available. D, Currents
elicited by the voltage protocols detailed in C in a
cell expressing hNE channels. After the 50 msec ramp, much of the hNE
current remains available for activation, and after the 250 msec ramp,
more than one-third of the current is still available.
Inset, The end of the 50 msec ramp depolarization at
higher gain. The arrow indicates a region where the ramp
depolarization elicits an inward current before the step
depolarization.
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Functional significance of slow development of inactivation
The slow rate for the development of closed-state inactivation in
hNE channels can also have important consequences. Because closed-state
inactivation occurs so slowly, especially at voltages from 90 to 50
mV, short prepulses are not sufficient to reach steady-state conditions
for hNE channels. For example, a 50 msec prepulse to 80 mV only
allows 30% of the channels to inactivate, compared with 97% for a 500 msec prepulse. As a consequence of this, if "steady-state
inactivation" is measured using 50 msec prepulses, the estimated
midpoint of the h curve is significantly more
positive for hNE channels (56 ± 2 mV) than for hSkM1 channels (70 ± 1 mV).
Based on this observation, we predicted that a large fraction of hNE
channels, but not hSkM1 channels, would remain available for activation
during slow ramp depolarizations. To test this, cells were slowly
depolarized from 100 to 50 mV and then stepped to 0 mV to determine
how much current remained available for activation. For hSkM1 channels
(Fig. 4C), 37 ± 19% (n = 9) of the
current remained available after a 50 msec (1 mV/msec) ramp, and only 10 ± 12% remained after a 250 msec (0.2 mV/msec) ramp. In
contrast, significantly more current remained available for hNE
channels (Fig. 4D), with 76 ± 10%
(n = 14) of the current available after the 50 msec
ramp and 36 ± 13% still available after the 250 msec ramp. Thus,
although neurons expressing faster repriming channels may be capable of
firing at higher frequencies, neurons expressing hNE channels should be
able to generate action potentials in response to slowly rising inputs
that do not elicit a regenerative response in neurons expressing
channels with fast closed-state inactivation.
Slow ramp currents can be evoked in hNE channels
As can be seen in the inset in Figure
4D, during some of the ramp and step experiments on
cells expressing hNE channels, a small inward current was observed
during the slow ramp. This was further examined using extended ramp
depolarizations that ranged from 100 to 40 mV. These slow ramps (0.23 mV/msec) elicited little or no current in cells expressing hSkM1
channels (Fig. 5A), but prominent currents were evoked in cells expressing hNE channels (Fig.
5B). The hNE ramp currents were 1.7 ± 0.2%
(n = 11) of the peak current amplitude (obtained in
response to stimulation with step depolarizations to 10 mV) and, when
compared with the scaled peak current-voltage relationship, reach
maximal amplitude at potentials ~20 mV more negative than the peak
currents (Fig. 5B). This type of ramp current, often
referred to as "subthreshold" or persistent current, has been
recorded in several different types of neurons (Stafstrom et al., 1985;
Brown et al., 1994; Cepeda et al., 1995; Chao and Alzheimer, 1995;
Fleidervish and Gutnick, 1996; Pennartz et al., 1997; Raman and Bean,
1997; Feigenspan et al., 1998; Parri and Crunelli, 1998). It has been
proposed that, because neuronal ramp currents occur near resting
potentials and are often larger than other voltage-gated currents at
these potentials, ramp currents may significantly influence
excitability (Crill, 1996). Indeed, it has been shown that persistent
sodium currents in the dendrites of neocortical neurons can help boost transmission of synaptic depolarizations (Schwindt and Crill, 1995).
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Figure 5.
