 |
 Previous Article | Next Article 
The Journal of Neuroscience, August 15, 2001, 21(16):5952-5961
Nav1.3 Sodium Channels: Rapid Repriming and Slow
Closed-State Inactivation Display Quantitative Differences after
Expression in a Mammalian Cell Line and in Spinal Sensory Neurons
Theodore R.
Cummins,
Fabio
Aglieco,
Mathurkrisnan
Renganathan,
Raimund I.
Herzog,
Sulayman D.
Dib-Hajj, and
Stephen G.
Waxman
Department of Neurology and Paralyzed Veterans of America/Eastern
Paralyzed Veterans Association Neuroscience Research Center, Yale
Medical School, New Haven, Connecticut 06510, and Rehabilitation
Research Center, Veterans Connecticut Healthcare Center, West Haven,
Connecticut 06516
 |
ABSTRACT |
Although rat brain Nav1.3 voltage-gated sodium channels have been
expressed and studied in Xenopus oocytes, these channels have not been studied after their expression in mammalian cells. We
characterized the properties of the rat brain Nav1.3 sodium channels
expressed in human embryonic kidney (HEK) 293 cells. Nav1.3 channels
generated fast-activating and fast-inactivating currents. Recovery from
inactivation was relatively rapid at negative potentials (< 80 mV)
but was slow at more positive potentials. Development of closed-state
inactivation was slow, and, as predicted on this basis, Nav1.3 channels
generated large ramp currents in response to slow depolarizations.
Coexpression of  3 subunits had small but significant effects on the
kinetic and voltage-dependent properties of Nav1.3 currents in HEK 293 cells, but coexpression of 1 and 2 subunits had little or no
effect on Nav1.3 properties. Nav1.3 channels, mutated to be
tetrodotoxin-resistant (TTX-R), were expressed in SNS-null dorsal root
ganglion (DRG) neurons via biolistics and were compared with the same
construct expressed in HEK 293 cells. The voltage dependence of
steady-state inactivation was ~7 mV more depolarized in SNS-null DRG
neurons, demonstrating the importance of background cell type in
determining physiological properties. Moreover, consistent with the
idea that cellular factors can modulate the properties of Nav1.3, the
repriming kinetics were twofold faster in the neurons than in the HEK
293 cells. The rapid repriming of Nav1.3 suggests that it contributes
to the acceleration of repriming of TTX-sensitive (TTX-S) sodium currents that are seen after peripheral axotomy of DRG neurons. The
relatively rapid recovery from inactivation and the slow closed-state inactivation kinetics of Nav1.3 channels suggest that neurons expressing Nav1.3 may exhibit a reduced threshold and/or a relatively high frequency of firing.
Key words:
ion channel; -subunits; spinal sensory neurons; biolistics; nerve injury; ramp current
| |
INTRODUCTION |
Voltage-gated sodium channels play a
critical role in excitable cells. Sodium channels underlie the rapid
action potentials that are characteristic of neurons and muscle cells
and may contribute to subthreshold currents that modulate excitability.
At least nine distinct voltage-gated sodium channels have been cloned
from mammals (Black and Waxman, 1996 ; Goldin et al., 2000), and mRNA for almost all of these different channels is found in neurons. Many of
these channels have specific developmental, tissue, or cellular
distributions. Expression of recombinant channels in Xenopus
oocytes and mammalian cells indicates that the different channels
also can have distinct kinetic and voltage-dependent properties.
For example, Smith and Goldin (1998) have shown that, whereas Nav1.1
and Nav1.2 channels both encode fast sodium currents, the voltage
dependence of activation and of steady-state inactivation is
significantly more positive for the Nav1.1 channels. Cummins et al.
(1998) showed that, whereas Nav1.7 and Nav1.4 channels expressed in
human embryonic kidney (HEK) 293 cells have similar voltage-dependent
properties, Nav1.7 channels display much slower closed-state
inactivation kinetics than Nav1.4 channels. This difference in
closed-state inactivation endows Nav1.7 channels with the ability to
generate ramp currents in response to slow depolarizations, a property
that may permit them to play an important role in boosting subthreshold
stimuli. Thus differences in the kinetic and voltage-dependent
properties of voltage-gated sodium channels may be an important
determinant of the integrative and firing properties of neurons.
Nav1.3 neuronal channels are expressed primarily at early stages during
development and are almost undetectable in the normal adult nervous
system (Felts et al., 1997). However, Nav1.3 channels are reexpressed
in adult neurons after some types of injury, such as axotomy (Waxman et
al., 1994) and kainate-induced seizures (Gastaldi et al., 1997). The
levels of Nav1.3 mRNA and protein are increased in sensory neurons
after transection of their peripheral, but not their central, axons
(Black et al., 1999). The increase in Nav1.3 protein and mRNA levels
after peripheral axotomy is paralleled by a switch from
tetrodotoxin-sensitive (TTX-S) current with slow recovery from
inactivation to TTX-S current that recovers fourfold faster in small
dorsal root ganglion (DRG) neurons (Cummins and Waxman, 1997), a change
that is expected to increase the ability of these neurons to sustain
repetitive firing (see, for example, Chahine et al., 1994; Yang et al.,
1994). These results suggest that the functional properties of Nav1.3
sodium channels may play an important role in the DRG neuron
hyperexcitability that can occur after nerve injury. Therefore, we were
interested in determining the voltage-dependent and kinetic properties
of Nav1.3 channels expressed in mammalian cells.
In this study we first examined the electrophysiological properties of
Nav1.3 channels expressed alone and with -subunits in a mammalian
cell line, HEK 293 cells. Because there are data suggesting that the
properties of sodium channels depend on the type of cell in which they
are expressed, we also wished to express Nav1.3 channels in DRG
neurons. To facilitate identification of the currents produced by these
channels, we created a tetrodotoxin-resistant (TTX-R) Nav1.3 construct
and used biolistics to express it in SNS-null DRG neurons in which
other TTX-R sodium currents are not expressed. Our results show that
the physiological properties of Nav1.3 channels depend on the cell type
in which they are expressed and indicate that Nav1.3 channels
contribute to rapidly repriming TTX-S current in axotomized DRG neurons.
| |
MATERIALS AND METHODS |
Construction of mammalian expression vectors encoding
neuronal rat Nav1.3 (rNav1.3) channel. The insert of rNav1.3 was
moved from the bacterial expression pBluescript
SK
(pBS-SK) plasmid (Joho et al., 1990)
into a mammalian expression vector pcDNA3.1 (Invitrogen, San Diego, CA)
that was modified to render it a low copy number plasmid (Klugbauer et
al., 1995). The open reading frame of rNav1.3 was removed from the
bacterial expression plasmid by a SalI/NotI
double digest. The SalI end was polished to produce a blunt
end, and the fragment was cloned into the modified pcDNA3 vector cut
with HindIII and NotI. The HindIII
site of the vector was polished to receive the polished SalI
site of the insert. The insert was sequenced to confirm its integrity.
