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The Journal of Neuroscience, August 15, 1998, 18(16):6093-6102
Functional Analysis of the Mouse Scn8a Sodium Channel
Marianne R.
Smith1,
Raymond D.
Smith1,
Nicholas
W.
Plummer2,
Miriam H.
Meisler2, and
Alan L.
Goldin1
1 Department of Microbiology and Molecular Genetics and
Physiology and Biophysics, University of California, Irvine, California
92697-4025, and 2 Department of Human Genetics, University
of Michigan, Ann Arbor, Michigan 48109-0618
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ABSTRACT |
The mouse Scn8a sodium channel and its ortholog Na6 in the rat are
abundantly expressed in the CNS. Mutations in mouse Scn8a result
in neurological disorders, including paralysis, ataxia, and dystonia.
In addition, Scn8a has been observed to mediate unique persistent and
resurgent currents in cerebellar Purkinje cells (Raman et al., 1997 ).
To examine the functional characteristics of this channel, we
constructed a full-length cDNA clone encoding the mouse Scn8a sodium
channel and expressed it in Xenopus oocytes. The
electrophysiological properties of the Scn8a channels were compared
with those of the Rat1 and Rat2 sodium channels. Scn8a channels were
sensitive to tetrodotoxin at a level comparable to that of Rat1 or
Rat2. Scn8a channels inactivated more rapidly and showed differences in
their voltage-dependent properties compared with Rat1 and Rat2 when
only the  subunits were expressed. Coexpression of the
 1 and 2 subunits modulated the properties
of Scn8a channels, but to a lesser extent than for the Rat1 or Rat2
channels. Therefore, all three channels showed similar voltage
dependence and inactivation kinetics in the presence of the subunits. Scn8a channels coexpressed with the subunits exhibited a
persistent current that became larger with increasing depolarization,
which was not observed for either Rat1 or Rat2 channels. The unique
persistent current observed for Scn8a channels is consistent with the
hypothesis that this channel is responsible for distinct sodium
conductances underlying repetitive firing of action potentials in
Purkinje neurons.
Key words:
sodium channel; cloning; expression; Xenopus
oocytes; brain; RT-PCR; Purkinje cells; resurgent current; persistent
current
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INTRODUCTION |
Voltage-gated sodium channels play a
critical role in the generation of action potentials in excitable cells
throughout the nervous system (Catterall, 1992). Many sodium channel
isoforms have been identified in the CNS, including Rat1 (Noda et al., 1986), Rat2 (Noda et al., 1986), a splice variant termed Rat2A (Auld et
al., 1988), Rat3 (Kayano et al., 1988; Joho et al., 1990), Rat6
(Schaller et al., 1995), and the Rat6 mouse ortholog Scn8a (Burgess et
al., 1995). Scn8a/Rat6 is abundantly expressed in neurons throughout
the brain and spinal cord (Schaller et al., 1995) and is the major
contributor to sodium current in postnatal motor neurons (Garcia et
al., 1998). Rat1 is expressed predominantly in the caudal regions and
spinal cord, whereas Rat2 is found most abundantly in the rostral
regions of the CNS (Gordon et al., 1987; Beckh et al., 1989). In the
cerebellum, both Rat1 and Rat6 are found at high levels in the Purkinje
cells, whereas Rat2 is expressed in granule cells and Purkinje cells
(Furuyama et al., 1993; Black et al., 1994; Schaller et al., 1995;
Felts et al., 1997; Vega-Saenz de Miera et al., 1997). The
1 and 2 subunits are also present in the
majority of these cells and can modulate the electrophysiological properties of the channels (Isom et al., 1992, 1995; Smith and Goldin,
1998).
The Scn8a/Rat6 and Rat1 channels may be responsible for distinct sodium
conductances in Purkinje cells. Vega-Saenz de Miera et al. (1997)
suggested that inactivating and persistent sodium conductances in
Purkinje cells result from expression of Rat1 and Rat6, respectively.
Raman et al. (1997) compared normal Purkinje cells with those from
Scn8a null mice. In Scn8a null cells, there was a reduction in
persistent, noninactivating current compared with transient current,
suggesting that Scn8a may be predominantly responsible for the
persistent current. They also identified a resurgent current that was
elicited by an action potential-like waveform in normal Purkinje cells,
and this current was significantly decreased in cells from Scn8a null
mice.
Several mutations in Scn8a have been characterized, resulting in mice
with a variety of symptoms ranging from mild ataxia to dystonia,
paralysis, and juvenile lethality (Meisler et al., 1997). These
mutations include med and medtg, both of
which result in complete disruption of the Scn8a gene (Burgess et al.,
1995; Kohrman et al., 1995, 1996b). Another mutation, medjo, is a single point mutation of Ala to Thr in
the domain III S4-S5 linker (Kohrman et al., 1996a). This mutation
produces an ataxic phenotype, which may be caused by changes in the
voltage-dependent properties of the Scn8a channel (Kohrman et al.,
1996a). Both the null and mis-sense mutation have been shown to disrupt
spontaneous and repetitive firing of Purkinje cells (Dick et al., 1985;
Harris et al., 1992; Raman et al., 1997), suggesting that Scn8a plays a
critical role in the firing properties of Purkinje neurons.
Given the fundamental importance of the Scn8a channel, we constructed a
full-length cDNA clone encoding Scn8a to characterize its functional
properties in Xenopus oocytes. Most of the
electrophysiological properties of Scn8a sodium channels were similar
to those of Rat1 and Rat2 channels and were modulated by the accessory
subunits. However, Scn8a sodium channels demonstrated a persistent
current that was distinctly different from Rat1 or Rat2 currents.
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MATERIALS AND METHODS |
Reverse transcription and PCR. Total RNA was isolated
from C57BL/6J adult mice brains using the Trizol reagent (Life
Technologies, Gaithersburg, MD). RNA was suspended in sterile,
RNase-free water at a concentration of 1.36 mg/ml and stored at
 75°C. Mouse brain RNA was reverse-transcribed using an
oligo-dT18 primer. The Scn8a coding region was amplified by
PCR using primers that hybridize to the 5' and 3' ends of the published
sequence (Burgess et al., 1995). For the upstream primer, a T7 RNA
polymerase recognition sequence was incorporated 5' to the Scn8a
sequence so that RNA could be transcribed by T7 RNA polymerase directly
from the PCR product in vitro. The two PCR primers were 5'
end:
5'-GCGCGC(GAATTC)[TAATACGACTCACTATA]GAGAAGATGGCAGCGCGGG-3'; and 3' end: 5'-GCG(GACGTC)TCTGTGTCCGTGAGATTCGG-3'. The 5' primer contains a six-base G-C clamp, an EcoRI restriction
site (in parentheses), a T7 promoter (in brackets), and sense Scn8a DNA
sequence consisting of five bases upstream of the Scn8a coding region
and 13 bases of coding sequence beginning with the translational start
codon (underlined). The 3' primer contains a three-base G-C clamp, an AatII restriction site (in parentheses), and antisense DNA
sequence 67 bases downstream of the Scn8a stop codon.