Characterization of ramp currents. Ramp currents
were examined using 600 msec voltage ramps from 100 to +40 mV
(~0.23 mV/msec). A, The average ramp current recorded
in cells expressing hSkM1 channels (n = 12). The
thick solid line at the bottom
illustrates the ramp voltage protocol. The ramp current is plotted as a
percentage of the peak sodium current elicited with step
depolarizations from 100 mV. The error bar indicates the SE at 45
mV. B, The average ramp current recorded in cells
expressing hNE channels (n = 11). The ramp current
is plotted as a percentage of the peak sodium current elicited with
step depolarizations from 100 mV. The error bar indicates the SE at
45 mV. The dotted curve shows the averaged
current-voltage (I-V) relationship for the peak
current elicited with step depolarizations in these cells scaled to the
amplitude of the ramp current. The filled squares show
the peak I-V data at full scale. Only the foot of the
curve can be seen at this scale. The step depolarizations to 60 mV
elicited ~1.2% of the current elicited by the step depolarizations
to 15 mV. C, Current traces elicited in
an hNE cell by 600 msec ramps. The current (plotted as a percentage of
peak current) is shown before and after the addition of 250 nM TTX to the extracellular solution. TTX blocks the ramp
current. D, Current traces elicited in an
hNE cell by 600 msec ramps shown before and after the addition of
increasing concentrations of cadmium (50-500 µM
Cd2+) to the extracellular solution. Cadmium
increases the amplitude of the ramp current in hNE cells.
E, The average currents elicited by 600 msec ramps in
cells expressing hSkM1 channels shown before and after addition of 200 µM cadmium to the extracellular solution
(n = 4). Cadmium does not induce hSkM1 ramp
currents. F, The average currents elicited by 600 msec
ramps in cells expressing hNE channels shown before and after addition
of 200 µM cadmium to the extracellular solution
(n = 5). Cadmium increased the amplitude of the
ramp current by ~150%, and all of the ramp current in cadmium was
blocked by 250 nM TTX. Error bars indicate SE at 40
mV.
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The narrow voltage range in which hNE ramp currents are recorded is
very similar to that for ramp currents recorded in neurons. The
mechanism underlying the distinct voltage dependence of neuronal ramp
currents has not been completely understood. Because ramp currents seem
to be activated at more negative potentials than the currents elicited
with step depolarizations, it has been suggested that distinct channel
populations might underlie ramp currents and transient currents in
neurons. In the HEK293 cells the ramp and transient currents are
probably generated by the same channel isoform. Indeed, the apparent
difference in voltage dependence, at least for hNE channels, is an
artifact that results from the scaling of the peak current-voltage
curve to the size of the ramp current. The ramp current at 55 mV and
the peak current elicited with a step depolarization to 55 mV are
both ~1% of the maximum peak current (measured with step
depolarizations). When the peak current data are plotted at full scale
(Fig. 5B, solid squares), it is clear that
the foot of the peak current-voltage relationship closely matches the
voltage dependence of the onset of the ramp current.
Our data indicate that the ramp currents are observed at 60 mV
because hNE channels, with slow closed-state inactivation kinetics, do
not all inactivate during slow ramps and therefore some remain
available for activation. Conversely, although a step depolarization to
55 mV activates 1.9 ± 0.6% (n = 10) of the peak current for hSkM1 channels, almost no hSkM1 current is observed during slow ramp depolarizations because the hSkM1 channels undergo rapid closed-state inactivation and are inactivated during slow depolarizations before reaching the open state. The decay of the ramp
currents at more depolarized voltages probably reflects channels undergoing open-state inactivation, which is still relatively fast in
hNE channels. Therefore, our results suggest that the distinct voltage
dependence of ramp currents does not necessarily arise from unique
activation properties of the underlying sodium channels but rather from
their distinctive inactivation kinetics.
hNE ramp currents are differentially sensitive to TTX
and cadmium
To confirm that the ramp currents recorded in hNE cells were
sodium currents, we tested the pharmacology of the ramp currents with
nanomolar concentrations of TTX, which blocks hNE channels, and
micromolar concentrations of cadmium, which does not block hNE channels
(Klugbauer et al., 1995) but does block voltage-gated calcium channels.