When compared with the published sequence (Kayano et al., 1988),
multiple nucleotide substitutions were detected as described previously
(Joho et al., 1990). The construct was modified later to delete the
3'-untranslated region downstream from the translation termination
codon TAA.
A PCR-based mutagenesis and cloning method (Horton et al., 1993) was
used to replace the amino acid tyrosine at position 384 with serine
(Y384S) to make this channel resistant to TTX. One wild-type primer
pair (T7 in the vector and R1) and one mutagenic pair (M1 and M2) were
used to introduce the Y384S substitution. The T7 primer, upstream of
the cloning site in the vector, was used as the forward wild-type
primer. The wild-type reverse primer R1
(5'-CGACTTGGGAAACCTGTCTCCATCG-3') corresponds to nucleotides 1991-1967. Forward mutagenic primer M1
(5'-ACTCAGGACTCCTGGGAGAATCTTTAC-3'; the A-to-C substitution,
in boldface type, changes the tyrosine TAC codon to serine codon TCC)
corresponds to nucleotides 1554-1580. Reverse mutagenic primer M2
(5'-ATTCTCCCAGGAGTCCTGAGTCATGAG-3'; a T-to-G substitution,
in boldface type, complements the change in the mutagenic forward
primer) corresponds to nucleotides 1574-1548. PCR was performed as
described previously (Dib-Hajj et al., 1997). The final PCR product
carrying the Y384S substitution was cut with the restriction enzymes
NheI (in the vector) and SacII in the insert;
then the fragment was used to replace the corresponding fragment from
the wild-type insert. The mutation was confirmed by sequencing the
whole Nav1.3 insert. Sequencing of the mutant insert revealed two
additional mutations in the S5-S6 linker upstream of the Y384
position: a glycine-to-arginine change at position 349 (G349R) and a
conservative change of isoleucine to leucine at position 351 (I351L).
These changes are not expected to affect the TTX phenotype of the channel.
Construction of mammalian expression vectors encoding human 2
and rat 3 subunits. A PCR fragment encoding the sodium channel 2 subunit (Eubanks et al., 1997) was amplified from a human DRG template by using a forward primer (5'-CTGAAAATGCACAGAGATGCCTGG-3', which corresponds to nucleotides 167-190) and reverse primer
(5'-CACTACTTGGCGCCATCATCCG-3', which corresponds to nucleotides
822-801). The PCR product first was cloned into pGEM-T (Promega,
Madison, WI) and later moved as an EcoRI insert into
pcDNA3.1 (Invitrogen) for expression in mammalian cells. The sequence
of the insert matched the published sequence except for a T-to-C
nucleotide substitution that replaced serine at position 44 of the
polypeptide by a proline (S44P). It is not clear whether this change
was introduced by the amplification/cloning process or whether it
reflects a naturally occurring polymorphism.
A PCR fragment encoding the sodium channel 3 subunit (Morgan et al.,
2000) was amplified from a rat DRG template with a forward primer
(5'-AAGATGCCTGCCTTCAACAGATTGCTTC-3', which corresponds to nucleotides
3-30) and reverse primer (5'-CACCACATTATTCCTCCACAGGTAC-3', which
corresponds to nucleotides 670-647). The PCR product was cloned into
pTarget (Promega). The sequence of the insert matched the published
sequence. The cloning of the 1 subunit construct has been described
previously (Tong et al., 1993; Bendahhou et al., 1995).
Transfection of HEK 293 cells. Transfections of HEK 293 cells were performed via the calcium phosphate precipitation technique as described previously (Cummins et al., 1998). Green fluorescent protein (GFP) was used to select for transfected cells, which subsequently were tested for channel expression by whole-cell patch-clamp recording techniques.
Axotomized DRG neurons. Adult rats were anesthetized with
sodium pentobarbital (60 mg/kg of body weight), and the right sciatic nerves were exposed at the mid-thigh level, ligated with 4-0 silk sutures, and transected; the proximal stumps were placed in silicon cuffs to prevent regeneration (Waxman et al., 1994). Hydroxystilbamine methanesulfonate (4% w/v; Molecular Probes, Eugene, OR), the active component of Fluorogold and a retrogradely transported fluorescent label, was placed in all cuffs before insertion of the nerve stump. The
fluorescent label identified neurons that gave rise to axons that were transected.
Culture of DRG neurons. Axotomized DRG cells were studied
after short-term culture (12-24 hr). The short-term culture provided cells with truncated axonal processes that can be voltage clamped readily and reliably and also allowed the cells sufficient time to
adhere to the glass coverslip. Adult rat DRG neurons maintained in vitro for 24 hr display a profile of sodium channel mRNA
expression similar to that for DRG neurons in situ,
indicating that short-term culture does not alter the expression of
sodium channel mRNAs substantially in these cells (Black et al., 1996).
Briefly, the L4 and L5 DRG ganglia were harvested from adult male
Sprague Dawley rats. The DRG were treated with collagenase A (1 mg/ml)
for 25 min and collagenase D (1 mg/ml) and papain (30 U/ml) for 25 min, 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.
Biolistic transfection of SNS-null DRG neurons. The Helios
Gene Gun System (Bio-Rad Laboratories, Hercules, CA) was used for the
biolistic transfection of neurons with DNA-coated gold particles. In
the presence of a 0.05 M solution of the polyanion
spermidine, 10 µg of Nav1.3-TTX-R DNA was mixed with 5 µg of GFP
DNA and coprecipitated onto 1 µm gold particles with
CaCl2. The DNA gold suspension was washed twice
in 100% ethanol, resuspended in 0.05% polyvinylpyrrolidone in
ethanol, and used for coating the inner wall of 10 inches of Tefzel
tubing (Bio-Rad Laboratories). The tubing was dried by using ultrapure
nitrogen and was cut into ~20 cartridges for the Helios Gene Gun.
This process resulted in a density of 1 mg of gold particles per shot
and 0.75 µg of total DNA per cartridge.
DRG neurons were isolated from SNS-null mice (Akopian et al., 1999) by
the same procedure described for the axotomized rat DRG neurons, with
the exception that the SNS-null neurons were kept under standard tissue
culture conditions for 3-5 d before biolistic transfections. We have
shown previously that SNS-null DRG neurons express persistent TTX-R
sodium currents (Cummins et al., 1999), but these currents are
typically <1 nA after several days in culture and run down quickly
(<10 min) in whole-cell recording configuration; therefore, these
persistent TTX-R currents are not significant under the culture and
recording conditions that were used in the present study. Just before
biolistic transfection the culture medium was removed from the Petri
dish. The gene gun was held 1 cm above the cells, and a pressure of
~120 psi was used to deliver the gold particles to the cells. A 70 µm nylon mesh (Small Parts, Miami, FL) was placed just in front of
the Helios Gene Gun barrel liner to achieve a more uniform distribution of gold particles (Wellmann et al., 1999).