Total brain RNA (2.8 µg) was heat-denatured at 65°C for 5 min,
followed by rapid cooling on ice. Reverse transcription was performed
with 0.5 mM deoxynucleotide triphosphates, 10 mM dithiothreitol, 100 pmol of oligonucleotide primers, 40 U of RNasin (Promega, Madison, WI), and 500 U of Moloney murine
leukemia virus reverse transcriptase (Life Technologies) in a
total volume of 50 µl. Reactions were incubated at room temperature
for 5 min and then at 37°C for 2 hr. Reaction products were purified
by phenol and chloroform extraction and ethanol precipitation and were
resuspended in 10 µl of distilled water.
The RT product was amplified using the PCR primers described above. One
microliter ( ) of the RT product was combined with 1 mM MgCl2, 200 µmol of deoxynucleotide
triphosphates, a 0.2 µM concentration of each primer, and
2.5 U of LA Taq Polymerase (PanVera, Madison, WI). Thermal
cycle parameters were one cycle consisting of denaturation at 94°C
for 4 min, annealing at 53°C for 3 min, and polymerase extension at
72°C for 6 min, followed by 30 cycles consisting of denaturation at
95°C for 30 sec, annealing at 53°C for 1 min, and polymerase
extension at 72°C for 6 min. A single PCR fragment of ~6 kb in size
was extracted with phenol and chloroform, precipitated with ethanol,
and resuspended in RNase-free water.
Cloning of Scn8a. To ensure that the PCR product encoded a
functional sodium channel, RNA was transcribed directly from a sample
of the product using a T7 Message Machine RNA transcription kit
(Ambion, Austin, TX). Injection of RNA into Xenopus oocytes resulted in expression of up to 2 µA of whole-cell tetrodotoxin (TTX)-sensitive inward sodium current, as measured with a two-electrode voltage clamp.
A full-length plasmid containing the Scn8a sequence was constructed by
cutting the functionally confirmed PCR product with EcoRI
and AatII, and ligating the fragment into pLCT1-A, a
modified version of pLCT1 (Smith and Goldin, 1998). pLCT1 was modified by inserting unique EcoRI and AatII sites into the existing
unique BglII site. The resulting plasmid, pNaScn8a, was
linearized with NotI and transcribed using a T7 Message
Machine transcription kit. Injection of undiluted RNA into oocytes
resulted in currents as large as 100 µA after 48 of incubation at
20°C. The DNA sequence of pNaScn8a was determined at the University
of Michigan DNA Sequencing Core Facility (R. Lyons, Director) using the
dideoxy chain termination method and the Taq dye terminator
cycle sequencing kit with Applied Biosystems (Foster City, CA) 373 Stretch and 377 sequencers.
Expression and electrophysiology. RNA transcripts were
synthesized from NotI-linearized DNA templates using a T7
RNA polymerase Message Machine transcription kit (Ambion). The yield of
RNA was estimated by glyoxal gel analysis, and pNaScn8a RNA was diluted 1:2000 to result in currents in the range of 2-5 µA. Stage 5 oocytes were removed from adult female Xenopus laevis frogs and
prepared as described previously (Goldin, 1991) and incubated in ND96
media, which consists of (in mM): 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES, pH
7.5, supplemented with 0.1 mg/ml gentamycin, 0.55 mg/ml
pyruvate, and 0.5 mM theophylline. Scn8a and Rat2 sodium channel RNA were injected at 100 pg/oocyte, and Rat1 RNA was injected at 50 ng/oocyte. The oocytes were incubated for 24-48 hr at 20°C in
ND96.
Sodium currents were recorded using the cut-open oocyte technique
(Taglialatela et al., 1992) with the CA-1 high-performance oocyte
voltage clamp (Dagan, Minneapolis, MN) and Digidata 1200A interface
(Axon Instruments, Foster City, CA) and pCLAMP 6.0.3 software (Axon
Instruments), as described previously (Kontis et al., 1997).
Temperature was maintained at 20°C using a HCC-100A temperature
controller (Dagan). The intracellular solution consisted of (in
mM): 88 K2SO4, 10 EGTA, 10 HEPES, and 10 Na2SO4, pH 7.5, and the
extracellular solution consisted of (in mM): 120 sodium MES, 10 HEPES, and 1.8 Ca-MES, pH 7.4. Capacitive transients and leak
currents were corrected by P/4 subtraction. Sodium current amplitudes
were between 1 and 5 µA.
For analysis of recovery from inactivation, voltage dependence of
persistent current, and the resurgent current, a two-electrode voltage
clamp was used at room temperature as described previously (Patton and
Goldin, 1991). Although this voltage clamp does not provide the fast
time resolution of the cut-open oocyte clamp, it was used for two
reasons. First, the oocytes are more stable over long periods of time,
which was essential for analyzing recovery from inactivation. Second,
it was possible to subtract comparable records obtained in the presence
of 400 nM TTX to eliminate capacitive and leak currents.
TTX subtraction was particularly important when measuring persistent
and resurgent currents, because outward currents could easily obscure
the small persistent and resurgent currents. The bath solution
consisted of ND96.
Data analysis. Analysis was performed using pCLAMP 6.0.3 software (Axon Instruments), Excel 7.0 (Microsoft, Redmond, WA), and
Sigmaplot 4.0 (Jandel, San Rafael, CA). Inactivation time constants
were determined using the Chebyshev method to fit current traces with
a single exponential equation: I = Aslow*exp[(t k)/ slow] + C, or a double
exponential equation: I = Afast*exp[(t K)/fast]+ Aslow*exp[ (t K)/slow] + C, where I is the
current, Afast and Aslow
represent the percentage of channels inactivating with time constants
fast and slow, K is the
time shift, and C is the steady-state asymptote. The time
shift was manually selected by fitting the traces at the time when the
currents were just starting to exponentially decrease. Recovery data
were fit using a single, double, or triple exponential equation of the
form I = 1 [A1*exp(t/1),
I = 1 [A1*exp(t/1) + A2*exp(t/2)], and I = 1 [A1*exp(t/1) + A2*exp(t/2) + A3*exp(t/3)],
where A1,
A2, and A3 are the
relative proportions of current recovering with time constants
1, 2, and
3, and t is the recovery interval.
Conductance values were calculated using the formula G = I/(V Vr),
where G is conductance, I is current amplitude,
V is the depolarized membrane potential, and
Vr is the reversal potential. Reversal
potentials were individually estimated for each data set by fitting the
I-V data with an equation that includes terms for both the voltage dependence of conductance and the driving force:
I = [1 + exp(0.03937*z*(V V1/2))] 1*g*(V Vr), where z is the
apparent gating charge, g is a factor related to the number
of channels contributing to the macroscopic current, V is
equal to the voltage potential of the pulse, and V1/2 is the half-maximal voltage.