Figure 5C shows that the hNE ramp currents were blocked by
250 nM TTX. The effect of cadmium on hNE ramp currents is
illustrated in Figure 5D. Surprisingly, cadmium increased
the size of the ramp currents, at concentrations that are equal to or
lower than those routinely used in the isolation of sodium currents
(Brown et al., 1994; Cepeda et al., 1995; Fleidervish and Gutnick,
1996; Pennartz et al., 1997; Raman and Bean, 1997; Parri and Crunelli,
1998). In five cells expressing hNE channels, 200 µM
cadmium increased the size of the ramp currents by 160 ± 17%,
and the total ramp current was also blocked by TTX (Fig. 5F). By contrast, cadmium did not induce ramp
currents in cells expressing hSkM1 channels (Fig. 5E).
To understand how cadmium increased ramp currents, we examined the
effect of cadmium on the other properties of hNE and hSkM1 currents.
Cadmium (200 µM) had little effect on hNE peak current amplitude, which was decreased by 5 ± 5% (n = 8). Cadmium also did not significantly alter noninactivating hNE
currents (0.35 ± 0.16% of peak, control; 0.29 ± 0.14% in
200 µM Cd2+; n = 6),
measured at 100 msec during a step depolarization to 0 mV. The
midpoints of activation (27 ± 2 mV, control; 27 ± 3 mV,
cadmium; n = 8) and steady-state inactivation
(77 ± 3 mV, control; 74 ± 4 mV, cadmium;
n = 8) for hNE currents were not significantly altered
by 200 µM cadmium. Similarly, cadmium did not alter these
properties in hSkM1 channels (data not shown).
However, as can be seen in Figure
6A, 200 µM cadmium did prolong the time course for the
development of closed-state inactivation in hNE channels. In seven
cells, 200 µM cadmium increased the time constant for the
development of inactivation at 80 mV by 61 ± 17% and increased
the time constant for recovery from inactivation at 80 mV by 46 ± 12%. These differences were significant (paired t test,
p < 0.005). In contrast, 200 µM cadmium
did not affect the time course for development of closed-state
inactivation in hSkM1 channels (Fig. 6B). Even at
higher concentrations (500 µM), cadmium had little effect
on hSkM1 channels, increasing the time constant for the development of
inactivation at 80 mV by only 7 ± 6% and the time constant for
recovery from inactivation at 80 mV by only 6 ± 4%. Although
Figure 6C shows that even 100 µM cadmium
greatly increased the inactivation time constants for hNE channels at
voltages ranging from 100 to 50 mV, Figure 6D demonstrates that 500 µM cadmium had little effect on the
inactivation time constants of hSkM1 channels in this voltage range.
This demonstrates that the lack of effect of cadmium on hSkM1 channels
was not simply a slight difference in sensitivity to cadmium.
Interestingly, cadmium had no effect on the rate of decay of
macroscopic currents (evoked by step depolarizations ranging from 30
to +30 mV) for either hNE channels (200 µM; Fig.
6E) or hSkM1 channels (500 µM; Fig.
6F). This indicates that cadmium primarily slows
closed-state inactivation but not open-state inactivation of hNE
channels. This also provides additional evidence to support the
hypothesis that slow closed-state inactivation of hNE channels
underlies the generation of ramp currents.
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Figure 6.
Cadmium modulates closed-state inactivation in hNE
but not in hSkM1 channels. A, Cadmium slows the
development of sodium current inactivation in a cell expressing hNE
channels. The time course for development of inactivation at 80 mV is
shown before (open squares) and after
(filled squares) addition of 200 µM
Cd2+ to the extracellular solution. The solid
curves are single-exponential functions fit to the hNE data
before ( = 96 msec) and after ( = 155 msec) cadmium.