Within 24 hr the cells usually showed expression of GFP, indicating a
successful biolistic transfection. Electrophysiological studies were
conducted 18-48 hr after transfection, and most of the cells that
expressed GFP also expressed fast-inactivating TTX-R sodium currents.
Because these currents are not observed in untransfected SNS-null
neurons or SNS-null neurons transfected with just GFP-coated gold
particles, this confirmed that most of the cells that expressed GFP
also had been cotransfected successfully with the Nav1.3-TTX-R channel.
Whole-cell patch-clamp recordings. Whole-cell patch-clamp
recordings were conducted at room temperature (~21°C) with an EPC-9 amplifier. Data were acquired on a Windows-based Pentium III computer with the Pulse program (v 8.1, HEKA Electronics, Lambrecht, Germany). Fire-polished electrodes (0.8-1.5 M ) were fabricated from 1.7 mm
capillary glass, using a Sutter P-97 puller (Novato, CA). To minimize
space-clamp problems, we selected only isolated cells with a
soma diameter of <25 µm for recording. Cells were not considered for
analysis if the initial seal resistance was <2 G, if they had high
leakage currents (holding current >0.1 nA at 80 mV for HEK 293 cells; >0.5 nA for DRG neurons), or an access resistance >4 M. The
average access resistance was 1.7 ± 0.6 M (mean ± SD).
Voltage errors were minimized by using 80-90% series resistance compensation, and the capacitance artifact was canceled by 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 usually
were 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 the cells
were patched.
Data analysis. Data were analyzed with the Pulsefit (HEKA
Electronics) and Origin (Microcal Software, Northampton, MA) software programs. Unless otherwise noted, statistical significance was determined by p < 0.05, using an unpaired Student's
t test. Results are presented as mean ± SEM, and error
bars in the figures represent SE. The curves in the figures are drawn
to guide the eye unless otherwise noted. Time course data were fit with
single exponential functions.
| |
RESULTS |
Sodium current activation
We compared the kinetic and voltage-dependent properties of TTX-S
sodium currents from axotomized DRG neurons in which Nav1.3 sodium
channel mRNA and protein are known to be upregulated (Dib-Hajj et al.,
1996; Black et al., 1999) with the currents from HEK 293 cells
transfected with recombinant rat brain Nav1.3 sodium channels. Fast-inactivating TTX-S sodium currents were observed in HEK 293 cells
transfected with Nav1.3 channels (Fig.
1A), and these currents appear to be similar to those recorded from axotomized DRG neurons (Fig. 1B). The threshold and voltage dependence of
activation for the peak sodium current were similar for Nav1.3 currents
and for TTX-S sodium currents in axotomized DRG neurons (Fig.
1C). The midpoint of activation was 25.5 ± 1.6 mV
(mean ± SEM, n = 24) for Nav1.3 currents and
29.1 ± 2.0 mV (n = 17) for axotomized DRG
currents. In HEK 293 cells, Nav1.3 currents have slightly slower
kinetics compared with sodium currents in axotomized DRG neurons (Fig.
1D). However, this simply may reflect a shifted voltage dependence of inactivation. Both the macroscopic open-state inactivation time constant versus voltage curve (Fig.
1E) and the steady-state inactivation curve (Fig.
1F) are shifted by approximately +10 mV for Nav1.3
channels in HEK 293 cells compared with TTX-S sodium currents in
axotomized DRG neurons. The midpoint of steady-state inactivation was
64.9 ± 1.5 mV (mean ± SEM, n = 25) for
Nav1.3 currents and 72.2 ± 1.3 mV (n = 19) for
axotomized DRG currents.
View larger version (21K):
[in this window]
[in a new window]
|
Figure 1.
Comparison of Nav1.3 currents in HEK 293 cells and
TTX-S currents in axotomized DRG neurons. A, Family of
traces from representative HEK 293 cells expressing rat Nav1.3
channels. B, Family of sodium current traces from
representative axotomized rat small DRG neuron. The currents were
elicited by 40 msec test pulses to various potentials from 80 to +40
mV. Cells were held at 120 mV. C, Normalized peak
current-voltage relationship for Nav1.3 channels (open
circles; n = 24) and axotomized DRG TTX-S
sodium currents (filled squares;
n = 17). D, Representative currents
from whole-cell recordings of an HEK 293 cell expressing Nav1.3
channels and an axotomized small DRG neuron from rat. Currents were
elicited by a step depolarization to 10 mV from a holding potential
of 120 mV and were scaled for comparison. The Nav1.3 current displays
slower kinetics. E, Inactivation kinetics as a function
of voltage. The macroscopic decay time constant is greater for Nav1.3
currents in HEK 293 cells (open circles;
n = 9) than for axotomized DRG TTX-S sodium
currents (filled squares; n = 11) 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. F,
Comparison of Nav1.3 (open circles;
n = 13) and axotomized DRG TTX-S sodium current
(filled squares;
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.
|
|
Development of closed-state inactivation
Recently, we demonstrated that Nav1.7 channels exhibit fivefold
slower closed-state inactivation than Nav1.4 channels in HEK 293 cells,
and we suggested that differences in closed-state inactivation might be
an important determinant for the generation of threshold ramp currents
(Cummins et al., 1998). Therefore, we compared the development of
inactivation kinetics of Nav1.3 channels in HEK 293 cells with that of
TTX-S sodium currents from axotomized small DRG neurons. At 70 mV the
development of inactivation was slow for heterologously expressed
Nav1.3 channels (Fig.
2A) and for TTX-S
sodium currents from axotomized small DRG neurons (Fig. 2B). The time constant for development of
inactivation was ~150 msec for both heterologously expressed Nav1.3
channels and TTX-S sodium currents from axotomized small DRG neurons at
70 mV (Fig. 2D).
View larger version (24K):
[in this window]
[in a new window]
|
Figure 2.
Development of closed-state inactivation is
similar for Nav1.3 channels expressed in HEK 293 cells and TTX-S sodium
currents in axotomized DRG neurons. A, B,
Family of current traces from HEK 293 cells expressing Nav1.3 channels
(A) and from axotomized DRG neurons
(B) showing the rate of development of
inactivation at 70 mV. C, The standard development of
inactivation voltage protocol. From a holding potential of 120 mV the
cells were prepulsed to 70 mV
(Vdev) for increasing durations
( t) and then stepped to 20 mV to determine the
fraction of current that was inactivated during the prepulse. The
duration of the inactivation prepulse for each data trace in
A and B is indicated. D,
Time course for the development of inactivation for the peak current.
Inactivation develops at the same rate at 70 mV for Nav1.3 channels
expressed in HEK 293 cells (open circles) and TTX-S
sodium currents in axotomized DRG neurons (filled
squares). The fraction of channels that inactivates at 70 mV
is lower for the Nav1.3 currents in HEK 293 cells; therefore, for
comparison the time course for the Nav1.3 currents is shown scaled to
that of the time course for TTX-S sodium currents in axotomized DRG
neurons (dotted curve).
|
|
The time constant for development of inactivation was estimated at
voltages ranging from 90 to 40 mV and was relatively large
throughout this voltage range for both heterologously expressed Nav1.3
channels and TTX-S sodium currents from axotomized small DRG neurons
(Fig. 3A). On the basis of
these estimates we predicted that the sodium currents in these two cell
types would generate inward currents during slow ramp depolarizations.