Conductance values were fit with a two-state Boltzmann equation,
G = 1/(1 + exp[0.03937*z*(V V1/2)]), with z equal to the
apparent gating charge, V equal to the pulse potential, and
V1/2 equal to the voltage required for
half-maximal activation. The voltage dependence of fast inactivation
data were fit with a two-state Boltzmann equation, I = 1/(1 + exp[(V V1/2)/a]), with I
equal to the current amplitude measured during the test depolarization,
V equal to the inactivating depolarization potential,
a equal to the slope factor, and
V1/2 equal to the voltage depolarization
required for half-maximal inactivation.
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RESULTS |
Construction of a full-length cDNA clone encoding the Scn8a
sodium channel
The full-length coding region of Scn8a was amplified and cloned
from mouse RNA using RT-PCR, as described in Materials and Methods. The
sequence closely matches the previously published sequence for Scn8a
(Burgess et al., 1995), except in the region of the I-II linker, and
is shown schematically in Figure
1A. The sequence that
was published by Burgess et al. (1995) contained a short cytoplasmic
linker consisting of only 87 amino acids between domains I and II. The
sequence that we obtained for the I-II linker contains 333 amino acids
(Fig. 1B, GenBank accession number AFO49617). This
linker sequence is comparable in length to that previously obtained for
the Rat6 channel (Schaller et al., 1995) and is identical to the
sequence later determined by N. W. Plummer, J. Galt, J. M. Jones, D. L. Burgess, L. K. Sprunger, D. C. Kohrman, and M. H. Meisler (unpublished
observations) for the Scn8a channel. In addition, this sequence differs
at only one position (E in Scn8a compared with D in Rat6 at position
684) from the sequence for the Rat6 channel (also called PN4) that was
determined by Dietrich et al. (1998). Both Plummer, Galt, Jones,
Burgess, Sprunger, Kohrman, and Meisler (unpublished observations) and
Dietrich et al. (1998) observed different alternatively spliced forms
of the I-II linker. The current cDNA clone contains the major form of
the transcript, which lacks 10 amino acids following residue 664 in the
I-II linker (Fig. 1A, arrow, B, boxed
residues). In addition to the differences in the I-II linker,
there are eight nucleotide differences in the remainder of the
sequence. Three of the differences are silent and are indicated in
Figure 1A by solid circles. Five of the
nucleotide differences change the amino acid sequence and are indicated
in Figure 1A by the circled residues. Two
of the amino acid differences (V5L and K15R) are in the N terminus, two
are in or near domain IS1 (I142T and N153T), and the final difference
is in domain IIS6 (V958A).
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Figure 1.
Diagram of the Scn8a sodium channel.
A, Diagram of the Scn8a sodium channel, showing the
differences between the sequence of the cDNA clone described in this
work and the previously published sequence (Burgess et al., 1995). The
five amino acid differences are represented by the circled
residues, which indicate the sequence of the cDNA clone
described here, compared with the originally published sequence shown
in parentheses. The positions of the silent nucleotide
differences are shown by the solid circles. The position
of the 10-amino acid deletion resulting from alternative splicing is
shown by the arrow. B, Amino acid
sequence of the cytoplasmic linker between domains I and II. This
sequence was not included in the work by Burgess et al. (1995), but it
is identical to the sequence that was later determined for the mouse
Scn8a channel by Plummer, Galt, Jones, Burgess, Sprunger, Kohrman, and
Meisler (unpublished observations), and it differs at only one position
(E in Scn8a compared with D in Rat6 at position 684) compared with the
Rat6 (PN4) channel sequence that was determined by Dietrich et al.
(1998). The 10 amino acids that are deleted in this spliced form are
enclosed in a box.
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Functional expression of Scn8a sodium channels and modulation by
1 and 2
Previous studies suggested that Scn8a mediates persistent and
resurgent sodium conductances in addition to the transient sodium current, which might reflect unique kinetic or voltage-dependent properties for this channel (Raman et al., 1997). To examine this possibility, the functional properties of Scn8a were determined by
expression in Xenopus oocytes and compared with those of
Rat1 and Rat2 sodium channels. Sodium currents were recorded using the
cut-open voltage clamp, which provides sufficient time resolution to
accurately compare the inactivation properties of the three channels.
Injection of RNA encoding Scn8a resulted in sodium currents that were
blocked by TTX with a Kapp of 6.4 ± 0.6 nM, which is similar to the values that Smith and Goldin
(1998) previously obtained for Rat1 (9.6 ± 3.2 nM)
and Rat2 (8.8 ± 4.0 nM). Figure 2A shows current traces
for channels consisting of only the subunits of Scn8a, Rat1, and
Rat2. The records were obtained during depolarizations from 65 to +25
mV in 10 mV increments from a holding potential of 100 mV. Scn8a
channels inactivated more rapidly than either Rat1 or Rat2 channels
when only the subunit was present. Figure 2B
shows representative current traces for Scn8a, Rat1, and Rat2 subunits coexpressed with the 1 and 2 subunits. Coexpression with the two subunits accelerated the inactivation kinetics of all three channels. This has been shown previously to be the case for both Rat1 (Smith and Goldin, 1998) and
Rat2 (Isom et al., 1992, 1995; Smith and Goldin, 1998). However, the
subunits had less of an effect on Scn8a than on either Rat1 or
Rat2, possibly because Scn8a subunit channels already inactivated faster than either Rat1 or Rat2 subunit channels.
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Figure 2.
Representative current traces for Scn8a, Rat1, and
Rat2. Channels consisting of subunits alone
(A) or subunits coexpressed with
1 and 2 subunits
(B) were expressed in Xenopus
oocytes as described in Materials and Methods. Currents were recorded
using the cut-open oocyte voltage clamp at 20°C as described in
Materials and Methods. Currents were elicited by membrane
depolarizations ranging from 65 to +25 mV in 10 mV increments from a
holding potential of 100 mV. The currents from subunit channels
are shown with a longer time axis to demonstrate inactivation kinetics
of these channels, which was much slower than for the channels
expressed with the subunits. Calibration: 5 msec, 0.5 µA.
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Scn8a channels inactivate more rapidly than Rat1 or
Rat2 channels
To quantify the differences in inactivation kinetics between the
three channels, the time constants of inactivation were determined for
the subunits alone (Fig.
3A) and for the subunits
coexpressed with the subunits (Fig. 3B). Currents were
recorded during depolarizations from 20 to +45 mV in 5 mV increments
from a holding potential of 100 mV. Inactivation time constants were
determined by fitting the current traces with either a single or double
exponential equation, as described in Materials and Methods. In Figure
3 the top and middle panels show the slow
(slow) and fast (fast)
inactivation time constants, respectively, for Scn8a, Rat1, and Rat2.
The bottom panel shows the percent of current
inactivating with fast for each channel.
slow of Scn8a was very similar to slow
for Rat2, and slightly smaller than slow for Rat1, over
the voltage range from +10 to +45 mV. At more negative depolarizations,
slow was similar among the three channels. However,
fast was significantly smaller for Scn8a compared with
Rat1 and Rat2. In addition, during depolarizations from 10 to 5 mV,
Scn8a inactivated with both a fast and slow component, whereas Rat1 and
Rat2 inactivated with only a slow component. At more positive
depolarizations (+10 to +45 mV), the percent of current that
inactivated with fast was similar for all three
channels.