B, Cadmium does not slow the development of sodium
current inactivation in a cell expressing hSkM1 channels. The time
course for development of inactivation at 80 mV is shown before
(open circles; = 29 msec) and after
(filled circles; = 26 msec) addition of 200 µM Cd2+ to the extracellular solution.
Please note the difference in the x-axis scales between
A and B. C, The
inactivation time constants between 140 and 40 mV for
Na+ currents in cells expressing hNE cells
(n = 5) are shown before (open
squares) and after (filled squares)
addition of 100 µM Cd2+ to the
extracellular solution. At voltages at which both development of
inactivation and recovery from inactivation were measured (i.e.,
between 60 and 90 mV), the inactivation time constant was estimated
by averaging the development of inactivation and recovery from
inactivation time constants. D, The inactivation time
constants between 140 and 40 mV for Na+ currents
in cells expressing hSkM1 cells (n = 4) are shown
before (open circles) and after (filled
circles) addition of 500 µM
Cd2+ to the extracellular solution. Note the
difference in the y-axis scales between C
and D. E, The inactivation time constants
for open-state inactivation (h) measured from
single-exponential fits to the decay of currents elicited by step
depolarizations to voltages between 30 and +30 mV for
Na+ currents in cells expressing hNE channels
(n = 6) are shown before (open
squares) and after (filled squares)
addition of 200 µM Cd2+ to the
extracellular solution. Inset, Current
traces are from a representative hNE cell before
(solid trace) and after (dashed trace)
cadmium. F, Plots of h
measured at voltages between 30 and +30 mV for Na+
currents in cells expressing hSkM1 channels (n = 4)
are shown before (open circles) and after
(filled circles) addition of 500 µM
Cd2+ to the extracellular solution.
Inset, Current traces are from a
representative hSkM1 cell before (solid trace) and after
(dashed trace) cadmium.
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Some, but not all, divalent cations modulate hNE channels, and this is
illustrated in Figure 7. Zinc, like
cadmium, increased hNE ramp currents (Fig. 7A) and also
increased the inactivation time constants for closed-state inactivation
at negative potentials (Fig. 7B). Cobalt (200 µM) had a slightly less pronounced effect (data not
shown) then did cadmium and zinc, whereas barium (200 µM)
had virtually no effect on hNE ramp currents (Fig. 7C) and on hNE inactivation time constants (Fig. 7D). These data
also support the link between slow closed-state inactivation and the generation of ramp currents.
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Figure 7.
Zinc, but not barium, also modulates hNE
closed-state inactivation and increases hNE ramp currents.
A, Current traces elicited in an hNE cell
by 600 msec ramps are shown before and after the addition of increasing
concentrations of zinc (50-500 µM) to the extracellular
solution. Zinc increases the amplitude of the ramp current in hNE
cells. B, The inactivation time constants between 140
and 40 mV for Na+ currents in cells expressing hNE
cells (n = 4) are shown before
(filled squares) and after (open
diamonds) addition of 100 µM
Zn2+ to the extracellular solution.
C, Currents elicited by 600 msec ramps in an hNE cell
are shown before and after addition of 200 µM barium to
the extracellular solution. Barium does not increase hNE ramp currents
(n = 4). D, The inactivation time
constants between 140 and 40 mV for Na+ currents
in cells expressing hNE (n = 6) are shown before
(filled squares) and after (open
triangles) addition of 200 µM
Ba2+ to the extracellular solution.
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Ramp currents and slow closed-state inactivation in
DRG neurons
Because hNE is expressed in a majority of small DRG neurons (Black
et al., 1996) and the TTX-S sodium current in small DRG neurons has
slow closed-state inactivation kinetics (Elliott and Elliott, 1993;
Cummins and Waxman, 1997), we tested whether cadmium also modulated DRG
TTX-S currents. We used 100 µM cadmium for these
experiments because this concentration was used in studies by others
(Parri and Crunelli, 1998) and in our previous studies on repriming
kinetics in small DRG neurons (Cummins and Waxman, 1997). Figure
8, A and B, shows
that cadmium slowed the development of inactivation for the TTX-S
current in small DRG neurons. Cadmium had a dramatic effect on the time
constants of inactivation for DRG TTX-S channels at potentials ranging
from 100 to 60 mV (Fig. 8C). Small DRG neurons also
displayed ramp currents that were increased by cadmium and blocked by
TTX (Fig. 8D). In four cells, 100 µM
cadmium increased the amplitude of the ramp current by 45 ± 6%.