Indeed, large currents were evoked by slow ramps (100 to +40 mV over
600 msec) in HEK 293 cells expressing Nav1.3 channels (Fig.
3B), and these ramps elicited TTX-S sodium currents from
axotomized small DRG neurons (Fig. 3C). In 11 HEK 293 cells
expressing Nav1.3 channels, the ramp currents elicited by 600 msec ramp
depolarizations averaged 7.0 ± 0.9% of the peak current
amplitude. By contrast, the ramp currents in axotomized small DRG
neurons were smaller, averaging 4.1 ± 0.5% of the peak TTX-S
current amplitude (n = 8). The ramp currents generated
by Nav1.3 in HEK 293 cells activated ~10 mV more negatively than the
ramp currents in axotomized small DRG neurons (Fig. 3D).
View larger version (23K):
[in this window]
[in a new window]
|
Figure 3.
Nav1.3 currents exhibit slow closed-state
inactivation and generate large ramp currents. A, The
time constants for the development of inactivation are plotted as a
function of voltage. Time constants were estimated from single
exponential fits to time courses measured with the protocol shown in
Figure 2C for HEK 293 cells expressing Nav1.3 channels
(open circles; n = 8) and TTX-S
sodium currents in axotomized DRG neurons (filled
squares; n = 8). The inactivation voltage
(Vdev) was varied from 90 to 40
mV. B, Current elicited in a HEK 293 cell expressing
Nav1.3 channels by a 600 msec ramp depolarization from 100 to +40 mV.
C, Current elicited in a axotomized rat small DRG
neurons by a 600 msec ramp depolarization from 100 to +40 mV.
D, Comparison of averaged ramp currents from HEK 293 cells expressing Nav1.3 channels (n = 6) and
axotomized rat small DRG neurons (n = 3). Currents
were normalized and averaged for a comparison of voltage
dependence.
|
|
Recovery from inactivation
In small DRG neurons the increase in Nav1.3 channel mRNA
expression after axotomy of the sciatic nerve is paralleled by a change
from TTX-S sodium currents with slow recovery from inactivation to
TTX-S sodium currents with more rapid recovery from inactivation in
small DRG neurons (Cummins and Waxman, 1997; Black et al., 1999).
Therefore, we compared the recovery from inactivation (repriming) kinetics of Nav1.3 channels in HEK 293 cells with that of TTX-S sodium
currents from axotomized small DRG neurons. At 100 mV the repriming
rates were similar (Fig.
4A), but at 70 mV the Nav1.3 currents in HEK 293 cells reprimed more slowly than sodium currents from axotomized DRG neurons (Fig. 4B). As
Figure 4D shows, the repriming time constants were
similar at negative potentials (less than or equal to 90 mV) but
significantly different between 80 and 60 mV.
View larger version (20K):
[in this window]
[in a new window]
|
Figure 4.
Recovery from inactivation kinetics diverges for
Nav1.3 channels in HEK 293 cells and TTX-S currents in axotomized DRG
neurons. A, Family of current traces from representative
HEK 293 cell expressing Nav1.3 channels and an axotomized DRG neuron
showing the rate of recovery from inactivation at 100 mV. The time
course for recovery from inactivation of peak currents at 100 mV is
shown at right. Recovery is similar for Nav1.3 channels
expressed in HEK 293 cells (open circles) and TTX-S
sodium currents in axotomized DRG neurons (filled
squares). B, Family of current traces from HEK
293 cell expressing Nav1.3 channels or axotomized DRG neuron showing
the rate of recovery from inactivation at 70 mV. The time course for
recovery from inactivation of peak currents at 70 mV is shown at
right. Recovery is slower for Nav1.3 channels expressed
in HEK 293 cells (open circles) than for TTX-S sodium
currents in axotomized DRG neurons (filled
squares). C, The standard recovery from the
inactivation voltage protocol is shown. The cells were prepulsed to
20 mV for 20 msec to inactivate all of the current and then brought
back to the recovery potential (Vrec)
for increasing recovery durations (t) before the test
pulse to 20 mV. The maximum pulse rate was 0.5 Hz. The times
indicated for each trace shown in A and B
correspond to the recovery duration for that trace. D,
The time constants for recovery from inactivation are plotted as a
function of voltage. Time constants were estimated from single
exponential fits to time courses measured at recovery potentials
ranging from 140 to 60 mV with the protocol shown in
C for HEK 293 cells expressing Nav1.3 channels
(open circles; n = 23) and TTX-S
sodium currents in axotomized DRG neurons (filled
squares; n = 17).
|
|
Effect of coexpression with -subunits
Neuronal sodium channel  -subunits are associated typically with
one or more -subunits (Isom, 2000), and the expression of sodium
channel -subunits changes with development (Sashihara et al., 1995)
and after nerve injury (Shah et al., 2000). To determine whether sodium
channel -subunits might alter the properties of Nav1.3 channels, we
cotransfected 1, 2, and 3 subunits with Nav1.3 in HEK 293 cells. Because 1 and 2 subunits are found to be coexpressed in
some neurons, we also coexpressed Nav1.3 with 1 and 2 subunits
(1+2). Fast-inactivating sodium currents were observed when the
-subunits were coexpressed with Nav1.3 (Fig.
5A-E). The voltage dependence
of activation was similar for Nav1.3, Nav1.3+1, Nav1.3+2, and
Nav1.3+1+2 (Fig. 5F). However, the voltage
dependence of activation was shifted by +7 mV for Nav1.3+3 (Fig.
5F) compared with Nav1.3, and this shift was
significant (p < 0.01).
View larger version (26K):
[in this window]
[in a new window]
|
Figure 5.
Coexpression of 3 subunit alters Nav1.3
activation. Shown are families of traces from representative HEK 293 cells expressing rat Nav1.3 channels alone (A)
and Nav1.3 channels coexpressed with the 1 subunit
(B), the 2 subunit (C),
the 1+2 subunits (D), and the 3 subunit
(E). The currents were elicited by 40 msec test
pulses to various potentials from 80 to +40 mV. Cells were held at
120 mV. F, Normalized peak current-voltage
relationship for Nav1.3 channels (open circles;
n = 24) and Nav1.3 channels coexpressed with the
1 subunit (filled circles;
n = 15), the 2 subunit (open
triangles; n = 21), the 1+2 subunits
(filled inverted triangles; n = 12), and the 3 subunit (filled squares;
n = 14). The 3 subunit shifted the voltage
dependence of activation by >5 mV in the depolarizing direction.
|
|
We also examined the effect of -subunit coexpression on the voltage
dependence of steady-state inactivation (Fig.