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Figure 3.
Time constants for fast inactivation of Scn8a,
Rat1, and Rat2. Currents were recorded from oocytes expressing Scn8a,
Rat1, or Rat2 sodium channels using the cut-open oocyte voltage clamp
as described in the legend to Figure 2. The kinetics of inactivation
were fit with a single- or double-exponential equation as described in
Materials and Methods, and the time constants representing the slow and
fast components are shown on a logarithmic scale in the
top and middle panels, respectively, for
subunits alone (A) and +1 + 2 (B). Scn8a is indicated by the
solid diamonds, Rat1 is indicated by open
squares, and Rat2 is indicated by open
circles. The bottom panel shows the percent of
current inactivating with the fast component of inactivation. In all
cases, the fraction of fast plus the fraction of
slow equals 1. Values represent averages, and error bars
indicate SDs. Sample sizes were Scn8a , n = 5;
Rat1 , n = 7; Rat2 , n = 9; Scn8a + 1 + 2,
n = 6; Rat1 + 1 + 2, n = 7; and Rat2 + 1 + 2, n = 6.
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Coexpression of the subunits did not have a significant effect on
slow of Scn8a compared with slow of the
subunit alone (Fig. 3B), but it did decrease the
magnitude of Scn8a fast at the most negative
depolarizations (25 and 20 mV). During depolarizations to 10 mV
or more positive, the subunits increased the percent of Scn8a
current inactivating with fast. Although coexpression of
the subunits had subtle effects on the kinetics of Scn8a inactivation, it significantly accelerated the kinetics of Rat1 and
Rat2 inactivation, so that all three channels inactivated with similar
kinetics in the presence of the two subunits.
Recovery from inactivation is initially slower for Scn8a compared
with Rat1 and Rat2 sodium channels
Because Scn8a subunit channels inactivated more rapidly than
either Rat1 or Rat2 channels, it was possible that there might also be
differences in the kinetics of recovery from inactivation. To examine
recovery, currents were recorded using a two-electrode voltage clamp
with a protocol consisting of a 50 msec depolarization to 10 mV to
inactivate the channels, followed by a variable recovery interval from
1 to 3000 msec at 100 mV and a test depolarization to 10 mV to
determine the fraction of current that had recovered. Fractional
recovery versus time for Scn8a, Rat1, and Rat2 subunit channels is
shown on a log scale in Figure
4A. For the subunit alone, Scn8a recovery was best fit with a double exponential equation, whereas recovery of Rat1 and Rat2 was best fit with a triple
exponential equation. Scn8a initially recovered with a slower time
constant (larger 1) compared with Rat1 and Rat2
(Fig. 4A, Table 1).
However, Scn8a channels did not demonstrate an intermediate component
of recovery. There were only two time constants for recovery of Scn8a channels, and the second time constant (2) was of
the same order of magnitude as 3 for Rat1 and Rat2
channels. In addition, the percentage of current recovering with the
fast time constant was greater for Scn8a than for either Rat1 or Rat2.
Therefore, although Scn8a channels initially recovered more slowly than
Rat1 and Rat2 channels, they recovered more quickly during the interval
from 8 to 40 msec. The slower time constant for Scn8a
(2) was larger than 3 for Rat1 but
smaller than 3 for Rat2.
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Figure 4.
Recovery from fast inactivation of Scn8a, Rat1,
and Rat2 sodium currents. Recovery from inactivation was measured with
a two-electrode voltage clamp using a two-pulse protocol consisting of
an initial conditioning pulse to 10 mV for 50 msec (which inactivated
>95% of the channels), a variable recovery interval, and a test pulse
to 10 mV to measure the amount of current that had recovered.
Fractional recovery was calculated by dividing the current amplitude
during the test pulse by the amplitude during the corresponding
conditioning pulse. Fractional recovery is plotted on a log scale as a
function of recovery time for subunits alone
(A) and + 1 + 2
(B). Scn8a is indicated by the solid
diamonds, Rat1 is indicated by open squares, and
Rat2 is indicated by open circles. Values represent
averages, and error bars indicate SDs. The data were fit with a
single-, double-, or triple-exponential equation as described in
Materials and Methods, and the parameters of the fits and sample sizes
are shown in Table 1.
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Fractional recovery for Scn8a, Rat1, and Rat2 channels coexpressed with
the subunits is shown versus time on a log scale using a shorter
maximum time interval (300 msec) in Figure 4B. All of
the channels recovered more rapidly in the presence of the subunits. Recovery of Scn8a channels was monoexponential, whereas
recovery of Rat1 and Rat2 channels was best fit with a double-exponential equation. Initially, Scn8a recovered more slowly than Rat1 and Rat2, which was reflected in a 1 value
that was larger than that for Rat1 or Rat2 channels (Fig.
4B, Table 1). However, recovery of Scn8a channels was
similar or faster than that of Rat1 or Rat2 channels after a recovery
period of ~8 msec, because there was no slower time constant of
recovery for Scn8a channels.
The voltage dependence of activation is more positive for Scn8a
compared with Rat1 or Rat2
To examine whether Scn8a has unique voltage-dependent properties,
the voltage dependence of conductance was measured and fit with a
two-state Boltzmann equation, as described in Materials and Methods.
When the subunits were expressed alone, the voltage dependence of
conductance for Scn8a had a V1/2 that was
~9 mV more positive than that of Rat1 or Rat2 (Fig.
5A, Table 1). There were no
significant differences in the slopes of the conductance curves for the
three channels. Coexpression of the subunits with Scn8a resulted in
a negative shift of ~9 mV in the V1/2 of conductance (Fig. 5B, Table 1). This shift resulted in a
V1/2 for the voltage dependence of
conductance that was very similar to that of Rat1 and Rat2 coexpressed
with the subunits, because the subunits shifted the voltage
dependence of Scn8a conductance to a greater extent than that of Rat1
and Rat2. In summary, a more positive depolarization was necessary to
activate Scn8a subunit channels compared with Rat1 or Rat2, whereas
the voltage dependence of the three channels was comparable in the
presence of the subunits.
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Figure 5.
Voltage dependence of activation and
inactivation for Scn8a, Rat1, and Rat2 sodium channels. The voltage
dependence of activation is shown for subunits
(A) alone and + 1 + 2 (B). Sodium currents were
recorded using a cut-open oocyte clamp and elicited by depolarizing
pulses from a holding potential of 100 mV to potentials ranging from
55 to +30 mV in 5 mV increments. Conductance values were calculated
by dividing the peak current amplitude by the driving force at each
potential and normalizing to the maximum conductance, as described in
Materials and Methods. Scn8a is indicated by the solid
diamonds, Rat1 is indicated by open squares, and
Rat2 is indicated by open circles. Values represent
averages, and error bars indicate SDs. The data were fit with a
two-state Boltzmann equation as described in Materials and Methods, and
the parameters of the fits and sample sizes are shown in Table 1. The
voltage dependence of inactivation is shown for subunits alone
(C) and + 1 + 2
(D). The voltage dependence of inactivation was
determined using a two-step protocol in which a 500 msec conditioning
pulse to potentials ranging from 90 to +5 mV was followed by a 25 msec test pulse to 10 mV to measure peak current amplitude. The peak
current amplitude during the test pulse was normalized to the
maxi-mum current amplitude and is plotted as a function of the
conditioning pulse potential. Scn8a is indicated by the solid
diamonds, Rat1 is indicated by open squares, and
Rat2 is indicated by open circles. Values represent
averages, and error bars indicate SDs. The data were fit with a
two-state Boltzmann equation as described in Materials and Methods, and
the parameters of the fits and sample sizes are shown in Table 1.