The ramp current recorded in 100 µM cadmium was 1.9 ± 0.3% (n = 6) of the peak TTX-S fast sodium current
in small DRG neurons, compared with 1.7 ± 0.2%
(n = 9) for hNE ramp currents in 100 µM
cadmium.
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Figure 8.
Cadmium modulates closed-state inactivation and
ramp currents in DRG neurons. A, Families of current
traces from a small DRG neuron (21 µm in diameter)
before (left) and after (right)
application of 100 µM Cd2+ to the
extracellular solution show that the rate of development of
inactivation at 80 mV is slowed by Cd2+. The TTX-S
peak current amplitude was 98% of the total peak current amplitude in
this cell. B, Time course for development of
inactivation of the peak current before (filled
squares) and after (open circles) application of
100 µM Cd2+. Data are from the
currents shown in A. C, The inactivation
time constants between 140 and 40 mV for TTX-S
Na+ currents in small (18-25 µm) DRG neurons from
adult rats (n = 6) are shown before
(filled squares) and after (open
squares) addition of 100 µM
Cd2+ to the extracellular solution. Data were
obtained with the same two-pulse protocol described in Figure
3C. The TTX-S peak current amplitude was 75 ± 4%
(n = 6) of the total peak current amplitude for
these cells. D, Ramp current recorded in a small DRG
neuron before (left) and after (right)
addition of 100 µM Cd2+ to the
extracellular solution. Cd2+ increases the ramp
current component that peaks near 40 mV. This component is blocked by
250 nM TTX (right). A second component
(left; marked by the asterisk) is
apparently blocked by Cd2+ and may therefore be a
calcium current. The ramp, which extended from 100 to +40 mV, was 600 msec long. The scale bar indicates the percentage of the peak current
amplitude measured with a step depolarization to 10 mV.
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There is an apparent difference between the voltage dependence of DRG
TTX-S peak current elicited with step depolarizations and that of the
ramp current (Fig. 9A).
However, as was shown for hNE currents in Figure 5B, this
apparent difference is an artifact that results from the scaling of the
peak current curve to the size of the ramp currents. The solid
squares in Figure 9A show that when the peak
current data are plotted at full scale, the threshold for activation of
DRG TTX-S currents elicited with step depolarizations is similar to
that for the DRG TTX-S ramp current. Although multiple sodium channels
probably contribute to the excitability of DRG neurons, these data
support the hypothesis that a single sodium channel isoform can
underlie both a peak transient TTX-S current and a TTX-S ramp current
and argue against the need to invoke multiple channel populations to
account for these currents. Figure 9B shows that the voltage
dependences of the TTX-S ramp and peak currents in small DRG neurons
were nearly identical to those of the hNE currents recorded in HEK293
cells under the same conditions. This is consistent with the data
indicating that the hNE transcript is expressed in a majority of small
neurons. Because hNE channels and DRG TTX-S currents both exhibit slow closed-state inactivation and because cadmium can modulate closed-state inactivation and increase ramp currents in both DRG neurons and in
HEK293 cells expressing hNE channels, a similar mechanism probably underlies the ramp currents in both preparations.
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Figure 9.