6A). The 3 subunit
shifted steady-state inactivation for Nav1.3 in the depolarizing
direction by 7 mV (p < 0.01), and coexpression
of both the 1 and 2 subunits shifted steady-state inactivation by
5 mV (p < 0.05). Expression of either the 1
or 2 subunit did not alter the time constants significantly for
macroscopic open-state inactivation, but the Nav1.3 channels
coexpressed with the 3 subunit showed slower rates of inactivation
at test potentials ranging from 40 to 10 mV (Fig.
6B). This indicates that the 3 subunit has larger
effects on the inactivation properties of Nav1.3 channels than the 1
or 2 subunits.
View larger version (33K):
[in this window]
[in a new window]
|
Figure 6.
Coexpression of -subunits has small effects on
the inactivation properties of Nav1.3 currents. Nav1.3 channels were
expressed in HEK 293 cells alone (open circles;
n = 21) or coexpressed with the 1 subunit
(filled circles;
n = 16), the 2 subunit (open
triangles; n = 21), the 1+2 subunits
(filled inverted triangles; n = 13), and the 3 subunit (filled squares;
n = 15). A, Comparison of
steady-state inactivation for Nav1.3 expressed alone or coexpressed
with -subunits. 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.
B, Open-state inactivation kinetics as a function of
voltage shown for Nav1.3 expressed alone or coexpressed with
-subunits. The macroscopic decay time constants were estimated from
single exponential fits to the decay phase of currents elicited by 100 msec step depolarizations to the indicated potential. Coexpression of
the 3 subunit with Nav1.3 slowed macroscopic inactivation at
potentials ranging from 40 to 10 mV. C, The time
constants for recovery from inactivation are plotted as a function of
voltage for Nav1.3 expressed alone or coexpressed with -subunits.
Time constants were estimated from single exponential fits to time
courses measured with the protocols shown in Figure 4C.
Coexpression of the 1 subunit and the 3 subunit increased the
rate of recovery from inactivation for Nav1.3 channels expressed in HEK
293 cells (p < 0.05 at 80 mV).
D, The time constants for development of closed-state
inactivation are plotted as a function of voltage for Nav1.3 expressed
alone or coexpressed with -subunits. Time constants were estimated
from single exponential fits to time courses measured with the
protocols shown in Figure 2C.
|
|
Because Nav1.3 channels exhibited different repriming kinetics from
TTX-S sodium currents in axotomized neurons, we asked whether
coexpression of -subunits altered repriming kinetics. The time
constants for recovery from inactivation were measured in cells
coexpressing Nav1.3+1, Nav1.3+2, Nav1.3+1+2, and Nav1.3+3 (Fig. 6C). At 80 mV the recovery from
inactivation was ~50% faster for Nav1.3+1 and Nav1.3+3 than
for Nav1.3 expressed alone (p < 0.05). However,
at 70 and 60 mV, the time constants for recovery from inactivation
were still substantially smaller for sodium currents from axotomized
DRG neurons (dotted curve) than for Nav1.3 channels
coexpressed with -subunits (Fig. 6C). Development of
closed-state inactivation was not altered significantly by the
coexpression of -subunits with Nav1.3 channels (Fig.
6D).
Comparison of Nav1.3 channels expressed in HEK and DRG neurons
Recent data suggest that sodium channel properties can depend on
the cell type in which the channel is expressed. For example, Nav1.6
channels appear to underlie resurgent currents in cerebellar Purkinje
neurons, but not in hippocampal CA3 neurons (Raman et al., 1997) or
cultured spinal neurons (Pan and Beam, 1999). Therefore, we asked
whether recombinant Nav1.3 channels had different properties when
expressed in HEK 293 cells and DRG neurons. Because it would be
difficult to identify wild-type Nav1.3 channels expressed in DRG
neurons in the presence of large endogenous TTX-S currents, we created
a TTX-R Nav1.3 construct by replacing the tyrosine at position 384 with
a serine. These channels were not inhibited by 1 µM TTX
(data not shown), permitting us to identify the currents produced by
recombinant Nav1.3 channels after TTX-S currents were blocked with TTX.
When we created the Nav1.3-TTX-R channel, two additional mutations
(see Materials and Methods) also were inserted inadvertently into the
Nav1.3 construct. Efforts to correct these additional mutations were
not successful. The voltage dependence of activation and of
steady-state inactivation was ~10 mV more depolarized for the
Nav1.3-TTX-R channels expressed in HEK 293 cells than for wild-type
Nav1.3-TTX-R channels expressed in HEK 293 cells. However, because our
goal was to assess the effects of expression of recombinant Nav1.3
channels in HEK 293 cells versus DRG neurons, we felt that the mutant
Nav1.3-TTX-R channels would be useful nevertheless because they
permitted us to compare expression in the two cell types.
To minimize the possible confounding influence of endogenous TTX-R
currents in DRG neurons, we used neurons that had been cultured from
SNS-null mutant mice in which the slowly inactivating TTX-R SNS
(Nav1.8) channels (Akopian et al., 1996; Sangameswaran et al., 1996)
that are rapidly repriming (Elliott and Elliott, 1993) are not
expressed (Akopian et al., 1999; Cummins et al., 1999). After several
days in culture SNS-null neurons express very small TTX-R persistent
currents (0.33 ± 0.6 nA; n = 10). We were unable
to transfect DRG neurons with the standard calcium phosphate technique.
Therefore, biolistic techniques (Wellmann et al., 1999) were used to
transfect the SNS-null neurons. The SNS-null neurons were shot with GFP
alone or with GFP plus Nav1.3-TTX-R plasmid after 3-5 d in
vitro, and sodium currents were recorded in the presence of 500 nM TTX 1-2 d after transfection. The input resistance of neurons transfected with GFP plus Nav1.3-TTX-R plasmid (492 ± 129 M) was not significantly different from that of
control SNS-null neurons (256 ± 46 M), and biolistically
transfected SNS-null neurons appeared to be morphologically normal
(Fig. 7A,B).
View larger version (50K):
[in this window]
[in a new window]
|
Figure 7.
Transfection of SNS-null neurons and HEK 293 cells
with Nav1.3-TTX-R channels. A, Photomicrograph of
SNS-null neurons after biolistic transfection with GFP plus sodium
channel plasmid. Scale bar, 20 µm. Many gold particles (~1 µm
black particles) are visible throughout the field. Only one of the five
neurons in this field was transfected, indicated by the white
arrowhead. This neuron exhibited GFP fluorescence
(B). C, Family of traces from a
representative HEK 293 cell expressing rat Nav1.3-TTX-R channels.
D, Family of sodium current traces from a representative
SNS-null DRG neuron expressing rat Nav1.3-TTX-R channels after
biolistic transfection. E, Family of sodium current
traces from representative SNS-null DRG neuron after biolistic
transfection with GFP alone. For C-E, the extracellular
solution contained 500 nM TTX to block endogenous TTX-S
currents. The currents were elicited by 40 msec test pulses to various
potentials from 80 to +40 mV, and the cells were held at 120
mV.
|
|
Large fast-activating, fast-inactivating sodium currents were observed
in HEK 293 cells (Fig. 7C) (4.1 ± 0.8 nA,
n = 17) and SNS-null DRG neurons (Fig. 7D)
(24.9 ± 7.3 nA, n = 12) transfected with the
Nav1.3-TTX-R plasmid in the presence of 500 nM
TTX, but not in SNS-null neurons transfected with GFP alone (Fig.