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The voltage dependence of inactivation is more negative for Scn8a
compared with Rat1 and Rat2
The voltage dependence of steady-state inactivation examines the
availability of channels at a given potential, which can have profound
effects on the excitability of a cell. The voltage dependence of Scn8a
inactivation was determined and compared with that of Rat1 and Rat2
channels. Scn8a subunit channels demonstrated a
V1/2 for steady-state inactivation that
was more negative than that of Rat1 or Rat2 channels (Fig.
5C, Table 1). There were no significant differences in the
slopes of the steady-state inactivation curves among the three
channels. Coexpression of the subunits with Scn8a did not
significantly affect the voltage dependence of inactivation (Table 1).
In contrast, coexpression of the subunits markedly shifted the
V1/2 of Rat2 channels in the negative
direction, so that the curve for Rat2 channels with the subunits
was more negative than that for Scn8a channels (Fig. 5D).
Therefore, Scn8a subunit channels were inactivated at more negative
depolarizations than were Rat1 or Rat2 channels, but in the presence of
the subunits Rat2 channels were inactivated at more negative
potentials than Scn8a channels.
The persistent current of Scn8a increases with increasing voltage,
whereas that of Rat1 decreases
Previous studies have identified persistent and resurgent sodium
currents in cerebellar Purkinje neurons (Llinas and Sugimori, 1980;
Raman and Bean, 1997). In the rat, these cells have been shown to
express Rat1, Rat2, and Rat6, which is the Scn8a ortholog (Black et
al., 1994; Felts et al., 1995; Schaller et al., 1995; Vega-Saenz de
Miera et al., 1997). It has been suggested that Scn8a (or the
orthologous channel) mediates the persistent and resurgent currents
(Raman et al., 1997; Vega-Saenz de Miera et al., 1997). Therefore, we
examined the level of persistent current for Scn8a compared with that
for Rat1 and Rat2 channels. Persistent current
(Ipc) was measured 50 msec after the
beginning of a depolarization and normalized to the peak current
(Ipeak), as shown in Figure 6A for Scn8a + 1 + 2 during a depolarization to +25 mV.
To eliminate any contamination from other conductances, all analyses
were performed on current records from which had been subtracted
comparable records obtained in the presence of TTX. The levels of
persistent current for Scn8a, Rat1, and Rat2 channels coexpressed with
the subunits were examined over a voltage range from 20 to +25 mV
(Fig. 6B). Rat2 demonstrated a very small persistent
current over the entire voltage range. The percent of persistent
current for Rat1 decreased with more positive depolarizations. In
contrast, the percent of persistent current for Scn8a increased with
more positive depolarizations. Therefore, Rat1 demonstrated a greater
percent of persistent current at negative potentials (20 mV), and
Scn8a demonstrated a greater percent of persistent current at positive
potentials ( 0 mV). These data indicate that Scn8a channels mediate a
persistent sodium current that could be significant during the firing
of an action potential.
View larger version (17K):
[in this window]
[in a new window]
|
Figure 6.
Voltage dependence of persistent current for
Scn8a, Rat1, and Rat2 sodium channels. A, Representative
current trace using a two-electrode voltage clamp of Scn8a + 1 + 2 channels during a depolarization to
+25 mV. The percent of persistent current was calculated from
TTX-subtracted current traces by measuring the persistent current
remaining at 50 msec (Ipc) and
dividing by the peak current (Ipeak)
for each depolarization. B, The percent of persistent
current is shown for Scn8a (solid diamonds), for Rat1
(open squares), and for Rat2 (open
circles) over a voltage range from 20 to +25 mV in 5 mV
increments. Channels were coexpressed with the 1 and
2 subunits. Values represent averages, and error bars
indicate SDs. Sample sizes were Scn8a, n = 5; Rat1,
n = 5; and Rat2, n = 5.
|
|
Does Scn8a mediate resurgent current?
Raman and Bean (1997) and Raman et al. (1997) identified a small
transient current in Purkinje neurons that is elicited by an action
potential-like waveform. This current was termed a resurgent current,
and it was not present in Purkinje neurons from Scn8a null mice. To
examine the possibility that this current is an inherent property of
the Scn8a channel, we used a similar protocol to record from oocytes
expressing Scn8a, Rat1, or Rat2 subunits coexpressed with the subunits. Because the magnitude of the resurgent current was quite
small (~4%) compared with the peak current in the studies by Raman
and Bean (1997) and Raman et al. (1997), we used oocytes expressing
very high levels of peak sodium current (8-30 µA). Oocytes were
depolarized to +30 mV for 48 msec from a holding potential of 90 mV,
which resulted in a steady-state level of inactivation. This
depolarization was immediately followed by test depolarizations ranging
from 80 to +40 mV in 10 mV increments to elicit any resurgent current
(Fig. 7A). Figure
7B shows representative current traces for Scn8a currents
during test depolarizations from 60 to +40 mV in 20 mV increments,
after subtraction of comparable records obtained in the presence of
TTX. At negative depolarizations, there was an initial rapidly decaying
current followed by a persistent current. At depolarizations above +20
mV, the transient current was less pronounced.
View larger version (28K):
[in this window]
[in a new window]
|
Figure 7.
Possible resurgent current resulting from Scn8a,
Rat1, and Rat2 channels. Channels were coexpressed with the subunits as described in the legend to Figure 2. Oocytes expressing
current levels of 8-30 µA were used to attain larger and more easily
measurable potential resurgent currents, which were recorded using a
two-electrode voltage clamp. A, The protocol used to
elicit resurgent current consists of depolarization from a holding
potential of 90 mV to a membrane depolarization of +30 mV for 48 msec
followed by a range of depolarizations from 80 to +50 mV in 10 mV
increments for 25 msec. B, Representative TTX subtracted
current traces are shown for Scn8a channels during depolarizations from
60 to +40 mV in 20 mV increments. The vertical scale bar corresponds
to 10% of the initial current evoked during the depolarization to +30
mV, which is off the scale. C, The percent of the peak
potential resurgent current normalized to the peak initial current is
shown. D, The potential resurgent currents during each
depolarization were normalized to the peak current and plotted versus
voltage for Scn8a (solid diamonds), Rat1 (open
squares), and Rat2 (open circles). Values
represent averages, and error bars indicate SDs. Sample sizes were
Scn8a, n = 4; Rat1, n = 5; and
Rat2, n = 4.
|
|
Figure 7C shows the percent of the maximal transient current
relative to the peak current obtained during a single depolarization to
0 mV (10 mV for Rat1 and Rat2). Scn8a had a much larger percent of
transient current compared with Rat1 or Rat2. The level of transient
current for Scn8a was ~4% of the peak current. This level of current
is similar to the level of resurgent current observed in Purkinje cells
by Raman and Bean (1997) and Raman et al. (1997). The resurgent current
described by Raman et al. (1997) differed in voltage dependence
compared with the initial current, with a peak at 40 mV for the
resurgent current compared with 0 mV for the initial current.