The thresholds for activation of ramp currents and
peak transient currents are similar. A, The ramp current
recorded in a small DRG neuron expressing predominantly TTX-S sodium
current. The ramp current is plotted as a percentage of the peak sodium
current elicited with step depolarizations from 100 mV. The
dotted curve shows the current-voltage
(I-V) relationship for the peak current elicited
with step depolarizations in this cell scaled to the amplitude of the
ramp current. The filled squares show the peak
I-V data at full scale; only the foot of the curve can
be seen at this scale. The ramp, which extended from 100 to +40 mV,
was 600 msec long. The access resistance for this cell was 2 M, and
80% series resistance compensation was used. B,
Comparison of ramp currents in small DRG neurons that expressed
predominantly TTX-S currents (n = 5) and in HEK293
cells expressing hNE channels (n = 9). The
currents, elicited with 600 msec ramps that extended from 100 to +40
mV, were normalized for comparison of voltage dependence. The peak
current-voltage curves (dotted and dashed
lines) for the cells from which the ramp currents were
recorded are also shown. Note that both the DRG TTX-S and the hNE ramp
currents reach maximum amplitude ~20 mV more negative than did the
peak currents.
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DISCUSSION |
We have compared the functional properties of hNE and hSkM1 sodium
channels expressed in HEK293 cells. Our data show that although the
voltage dependences of activation and steady-state inactivation are
similar for hNE and hSkM1 channels, hNE channels display slower
open-state inactivation, slower closed-state inactivation, and slower
recovery from inactivation than do hSkM1 channels. We also observed
relatively large TTX-sensitive currents during ramp depolarizations in
cells expressing hNE, but not hSkM1, channels.
Mechanism underlying hNE ramp currents
Our results indicate that ramp currents arise in hNE channels
because the hNE channels have slow closed-state inactivation. Closed-state inactivation and recovery from inactivation were up to
500% slower for hNE channels than for hSkM1 channels. Because closed-state inactivation develops much more slowly for hNE than for
hSkM1 channels, hNE channels are less likely to inactivate during slow
subthreshold depolarizations and are more likely to be available to
open when the voltage reaches threshold. This mechanism is consistent
with the voltage dependence of the ramp currents. As Figure
5B shows, the ramp currents and the currents elicited with
step depolarizations have similar thresholds.
Although the generation of ramp currents by slow closed-state
inactivation can be described by a model such as that shown in Figure
3F, it should be noted that this model is probably
incomplete. For example, hNE channels also display slow inactivation
(data not shown), a kinetically and functionally distinct process from fast inactivation. TTX-S currents in rabbit Schwann cells (from which
NaS, the rabbit ortholog of hNE, was cloned) exhibit at least three
kinetically distinct types of inactivation (Howe and Ritchie, 1992).
Therefore it is likely that hNE, PN1, and NaS exhibit complex
inactivation characteristics in addition to the distinctive
closed-state inactivation properties described here.
Our data do not eliminate the possibility that persistent currents can
also arise from other sodium channel isoforms or from different
mechanisms. Several mechanisms have been proposed to underlie
persistent sodium currents in neurons: (1) window currents, (2)
modal gating, and (3) distinct channel isoforms (Crill, 1996). Indeed,
in DRG neurons persistent window currents can arise from TTX-resistant channels (Cummins and Waxman, 1997), and Alzheimer et al. (1993) demonstrated with single-channel recordings that CNS
sodium channels can generate persistent currents by switching between
different gating modes. Although these previously described mechanisms
may provide an explanation for some of the persistent sodium current in
neurons, our results show that persistent threshold currents can be
generated by a fourth mechanism, slow closed-state inactivation.
Indeed, this mechanism can account for the threshold ramp currents that
have been observed in many neuronal preparations without invoking
distinct channel populations.