7E) (0.28 ± 0.1 nA, n = 10). The
voltage dependence of activation was identical for the two cell types
(Fig. 8A). The voltage
dependence of steady-state fast-inactivation, on the other hand, was
~7 mV more depolarized for Nav1.3-TTX-R channels in SNS-null DRG
neurons than in HEK 293 cells (Fig. 8B). The time
constants for open-state inactivation (Fig. 8C) were similar
for Nav1.3-TTX-R channels in both cell types. The property that showed
the greatest difference between cell types was the rate of repriming.
The Nav1.3-TTX-R channels reprimed approximately twofold faster at
voltages from 100 to 60 mV when expressed in SNS-null DRG neurons
(Fig. 8D), and this difference was statistically
significant at 70 and 60 mV (p < 0.05).
Thus the recovery from inactivation kinetics of Nav1.3-TTX-R channels
seems to be sensitive to the cell background in which it is expressed
and is faster in DRG neurons. By contrast, the time constants for the
development of closed-state inactivation were similar for Nav1.3-TTX-R
channels in both cell types (Fig. 8E). Because
Nav1.3-TTX-R channels exhibited slow closed-state inactivation, we
predicted that these channels would generate ramp currents. Consistent
with this prediction, we found that slow ramp depolarizations can
elicit ramp currents in SNS-null DRG neurons transfected with
Nav1.3-TTX-R channels (Fig. 8F). However, the
voltage dependence of the Nav1.3-TTX-R ramp currents in SNS-null DRG
neurons was depolarized compared with the Nav1.3-TTX-R ramp currents
recorded in HEK 293 cells (Fig. 8F). The threshold for the Nav1.3-TTX-R ramp currents (defined as the voltage at which
the ramp current exceeds 10% of the peak amplitude) was 12 mV more
negative in the HEK 293 cells (59 ± 3 mV, n = 5) than in SNS-null DRG neurons (47 ± 5 mV, n = 3). Interestingly, this difference in voltage dependence is similar to
that observed when we compared Nav1.3 ramp currents in HEK 293 cells
with ramp currents recorded from axotomized DRG neurons (Fig.
3D). The ramp currents in axotomized DRG neurons and
Nav1.3-TTX-R currents expressed in SNS-null DRG neurons have a similar
voltage dependence (Fig. 9).
View larger version (24K):
[in this window]
[in a new window]
|
Figure 8.
Comparison of Nav1.3-TTX-R channels
expressed in HEK 293 cells and DRG neurons from SNS-null mice. The
Nav1.3-TTX-R channels were expressed in the DRG neurons by using the
Helios Gene Gun. The extracellular solution contained 500 nM TTX to block endogenous TTX-S currents.
A, Normalized peak current-voltage relationship for
Nav1.3-TTX-R channels expressed in HEK 293 cells (open
circles; n = 9) and SNS-null DRG neurons
(filled circles; n = 8). The
currents were elicited by 40 msec test pulses to various potentials
from 80 to +40 mV. Cells were held at 120 mV. B,
Comparison of steady-state inactivation for Nav1.3-TTX-R channels
expressed in HEK 293 cells (open circles;
n = 9) and SNS-null DRG neurons
(filled circles; n = 8).
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. C,
Open-state inactivation kinetics as a function of voltage. The
macroscopic decay time constants are similar for Nav1.3-TTX-R channels
expressed in HEK 293 cells (open circles;
n = 10) and SNS-null DRG neurons
(filled circles; n = 8). Time
constants were estimated from single exponential fits to the decay
phase of currents elicited by 100 msec step depolarizations to the
indicated potential. D, The time constants for recovery
from inactivation are plotted as a function of voltage for
Nav1.3-TTX-R channels expressed in HEK 293 cells (open
circles; n = 10) and SNS-null DRG neurons
(filled circles; n = 9). Time
constants were estimated from single exponential fits to time courses
measured with the protocol shown in Figure 4C. Recovery
from inactivation was faster for Nav1.3-TTX-R channels expressed in
SNS-null DRG neurons than for Nav1.3-TTX-R channels expressed in HEK
293 cells. E, The time constants for development of
closed-state inactivation are plotted as a function of voltage for
Nav1.3-TTX-R channels expressed in HEK 293 cells (open
circles; n = 10) and SNS-null DRG neurons
(filled circles; n = 6). Time
constants were estimated from single exponential fits to time courses
measured with the protocol shown in Figure 2C.
F, Current traces elicited in a representative SNS-null
DRG neuron and HEK 293 cell expressing Nav1.3-TTX-R channels by a 600 msec ramp depolarization from 100 to +40 mV. The traces were
normalized to compare the voltage dependence of the ramp
currents.
|
|
View larger version (17K):
[in this window]
[in a new window]
|
Figure 9.
Comparison of ramp currents from an axotomized DRG
neuron and a SNS-null neuron after biolistic transfection with
Nav1.3-TTX-R channels. The current traces were elicited by a 600 msec
ramp depolarization from 100 to +40 mV. The traces were normalized to
compare the voltage dependence of the ramp currents. So that the
Nav1.3-TTX-R ramp current could be recorded, the extracellular
solution contained 500 nM TTX to block endogenous TTX-S
currents.
|
|
| |
DISCUSSION |
We have characterized the kinetic and voltage-dependent properties
of the currents conducted by rat brain Nav1.3 sodium channels expressed
in HEK 293 cells. Although the differences were subtle, the Nav1.3
currents were distinctly different from the currents generated by other
TTX-S channels expressed in HEK 293 cells. Because voltage-gated sodium
channels can associate with auxiliary -subunits, we also examined
the consequences of the coexpression of Nav1.3 and the 1, 2, and
3 subunits. Coexpression of -subunits had different effects on
Nav1.3 current properties in HEK 293 than have been reported for Nav1.3
currents in Xenopus oocytes. Finally, we have compared the
properties of the currents produced by a TTX-R variant of Nav1.3
expressed in HEK 293 cells and DRG sensory neurons. Our data provide
insights into how sodium currents in neurons can be regulated by
altering the underlying -subunits and -subunits that are expressed.
Comparison of Nav1.3 to other Nav isoforms
Twenty years ago it was not uncommon to refer to the neuronal
sodium channel, and it was widely held that voltage-dependent neuronal
sodium currents served one primary function, to generate the rising
phase of the action potential. It is now known that at least eight
different voltage-gated sodium channel -subunits can be expressed in
neurons. To what extent these different -subunits have distinct
roles in electrogenesis is not entirely clear. However, it is clear
that different -subunits can have distinct voltage-dependent and
kinetic properties. Two TTX-R sodium channel isoforms have been
identified in peripheral neurons. Nav1.8 generates a TTX-R current with
macroscopic inactivation in HEK 293 cells that is ~10-fold slower
than the Nav1.3 current (our unpublished observations). Nav1.9 appears
to underlie a unique, persistent TTX-R current in small sensory neurons
that has very distinct properties compared with other sodium channels
(Cummins et al., 1999).