Therefore, we examined the voltage dependence of the transient current
by normalizing the current at each depolarization to the maximal
transient current (Fig. 7D). The transient current for all
three channels showed similar voltage dependence to the voltage
dependence of the peak current, with a peak at 0 mV for Scn8a and 10
mV for Rat1 and Rat2. Therefore, although the magnitude of the
transient current was similar to that previously observed for resurgent
current, the fact that the voltage dependence was equivalent to that of the peak current suggests that none of these channels mediates a true
resurgent current when expressed in Xenopus oocytes.
| |
DISCUSSION |
We have constructed a full-length cDNA clone encoding the Scn8a
sodium channel. Injection of RNA transcribed from this clone resulted
in functional sodium currents in Xenopus oocytes. There were
five amino acid differences between the sequence of the clone that we
obtained and the sequence previously determined for Scn8a (Burgess et
al., 1995) (Plummer, Galt, Jones, Burgess, Sprunger, Kohrman, and
Meisler, unpublished observations). One or more of the first four
differences might represent polymorphisms, because our data were
obtained from inbred C57BL/6J mice, and the first 257 residues reported
by Burgess et al. (1995) were obtained from strain ICR mice. Consistent
with this hypothesis, the leucine that we noted at position 5 is
present in the Rat6 sequence that was determined by Dietrich et al.
(1998) and in mouse strain DBA/2 (Kohrman et al., 1996b). However, it
is possible that at least some of the differences represent artifacts
of the PCR. The cytoplasmic linker connecting domains I-II in the cDNA
clone that we constructed is 10 amino acids shorter than the longest
alternatively spliced form that was detected in mice (Plummer, Galt,
Jones, Burgess, Sprunger, Kohrman, and Meisler, unpublished
observations) and rat (Schaller et al., 1995; Dietrich et al., 1998).
This shorter form represents the major form of the Scn8a transcript in
the brains of mice (Plummer, Galt, Jones, Burgess, Sprunger, Kohrman, and Meisler, unpublished observations) and rat (Dietrich et al., 1998).
The position and size of this variation corresponds to alternatively
spliced forms that were described previously for Rat1 (Schaller et al.,
1992).
The electrophysiological properties of the Scn8a sodium channel
differed somewhat from those determined by Dietrich et al. (1998) for
the Rat6 (PN4) channel. Specifically, Dietrich et al. (1998) observed
slightly faster inactivation kinetics and significantly more negative
V1/2 values for activation and
inactivation. At least some of these differences probably result from
different recording techniques, because Dietrich et al. (1998) used
macropatch recording and we used the cut-open oocyte voltage clamp.
Some of the electrophysiological properties that we observed for the Scn8a sodium channel also differed from those of the Rat1 and Rat2
channels, which are expressed in the adult CNS. The most dramatic
difference was that Scn8a subunit channels inactivated more rapidly
than either Rat1 or Rat2 subunit channels. With respect to voltage
dependence, Scn8a channels activated at more positive potentials than
Rat1 and Rat2 but demonstrated a more negative
V1/2 for steady-state inactivation. Based
on these characteristics alone, cells expressing only Scn8a should have decreased electrical excitability and a higher threshold for activation of action potentials compared with cells expressing Rat1 or Rat2.
However, sodium channels in the CNS are most likely associated with one
or both of the subunits, which modulate the properties of the Scn8a
sodium channel. The subunits accelerated the kinetics of
inactivation, but to much less of an extent than was observed for Rat1
and Rat2, so that all three channels demonstrated similar kinetics in
the presence of the subunits. Coexpression of the subunits
caused a large hyperpolarizing shift in the voltage dependence of
activation for Scn8a but had no significant effect on the voltage
dependence of steady-state inactivation. Recovery from inactivation was
accelerated by the presence of the subunits. Because of these
effects, the electrophysiological properties of Scn8a sodium channels
were very similar to those of Rat1 and Rat2 sodium channels when all
three channels were coexpressed with the subunits.
We observed that Scn8a with the subunits demonstrated a persistent
current that increased linearly with more positive membrane potentials.
In contrast, Rat1 demonstrated a persistent current that was large at
negative potentials and decreased with more positive membrane
potentials. These results suggest that Scn8a channels mediate a
persistent current that is largest when an action potential is fired,
which might play a critical role in the repetitive firing of action
potentials seen in Purkinje neurons. However, the persistent current
from Rat1 may also be involved in the excitability of Purkinje
neurons.
Raman et al. (1997) observed a persistent current in normal Purkinje
cells, and the magnitude of that current was greatly decreased in cells
from mice containing a Scn8a null mutation. Vega-Saenz de Miera et al.
(1997) used single-cell RT-PCR from guinea pig cerebellar slices and
in situ hybridization to show that Rat6 (the Scn8a ortholog)
and Rat1 are expressed in Purkinje neurons. There was no detectable
level of Rat2 mRNA in the studies by Vega-Saenz de Miera et al. (1997),
although Rat2 mRNA was detected in Purkinje neurons by Black et al.
(1994). Based on these results, Vega-Saenz de Miera et al. (1997)
suggested that Rat1 mediates a transient current in Purkinje neurons,
whereas Scn8a mediates noninactivating persistent current in Purkinje
neurons. Our data and the results of Raman et al. (1997) support a role
for Rat1 and Scn8a contributing to both transient and persistent
currents.
In addition to the persistent current, another unique sodium
conductance termed a resurgent current has been observed in cerebellar Purkinje neurons (Raman and Bean, 1997; Raman et al., 1997). The resurgent current is a small sodium current that is elicited by a
waveform that simulates an action potential. This current was observed
both in normal Purkinje cells and in cells containing the null Scn8a
mutation, but it was much larger in the normal Purkinje cells
expressing Scn8a. These results suggested that Scn8a might be the
primary sodium channel responsible for the resurgent current. Using
similar electrophysiological protocols to elicit resurgent current, we
observed a current that was ~4% of the peak transient current.
However, that current inactivated with a time course similar to that of
the initial transient current. In addition, the current that we
observed demonstrated a peak amplitude at 0 mV, whereas the resurgent
current in Purkinje cells demonstrated a peak at approximately 40 mV.