It is not clear which channel structures are responsible for the slow
closed-state inactivation in hNE. Several studies (Ji et al., 1996;
Dib-Hajj et al., 1997; Chen et al., 1998) have implicated the S3-S4
linker of domain 4 as being important in repriming kinetics. Interestingly, there is a threonine (T1590) in the domain 4 S3-S4 linker of hNE (and NaS) at a position where most of the other sodium
channel isoforms, including hSkM1 and rBII, have a lysine. However, rat
PN1 (Sangameswaran et al., 1997; Toledo-Aral et al., 1997) also has a
lysine at this position, suggesting that other parts of the channel
might determine the slow closed-state inactivation of hNE channels. The
only consistent difference between the hNE, NaS, and PN1 clones and
hSkM1 and rBII in a region previously identified as playing a prominent
role in inactivation is in the S4-S5 linker of domain 3 (Yang et al.,
1994). Although hNE, NaS, and PN1 have an isoleucine at position 1304, hSkM1 and rBII have a leucine. This is a conservative substitution, but
Smith and Goldin (1997) recently published compelling evidence
indicating that the nearby alanine at position 1302 (hNE numbering)
interacts directly with the putative inactivation particle for rBII.
Divalent modulation of closed-state inactivation and
ramp currents
The hypothesis that slow closed-state inactivation underlies ramp
currents is supported by our data demonstrating that hNE currents are
modulated by cadmium and zinc. These data show that cadmium and zinc
increase the time constants for closed-state inactivation of the hNE
sodium channels and increase the amplitude hNE ramp currents but have
little effect on other channel properties. Although cadmium had no
significant effect on hSkM1 currents, cadmium and zinc may modulate the
development of inactivation and recovery from inactivation for other
neuronal sodium channel isoforms. Indeed we have seen similar effects
on rbII channels expressed in Chinese hamster ovary cells (T. R. Cummins, J. R. Howe, and S. G. Waxman, unpublished
observations). Cadmium is commonly used in studies of sodium
currents because at 0.1-0.5 mM concentrations it blocks
calcium channels but not neuronal sodium currents (Klugbauer et al.,
1995; Fozzard and Hanck, 1996). Virtually all of the previous studies
on TTX-sensitive ramp currents in neurons used cadmium in the
extracellular solution. Our results show that cadmium should be used
cautiously when studying sodium currents.
In addition to the mechanistic implications of the cadmium and zinc
modulation, these effects might also have physiological relevance.
Cadmium has been shown to induce pathological changes in the CNS (Wong
and Klaassen, 1982) and in sensory ganglia and peripheral nerve
(Gabbiani et al., 1967; Sato et al., 1978). Although there is evidence
suggesting that the Cd2+-induced injury of white
matter may result from disruption of mitochondrial function (Fern et
al., 1996), our data raise the possibility that the enhancement of
threshold sodium currents [which are known to contribute to white
matter injury (Stys et al., 1993)] by cadmium might also contribute to
the toxicity of cadmium. The modulation of sodium currents by zinc is
intriguing for several reasons. Zinc can be coreleased with
neurotransmitters at synapses (Frederickson and Moncrieff, 1994),
raising the possibility that zinc could act as a modulator of dendritic
sodium currents. It has also been reported that zinc can affect
susceptibility to epileptic seizures (Fukahori and Itoh, 1990) and can
modulate nociceptive impulse trafficking (Izumi et al., 1995), which
involves small DRG neurons.
Functional consequences of slow closed-state inactivation and
ramp currents
We have shown that hNE currents are similar to the TTX-S current
in small DRG neurons, particularly with respect to the slow rate of
closed-state inactivation and the properties of the ramp currents. The
distinct properties of hNE sodium channels are expected to have
important consequences for cellular excitability. For example, a cell
expressing only hNE sodium channels would not be expected to be able to
sustain high repetitive firing rates that might be sustained by a cell
expressing hSkM1 channels. Conversely, the hNE cell would be expected
to respond to slow depolarizing inputs that the hSkM1 cell could not
respond too. Gilly and Armstrong (1984) proposed that threshold sodium
currents could play an important role in impulse initiation and
pacemaking. Because DRG TTX-S and hNE ramp currents can be evoked at
potentials close to the resting potential of DRG neurons, they might
contribute to the TTX-sensitive oscillations in resting membrane
potential that have been observed in these cells (Study and Kral, 1996;
Kapoor et al., 1997). Baker and Bostock (1997) proposed that persistent
threshold sodium currents in DRG neurons might alternatively play an
important role in amplifying depolarizing inputs. This is an intriguing
possibility, especially because hNE transcripts (in contrast to other
sodium channel transcripts) can be detected in virtually all DRG
neurons (Black et al., 1996) and PN1 (the rat ortholog of hNE)
immunoreactivity is reportedly highest in the growth cones of cultured
rat DRG neurons (Toledo-Aral et al., 1997), suggesting that hNE/PN1/NaS
is targeted to nerve terminals. This would situate it ideally for
amplifying excitatory inputs.