Although the TTX-R neuronal isoforms have very distinctive properties,
the TTX-S channels exhibit more subtle differences. Table
1 compares selected properties of Nav1.2,
Nav1.3, and Nav1.7 TTX-S neuronal sodium channel -subunits that are
expressed and characterized in HEK 293 cells. Although the Nav1.2
currents were characterized in a different laboratory (O'Leary, 1998),
we believe that the comparison is valid because both laboratories have
characterized the human skeletal muscle (Nav1.4) -subunit (Cummins
et al., 1998; O'Leary, 1998) and obtained nearly identical results
(see Table 1). Whereas the voltage dependence of activation and
steady-state inactivation are slightly more positive for Nav1.2 than
for Nav1.3, the voltage dependence of steady-state inactivation is
almost 15 mV more negative for Nav1.7 than for Nav1.3. The different isoforms also exhibit substantial differences in the development of,
and recovery from, inactivation. The time constant for recovery from
inactivation for Nav1.3 channels was intermediate between that for
Nav1.2 and Nav1.7. Because repriming kinetics may help to determine how
fast a neuron can fire repetitively, this suggests that cells
expressing Nav1.3 channels should be able to sustain higher firing
rates than cells expressing Nav1.7 channels, but not as high as in
cells expressing Nav1.2 channels. Development of inactivation was
slower for Nav1.3 and Nav1.7 channels than for Nav1.2 channels. This
suggests that cells expressing Nav1.3 and Nav1.7 channels may generate
more robust responses to slowly depolarizing inputs than cells
expressing Nav1.2 channels, because Nav1.3 and Nav1.7 channels are less
likely to undergo closed-state inactivation during slow
depolarizations.
View this table:
[in this window]
[in a new window]
|
Table 1.
Comparison of voltage-dependent properties of TTX-sensitive
sodium channel -subunits expressed in HEK 293 cells to TTX-sensitive
currents in axotomized DRG neurons
|
|
Effects of -subunit coexpression
-Subunits have been proposed to modulate properties of sodium
channel -subunits (Patton et al., 1994; Isom et al., 1995). In
Xenopus oocytes, coexpression of the 1 subunit with
Nav1.3 significantly increases the rate of open-channel inactivation and shifts steady-state inactivation by 11 mV (Patton et al., 1994).
1 has similar effects on the properties of Nav1.1, Nav1.2 (Smith and
Goldin, 1998), and Nav1.4 (Nuss et al., 1995) when coexpressed in
Xenopus oocytes. However, we found that coexpression of 1
with Nav1.3 channels in HEK 293 cells did not have a major effect on
open-channel inactivation and, if anything, slightly depolarized the
voltage dependence of steady-state inactivation for Nav1.3 channels
(Fig. 6A). 1 did increase the rate of recovery from inactivation for Nav1.3 channels, but, overall, 1 had only subtle effects on the properties of Nav1.3 currents in HEK 293 cells.
2 had no detectable effects on the properties that we examined of
Nav1.3 currents in HEK 293 cells. 3, on the other hand, had the
largest effect on Nav1.3 current properties, shifting the voltage
dependence of both activation and steady-state inactivation by 7 mV in
the depolarizing direction, slowing macroscopic open-channel inactivation, and accelerating recovery from inactivation. By contrast,
Morgan et al. (2000) reported that 1 had larger effects than 3 on
the properties of Nav1.2a channels expressed in Xenopus oocytes. This difference may reflect differences in the interaction of
-subunits with specific -subunits. Alternatively, -subunits might have different effects on sodium currents in Xenopus
oocytes and HEK 293 cells; however, Isom et al. (1995) reported similar effects for 1 on the properties of Nav1.2a channels in
Xenopus oocytes and Chinese hamster cells. Patton et al.
(1994) reported that 1 modulates Nav1.3 channels to a smaller extent
than Nav1.2a channels in Xenopus oocytes, and their data on
1 mRNA expression suggested that 1 may not associate with the
Nav1.3 sodium channel during development. Although the developmental
expression pattern of 3 is not known, Shah et al. (2000) recently
reported that high levels of mRNA for 3 are expressed in small DRG
neurons and that chronic constriction injury causes a significant
increase in 3 mRNA expression.
Comparison of Nav1.3 currents in HEK 293 cells and sodium currents
in DRG neurons
We compared the properties of Nav1.3 sodium currents in HEK 293 cells with the properties of the TTX-S sodium currents in axotomized
DRG neurons, which are known (Waxman et al., 1994; Black et al., 1999)
to express increased levels (compared with uninjured neurons) of Nav1.3
mRNA and protein. Although there were many similarities, there were
some obvious differences. Although the Nav1.3 currents in HEK 293 cells
exhibited slow closed-state inactivation like the TTX-S currents in
axotomized DRG neurons and large currents were elicited by ramp
depolarizations in both situations, the ramp currents generated by
Nav1.3 channels in HEK 293 cells activated ~10 mV more negatively
than the TTX-S ramp currents recorded from axotomized DRG neurons.
Furthermore, although the time constants for repriming were similar at
recovery potentials between 140 and 90 mV, repriming was slower for
the Nav1.3 currents in HEK 293 cells at more depolarized potentials. In
an effort to understand these differences, we expressed a TTX-R variant
of Nav1.3 in cultured DRG neurons and in HEK 293 cells and compared the
properties of the TTX-R currents in these cells. Ramp currents
generated by Nav1.3-TTX-R channels activated at more negative
potentials in the HEK 293 cells than in the DRG neurons, and repriming
was faster in the DRG neurons than in the HEK 293 cells. These results
indicate that at least some of the differences between Nav1.3 currents
in HEK 293 cells and TTX-S currents in DRG neurons are attributable to
the cell background, which may produce differences in interactions with
-subunit or other modulatory proteins.
The rapid repriming that we observed in Nav1.3 channels after
expression in DRG neurons suggests that Nav1.3 contributes to the
rapidly repriming TTX-S current in axotomized DRG neurons. However,
Nav1.3 is not the only contributor to the rapidly repriming TTX-S
current in axotomized neurons, because the repriming of Nav1.3-TTX-R
currents in DRG neurons was still slower at 60 mV than for TTX-S
currents in axotomized DRG neurons. Although in adult small sensory
neurons Nav1.7, which exhibits slow repriming kinetics, is thought to
be the predominant TTX-S channel and Nav1.3 expression is increased
after peripheral axotomy, these neurons also express detectable levels
of Nav1.1 and Nav1.6 mRNA (Black et al., 1996). These distinct isoforms
also may contribute to the rapidly repriming TTX-S currents in
axotomized DRG neurons.