Finally, currents from Rat1 and Rat2 channels demonstrated similar
"resurgent" currents, although the levels were approximately half
of that seen for Scn8a. It seems likely that the current that we
observed was not a true resurgent current but resulted from the large
persistent current observed for Scn8a at positive depolarizations. With
a large persistent current at +30 mV, depolarization to a more negative
potential would increase the driving force for sodium and result in a
significant increase in current amplitude. Although the current that we
identified was most likely not the resurgent current, it may contribute
to the resurgent current in Purkinje cells, particularly because the
magnitude was similar to that observed in Purkinje cells.
There are several possible reasons why we did not observe a true
resurgent current from Scn8a channels expressed in Xenopus oocytes. First, it is possible that the Scn8a channel undergoes post-translational modification in Purkinje neurons, which may change
some of the electrophysiological properties of the channel. Second,
there may be accessory proteins other than the subunits in Purkinje
neurons, and these could alter the properties of the channel. For
example, Ma et al. (1997) have shown that the Rat2 channel mediates
persistent current if that channel is coexpressed with G-protein 
subunits. A variation of this explanation is that the subunits
prevent resurgent current. However, we did not observe any resurgent
current when the Scn8a channel was expressed in the absence of the subunits (data not shown). Third, it is possible that multiple
different sodium channels have to be expressed to obtain resurgent
current. We consider this explanation unlikely, because we have not
observed resurgent current when Scn8a was coexpressed with Rat1 and/or
Rat 2 in oocytes (data not shown). Finally, it is possible that the
resurgent current may be a property of one specific splice variant of
the Scn8a sodium channel. Plummer, Galt, Jones, Burgess, Sprunger,
Kohrman, and Meisler (unpublished observations) have shown that
multiple splice variants of the Scn8a subunit exist. This
hypothesis can now be tested by constructing and expressing the various
splice variants.
| |
FOOTNOTES |
Received March 3, 1998; revised May 22, 1998; accepted May 27, 1998.
This work was supported by National Institutes of Health Grants NS26729
(A.L.G.) and NS34609 (M.H.M.). N.W.P. was supported by the
Organogenesis Center, University of Michigan. A.L.G. is an Established
Investigator of the American Heart Association. We thank Drs. Michael
Pugsley, Ted Shih, and Daniel Allen for helpful discussions during the
course of this work, Dr. Leslie Sprunger for critical review of this
manuscript, and Mimi Reyes for excellent technical assistance.
Correspondence should be addressed to Alan L. Goldin, Department of
Microbiology and Molecular Genetics and Physiology and Biophysics,
University of California, Irvine, CA 92697-4025.
Dr. R. Smith's present address: Department of Biology, University of
California, San Diego, La Jolla, CA 92093-0357.
Dr. Plummer's present address: Department of Genetics, Duke University
Medical Center, Durham, NC 27710.
| |
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C. Rosker, B. Lohberger, D. Hofer, B. Steinecker, S. Quasthoff, and W. Schreibmayer
The TTX metabolite 4,9-anhydro-TTX is a highly specific blocker of the Nav1.6 voltage-dependent sodium channel
Am J Physiol Cell Physiol,
August 1, 2007;
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[Abstract]
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Y. Kang, M. Saito, H. Sato, H. Toyoda, Y. Maeda, T. Hirai, and Y.-C. Bae
Involvement of Persistent Na+ Current in Spike Initiation in Primary Sensory Neurons of the Rat Mesencephalic Trigeminal Nucleus
J Neurophysiol,
March 1, 2007;
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[Abstract]
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J. R. A. Wooltorton, S. Gaboyard, K. M. Hurley, S. D. Price, J. L. Garcia, M. Zhong, A. Lysakowski, and R. A. Eatock
Developmental Changes in Two Voltage-Dependent Sodium Currents in Utricular Hair Cells
J Neurophysiol,
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[Abstract]
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E. Shirahata, H. Iwasaki, M. Takagi, C. Lin, V. Bennett, Y. Okamura, and K. Hayasaka
Ankyrin-G Regulates Inactivation Gating of the Neuronal Sodium Channel, Nav1.6
J Neurophysiol,
September 1, 2006;
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[Abstract]
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S. I. Levin, Z. M. Khaliq, T. K. Aman, T. M. Grieco, J. A. Kearney, I. M. Raman, and M. H. Meisler
Impaired Motor Function in Mice With Cell-Specific Knockout of Sodium Channel Scn8a (NaV1.6) in Cerebellar Purkinje Neurons and Granule Cells
J Neurophysiol,
August 1, 2006;
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E. Schiavon, T. Sacco, R. R. Cassulini, G. Gurrola, F. Tempia, L. D. Possani, and E. Wanke
Resurgent Current and Voltage Sensor Trapping Enhanced Activation by a beta-Scorpion Toxin Solely in Nav1.6 Channel: SIGNIFICANCE IN MICE PURKINJE NEURONS
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A. Van Wart and G. Matthews
Impaired firing and cell-specific compensation in neurons lacking nav1.6 sodium channels.
J. Neurosci.,
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C. Yue and Y. Yaari
Axo-Somatic and Apical Dendritic Kv7/M Channels Differentially Regulate the Intrinsic Excitability of Adult Rat CA1 Pyramidal Cells
J Neurophysiol,
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[Abstract]
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N. Astman, M. J. Gutnick, and I. A. Fleidervish
Persistent sodium current in layer 5 neocortical neurons is primarily generated in the proximal axon.