Belcher et al. (1995) and Sangameswaran et al. (1997) reported that
hNE/PN1/NaS mRNA can also be detected in CNS tissues, raising the
possibility that this isoform could underlie threshold currents in
other neuronal populations. Slow ramp depolarizations have been shown
to induce TTX-sensitive ramp currents in many CNS neurons, including
neocortical neurons (Stafstrom et al., 1985; Brown et al., 1994;
Fleidervish and Gutnick, 1996), thalamocortical neurons (Parri and
Crunelli, 1998), suprachiasmatic neurons (Pennartz et al., 1997),
neostriatal neurons (Cepeda et al., 1995; Chao and Alzheimer, 1995),
cerebellar Purkinje cells (Raman and Bean, 1997), and retinal amacrine
cells (Feigenspan et al., 1998). Although CNS sodium currents seem to
have predominantly fast repriming kinetics, slowly repriming sodium
currents have been recorded in CNS neurons (Martina and Jonas, 1997).
Sodium currents with slow repriming may be especially important in
dendrites of CNS neurons (Colbert et al., 1997; Jung et al., 1997).
However, some studies indicate that hNE or PN1 expression is restricted
to the peripheral nervous system (Felts et al., 1997; Toledo-Aral et al., 1997), and it is likely that other isoforms can contribute to the
ramp currents in CNS neurons. Indeed, recent evidence has indicated
that Na6 underlies a large proportion of the ramp current in cerebellar
Purkinje cells (Raman et al., 1997). It is not known whether Na6
channels, or any of the other channels isolated from brain, also have
slow closed-state inactivation.
Conclusion
We have studied Na+ currents produced by the
hNE or PN1 sodium channel and have shown that hNE channels have slow
closed-state inactivation. Our data show that this provides a
previously unrecognized mechanism for the generation of threshold ramp
currents and that these ramp currents are subject to modulation. Our
results also demonstrate the presence of ramp currents, with voltage
dependence and pharmacological characteristics very similar to those of
hNE currents, in DRG neurons, which express PN1. Because threshold ramp
currents might play a role in the amplification of synaptic inputs,
impulse initiation or the generation of pacemaker potentials in
neurons, expression of PN1 and this novel modulation could influence
the excitability of DRG neurons. Based on these observations, we
propose that the kinetics of sodium channel closed-state inactivation may be an important factor in determining the integrative and firing
properties of neurons.
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FOOTNOTES |
Received June 23, 1998; revised Sept. 11, 1998; accepted Sept. 11, 1998.
This work was supported in part by the Medical Research Service,
Department of Veterans Affairs, and by a grant from the National Multiple Sclerosis Society. T.R.C. was supported in part by a fellowship from the Paralyzed Veterans of America Spinal Cord Research Foundation.
Correspondence should be addressed to Dr. Stephen G. Waxman, Department
of Neurology, LCI 707, Yale University School of Medicine, 333 Cedar
Street, New Haven, CT 06510.
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REFERENCES |
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Akopian AN,
Sivilotti L,
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A tetrodotoxin-resistant voltage-gated sodium channel expressed by sensory neurons.
Nature
379:257-262
Top
Abstract
Introduction