Conclusions
Nav1.3 sodium channels are expressed at relatively high levels in
the developing nervous system but at lower levels in the mature nervous
system. Nav1.3 expression is, however, upregulated in some injured
neurons. Our observation, that Nav1.3 channels have different
physiological properties when expressed in different cell types, may
have methodological implications, especially with respect to the choice
of cell type for expression studies. This observation also may reflect
at least some degree of cell-specific heterogeneity of channel function
within the intact nervous system because many channel types, including
Nav1.3, are expressed in many different types of cells. Our data
indicate that Nav1.3 channels exhibit distinct properties that may have
important implications on cellular excitability. Nav1.3 currents, for
example, exhibit slow development of closed-state inactivation and
intermediate repriming kinetics and generate large currents in response
to slow ramp depolarizations. Our results suggest that Nav1.3 channels contribute to the accelerated repriming of TTX-S sodium currents that
is seen in axotomized DRG neurons. The distinct functional properties
of Nav1.3 may be important for developing neurons and also may
contribute to aberrant activity in injured neurons.
| |
FOOTNOTES |
Received Feb. 16, 2001; revised May 3, 2001; accepted May 31, 2001.
This work was supported in part by grants from the National Multiple
Sclerosis Society and the Medical Research Service and Rehabilitation
Research Service, Department of Veterans Affairs (S.G.W.). We thank the
Eastern Paralyzed Veterans Association and the Paralyzed Veterans of
America for support. We also thank W. Hormuzdiar and B. Toftness for
excellent technical support and Dr. A. L. Goldin for generously
providing the Nav1.3-pBS-SK plasmid.
Correspondence should be addressed to Dr. Stephen G. Waxman, Department
of Neurology, Yale University School of Medicine, 333 Cedar Street,
P.O. Box 208018, New Haven, CT 06520-8018. E-mail: Stephen.Waxman{at}Yale.Edu.
| |
REFERENCES |
-
Akopian AN,
Sivilotti L,
Wood JN
(1996)
A tetrodotoxin-resistant voltage-gated sodium channel expressed by sensory neurons.
Nature
379:257-262[Medline].
-
Akopian AN,
Souslova V,
England S,
Okuse K,
Ogata N,
Ure J,
Smith A,
Kerr BJ,
McMahon SB,
Boyce S,
Hill R,
Stanfa LC,
Dickenson AH,
Wood JN
(1999)
The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways.
Nat Neurosci
2:541-548[ISI][Medline].
-
Bendahhou S,
Cummins TR,
Potts JF,
Tong J,
Agnew WS
(1995)
Serine-1321-independent regulation of the µ1 adult skeletal muscle Na+ channel by protein kinase C.
Proc Natl Acad Sci USA
92:12003-12007[Abstract/Free Full Text].
-
Black JA,
Waxman SG
(1996)
Sodium channel expression: a dynamic process in neurons and non-neuronal cells.
Dev Neurosci
18:139-152[ISI][Medline].
-
Black JA,
Dib-Hajj S,
McNabola K,
Jeste S,
Rizzo MA,
Kocsis JD,
Waxman SG
(1996)
Spinal sensory neurons express multiple sodium channel -subunit mRNAs.
Mol Brain Res
43:117-132[Medline].
-
Black JA,
Cummins TR,
Plumpton C,
Chen YH,
Hormuzdiar W,
Clare JJ,
Waxman SG
(1999)
Upregulation of a silent sodium channel after peripheral, but not central, nerve injury in DRG neurons.
J Neurophysiol
82:2776-2785[Abstract/Free Full Text].
-
Chahine M,
George AL,
Zhou M,
Ji S,
Sun W,
Barchi RL,
Horn R
(1994)
Sodium channel mutations in paramyotonia congenita uncouple inactivation from activation.
Neuron
12:281-294[ISI][Medline].
-
Cummins TR,
Waxman SG
(1997)
Downregulation of TTX-resistant sodium currents and upregulation of a rapidly repriming TTX-sensitive sodium current in small spinal sensory neurons after nerve injury.
J Neurosci
17:3503-3514[Abstract/Free Full Text].
-
Cummins TR,
Howe JR,
Waxman SG
(1998)
Slow closed-state inactivation underlies tetrodotoxin-sensitive ramp currents in HEK 293 cells expressing hNE sodium channels and in dorsal root ganglion neurons.
J Neurosci
18:9607-9619[Abstract/Free Full Text].
-
Cummins TR,
Dib-Hajj SD,
Black JA,
Akopian AN,
Wood JN,
Waxman SG
(1999)
A novel persistent tetrodotoxin-resistant sodium current in small primary sensory neurons.
J Neurosci
19:RC43.
-
Dib-Hajj SD,
Black JA,
Felts P,
Waxman SG
(1996)
Down-regulation of transcripts for Na channel -SNS in spinal sensory neurons following axotomy.
Proc Natl Acad Sci USA
93:14950-14954[Abstract/Free Full Text].
-
Dib-Hajj SD,
Ishikawa K,
Cummins TR,
Waxman SG
(1997)
Insertion of a SNS-specific tetrapeptide in S3-S4 linker of D4 accelerates recovery from inactivation of skeletal muscle voltage-gated Na channel µ-1 in HEK 293 cells.
FEBS Lett
416:11-14[ISI][Medline].
-
Elliott AA,
Elliott JR
(1993)
Characterization of TTX-sensitive and TTX-resistant sodium currents in small cells from adult rat dorsal root ganglia.
J Physiol (Lond)
463:39-56[Abstract/Free Full Text].
-
Eubanks J,
Srinivasan J,
Dinulos MB,
Disteche CM,
Catterall WA
(1997)
Structure and chromosomal localization of the 2 subunit of the human brain sodium channel.
NeuroReport
8:2775-2779[Medline].
-
Felts PA,
Yokoyama S,
Dib-Hajj S,
Black JA,
Waxman SG
(1997)
Sodium channel -subunit mRNAs I, II, III, NaG, Na6, and hNE (PN1): different expression patterns in developing rat nervous system.
Brain Res Mol Brain Res
45:71-82[Medline].
-
Gastaldi M,
Bartolomei F,
Massacrier A,
Planells R,
Robaglia-Schlupp A,
Cau P
(1997)
Increase in mRNAs encoding neonatal II and III sodium channel -isoforms during kainate-induced seizures in adult rat hippocampus.
Brain Res Mol Brain Res
44:179-190[Medline].
-
Goldin AL,
Barchi RL,
Caldwell JH,
Hofmann F,
Howe JR,
Hunter JC,
Kallen RG,
Mandel G,
Meisler MH,
Netter YB,
Noda M,
Tamkun MM,
Waxman SG,
Wood JN,
Catterall WA
(2000)
Nomenclature of voltage-gated sodium channels.
Neuron
28:365-368[ISI][Medline].
-
Horton RM,
Ho SN,
Pullen JK
|
TOP
ABSTRACT
INTRODUCTION