J. Neurosci.,
March 29, 2006;
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N. Osorio, G. Alcaraz, F. Padilla, F. Couraud, P. Delmas, and M. Crest
Differential targeting and functional specialization of sodium channels in cultured cerebellar granule cells
J. Physiol.,
December 15, 2005;
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J.-Y. Lou, F. Laezza, B. R. Gerber, M. Xiao, K. A. Yamada, H. Hartmann, A. M. Craig, J. M. Nerbonne, and D. M. Ornitz
Fibroblast growth factor 14 is an intracellular modulator of voltage-gated sodium channels
J. Physiol.,
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C. Yue, S. Remy, H. Su, H. Beck, and Y. Yaari
Proximal Persistent Na+ Channels Drive Spike Afterdepolarizations and Associated Bursting in Adult CA1 Pyramidal Cells
J. Neurosci.,
October 19, 2005;
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S. Saleh, S. Y. M Yeung, S. Prestwich, V. Pucovsky, and I. Greenwood
Electrophysiological and molecular identification of voltage-gated sodium channels in murine vascular myocytes
J. Physiol.,
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[Abstract]
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K. Ptak, G. G. Zummo, G. F. Alheid, T. Tkatch, D. J. Surmeier, and D. R. McCrimmon
Sodium Currents in Medullary Neurons Isolated from the Pre-Botzinger Complex Region
J. Neurosci.,
May 25, 2005;
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[Abstract]
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A. M. Rush, S. D. Dib-Hajj, and S. G. Waxman
Electrophysiological properties of two axonal sodium channels, Nav1.2 and Nav1.6, expressed in mouse spinal sensory neurones
J. Physiol.,
May 1, 2005;
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R. M. Leao, C. Kushmerick, R. Pinaud, R. Renden, G.-L. Li, H. Taschenberger, G. Spirou, S. R. Levinson, and H. von Gersdorff
Presynaptic Na+ Channels: Locus, Development, and Recovery from Inactivation at a High-Fidelity Synapse
J. Neurosci.,
April 6, 2005;
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[Abstract]
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Z. M. Khaliq and I. M. Raman
Axonal Propagation of Simple and Complex Spikes in Cerebellar Purkinje Neurons
J. Neurosci.,
January 12, 2005;
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F. S. Afshari, K. Ptak, Z. M. Khaliq, T. M. Grieco, N. T. Slater, D. R. McCrimmon, and I. M. Raman
Resurgent Na Currents in Four Classes of Neurons of the Cerebellum
J Neurophysiol,
November 1, 2004;
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[Abstract]
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D. Johnson, M. L. Montpetit, P. J. Stocker, and E. S. Bennett
The Sialic Acid Component of the {beta}1 Subunit Modulates Voltage-gated Sodium Channel Function
J. Biol. Chem.,
October 22, 2004;
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E. K. Wittmack, A. M. Rush, M. J. Craner, M. Goldfarb, S. G. Waxman, and S. D. Dib-Hajj
Fibroblast Growth Factor Homologous Factor 2B: Association with Nav1.6 and Selective Colocalization at Nodes of Ranvier of Dorsal Root Axons
J. Neurosci.,
July 28, 2004;
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M. J. Craner, J. Newcombe, J. A. Black, C. Hartle, M. L. Cuzner, and S. G. Waxman
Molecular changes in neurons in multiple sclerosis: Altered axonal expression of Nav1.2 and Nav1.6 sodium channels and Na+/Ca2+ exchanger
PNAS,
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[Abstract]
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M. J. Craner, B. C. Hains, A. C. Lo, J. A. Black, and S. G. Waxman
Co-localization of sodium channel Nav1.6 and the sodium-calcium exchanger at sites of axonal injury in the spinal cord in EAE
Brain,
February 1, 2004;
127(2):
294 - 303.
[Abstract]
[Full Text]
[PDF]
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T. M. Grieco and I. M. Raman
Production of Resurgent Current in NaV1.6-Null Purkinje Neurons by Slowing Sodium Channel Inactivation with {beta}-Pompilidotoxin
J. Neurosci.,
January 7, 2004;
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R. I Herzog, T. R Cummins, F. Ghassemi, S. D Dib-Hajj, and S. G Waxman
Distinct repriming and closed-state inactivation kinetics of Nav1.6 and Nav1.7 sodium channels in mouse spinal sensory neurons
J. Physiol.,
September 15, 2003;
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[Abstract]
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R. I. Herzog, C. Liu, S. G. Waxman, and T. R. Cummins
Calmodulin Binds to the C Terminus of Sodium Channels Nav1.4 and Nav1.6 and Differentially Modulates Their Functional Properties
J. Neurosci.,
September 10, 2003;
23(23):
8261 - 8270.
[Abstract]
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[PDF]
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M. J. Craner, A. C. Lo, J. A. Black, and S. G. Waxman
Abnormal sodium channel distribution in optic nerve axons in a model of inflammatory demyelination
Brain,
July 1, 2003;
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1552 - 1561.
[Abstract]
[Full Text]
[PDF]
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Z. M. Khaliq, N. W. Gouwens, and I. M. Raman
The Contribution of Resurgent Sodium Current to High-Frequency Firing in Purkinje Neurons: An Experimental and Modeling Study
J. Neurosci.,
June 15, 2003;
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4899 - 4912.
[Abstract]
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T.R. Cummins, M. Renganathan, P.K. Stys, R.I. Herzog, K. Scarfo, R. Horn, S.D. Dib-Hajj, and S.G. Waxman
The pentapeptide QYNAD does not block voltage-gated sodium channels
Neurology,
January 28, 2003;
60(2):
224 - 229.
[Abstract]
[Full Text]
[PDF]
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T. R. Cummins, S. D. Dib-Hajj, S. G. Waxman, and D. F. Donnelly
Characterization and Developmental Changes of Na+ Currents of Petrosal Neurons With Projections to the Carotid Body
J Neurophysiol,
December 1, 2002;
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2993 - 3002.
[Abstract]
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K. J. Nielsen, M. Watson, D. J. Adams, A. K. Hammarstrom, P. W. Gage, J. M. Hill, D. J. Craik, L. Thomas, D. Adams, P. F. Alewood, et al.
Solution Structure of {micro}-Conotoxin PIIIA, a Preferential Inhibitor of Persistent Tetrodotoxin-sensitive Sodium Channels
J. Biol. Chem.,
July 19, 2002;
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J. Tan, Z. Liu, Y. Nomura, A. L. Goldin, and K. Dong
Alternative Splicing of an Insect Sodium Channel Gene Generates Pharmacologically Distinct Sodium Channels
J. Neurosci.,
July 1, 2002;
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[Abstract]
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T. M. Grieco, F. S. Afshari, and I. M. Raman
A Role for Phosphorylation in the Maintenance of Resurgent Sodium Current in Cerebellar Purkinje Neurons
J. Neurosci.,
April 15, 2002;
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3100 - 3107.
[Abstract]
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A. L. Goldin
Evolution of voltage-gated Na+ channels
J. Exp. Biol.,
March 1, 2002;
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575 - 584.
[Abstract]
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C. C. McIntyre, A. G. Richardson, and W. M. Grill
Modeling the Excitability of Mammalian Nerve Fibers: Influence of Afterpotentials on the Recovery Cycle
J Neurophysiol,
February 1, 2002;
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995 - 1006.
[Abstract]
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M. H. Meisler, J. Kearney, A. Escayg, B. T. Macdonald, and L. K. Sprunger
Sodium Channels and Neurological Disease: Insights from Scn8a Mutations in the Mouse
Neuroscientist,
April 1, 2001;
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136 - 145.
[Abstract]
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N. Maurice, T. Tkatch, M. Meisler, L. K. Sprunger, and D. J. Surmeier
D1/D5 Dopamine Receptor Activation Differentially Modulates Rapidly Inactivating and Persistent Sodium Currents in Prefrontal Cortex Pyramidal Neurons
J. Neurosci.,
April 1, 2001;
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K. A. Kazen-Gillespie, D. S. Ragsdale, M. R. D'Andrea, L. N. Mattei, K. E. Rogers, and L. L. Isom
Cloning, Localization, and Functional Expression of Sodium Channel beta 1A Subunits
J. Biol. Chem.,
January 14, 2000;
275(2):
1079 - 1088.
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F. Lehmann-Horn and K. Jurkat-Rott
Voltage-Gated Ion Channels and Hereditary Disease
Physiol Rev,
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J. Magistretti, D. S. Ragsdale, and A. Alonso
High Conductance Sustained Single-Channel Activity Responsible for the Low-Threshold Persistent Na+ Current in Entorhinal Cortex Neurons
J. Neurosci.,
September 1, 1999;
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[Abstract]
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Top
Abstract
Introduction
