BBRC
Biochemical and Biophysical Research Communications 319 (2004) 531–540
www.elsevier.com/locate/ybbrc
Role of auxiliary b1-, b2-, and b3-subunits and their interaction
with Nav 1.8 voltage-gated sodium channelq
Kausalia Vijayaragavan,a Andrew J. Powell,b Ian J. Kinghorn,b and Mohamed Chahinea,*
b
a
Department of Medicine, Laval University, 2725 Chemin Sainte-Foy, Sainte-Foy, Que. G1V 4G5, Canada
Gene Expression and Protein Biochemistry Department, GlaxoSmithKline, Stevenage, Hertfordshire SG1 2NY, UK
Received 19 April 2004
Available online 20 May 2004
Abstract
The nociceptive C-fibers of the dorsal root ganglion express several sodium channel isoforms that associate with one or more
regulatory b-subunits (b1 –b4 ). To determine the effects of individual and combinations of the b-subunit isoforms, we co-expressed
Nav 1.8 in combination with these b-subunits in Xenopus oocytes. Whole-cell inward sodium currents were recorded using the twomicroelectrode voltage clamp method. Our studies revealed that the co-expression b1 alone or in combination with other b-subunits
enhanced current amplitudes, accelerated current decay kinetics, and negatively shifted the steady-state curves. In contrast, b2 alone
and in combination with b1 altered steady-state inactivation of Nav 1.8 to more depolarized potentials. Co-expression of b3 shifted
steady-state inactivation to more depolarized potentials; however, combined b1 b3 expression caused no shift in channel availability.
The results in this study suggest that the functional behavior of Nav 1.8 will vary depending on the type of b-subunit that expressed
under normal and disease states.
Ó 2004 Elsevier Inc. All rights reserved.
Keywords: Voltage-gated sodium channels; Nav 1.8; b-Subunits; Dorsal root ganglion; C-fibers; Xenopus oocytes; Voltage clamp; Expression levels;
Steady-state properties; Decay kinetics
Voltage-gated Naþ channels are transmembrane
proteins responsible for the generation of action potentials in excitable cells and play an important role in
the modulation of electrical impulses. The mammalian
Naþ channels consist of a 260 kDa a-subunit that encodes the core, pore-forming protein of the channel. To
date, 10 different mammalian a-subunit isoforms have
been identified and at least 7 are expressed in the nervous system. Nav 1.1, Nav 1.2, Nav 1.3, and Nav 1.6 are
predominantly expressed in the central nervous system
(CNS) while Nav 1.7, Nav 1.8, and Nav 1.9 are principally
found in the peripheral nervous system (PNS) [1].
Many a-subunits associate with regulatory b-subunits
(32–36 kDa) either noncovalently (b1 -, b1A -, and/or
b3 -subunits) or covalently (b2 and b4 -subunits) [2,3]. The
b-subunits are structurally homologous and form single
q
Modulation of Nav1.8 by auxiliary b-subunits.
Corresponding author. Fax: +1-418-656-4509.
E-mail address: [email protected] (M. Chahine).
*
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2004.05.026
transmembrane glycoproteins with short intracellular
loops and immunoglobulin-like extracellular segments
[4]. The intracellular segment of the b1 -subunit is required for recruitment of cytosolic proteins such as
ankyrin G [5,6]. The extracellular Ig domain of the
b-subunits has cell adhesion properties that enable association with extracellular matrix proteins such as
tenascin-C, tenascin-R, and neurofascin [4,7–9]. The
association of Naþ channels with b-subunits, that consequently interact with intra- and extracellular proteins,
is important: (1) to promote and stabilize the density of
channels within the plasma membrane [10–13] and (2)
for the translocation/clustering of neuronal Naþ channels to specific and distinct domains within the neuron
such as the nodes of Ranvier axon initial segments [14].
The auxiliary subunits thus act as chaperones that regulate the expression levels of the channels. The transmembrane segment and parts of the intracellular regions
of the b1 -subunit have been suggested to modulate
expression levels of the channel [15].
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K. Vijayaragavan et al. / Biochemical and Biophysical Research Communications 319 (2004) 531–540
The extracellular Ig fold of the b1 -subunit interacts
with the outer pore segment of the a-subunit (SS2-S6
loop) and this interaction is required for the electrophysiological modulation of the channels [8,15–17]. The
influence of each b-subunit subtype on channel gating is
different. b2 - and b3 -subunits are observed to modulate
the gating of channels to a lesser extent than the b1 subunit in Xenopus oocytes [4,7]. Co-expression of b1
with many Naþ channels shifts steady-state activation
and inactivation; accelerates the time constant of current
decay and increases levels of expression in oocytes [7,18].
Furthermore, Qu et al. [26] reported that the brain
Nav 1.2 channels can mediate large persistent inward
Naþ currents when co-expressed with b3 , in tsA201 cells.
Zimmer and Benndorf [28] recently observed that different molecular segments of the b-subunits are involved
in modulation of different Naþ channel isoforms, where
the extracellular segment of the b1 -subunit is crucial for
modulation of the Nav 1.2 brain channel expression and
gating properties however the transmembrane segment
of the b1 -subunit regulates Nav 1.5 channel properties.
In situ hybridization and immunochemistry have
shown that the small and large fibers of the PNS dorsal
root ganglion (DRG) express 3 isotypes of b-subunits
namely the b1 -, b2 -, and b3 -subunits [7,19–21]. However,
the recently isolated b4 -subunit is only expressed in the
large and medium fibers and is absent in the small
C-fibers [3].
The present study examines the effects of co-expression of b1 -, b2 -, and b3 -subunits on Nav 1.8 a-subunit
expression and electrophysiological properties in Xenopus oocytes. Here we show that all three b-subunits are
able to interact with Nav 1.8 channel in oocytes. The coexpression of the b1 -subunit alone or in combination
with other b-subunits increased current amplitude, increased the time constant of current decay, and shifted
steady-state activation and inactivation of Nav 1.8 to
hyperpolarized potentials. In contrast, the b2 -subunit
had little effect on Nav 1.8 peak current levels but shifted
steady-state inactivation to more depolarized potentials
when expressed alone and in combination with b1 . The
b3 -subunit, on the other hand, positively shifted steadystate inactivation but did not modulate steady-state
activation or the time constant of inactivation or
increase functional expression of Nav 1.8.
Methods and materials
Molecular biology
Construction of full-length rat a-subunit Nav 1.8, b2 - and b3 -subunit
cDNAs. Total RNA was isolated from Sprague–Dawley rat dorsal root
ganglions (DRG) using the Trizol reagent (Life Technologies, Gaithersburg, MD). Rat DRG RNA was reverse-transcribed (RT) using
Superscript (Gibco-BRL) and the rat Nav 1.8 a-subunit cDNA was
isolated and subcloned into the pSP64T Xenopus oocyte expression
vector as described previously [13]. The pSP64T vector, initially
described by Krieg and Melton [22], contains a fragment of the globin
gene which when co-transcribed with the Nav 1.8 channel increases
stability of the injected cRNA in the Xenopus oocytes. The rat b2 and
b3 -subunit cDNAs were isolated from the DRG library and subcloned
into the pCDNA3a-topo and pGEM3z vectors, respectively. The
b1 -subunit was cloned into pGEM3z vector and kindly donated by R.
Kallen (University of Pennsylvania, Philadelphia, USA). Complementary RNAs (cRNAs) were prepared by the T7 (b1 –b3 ) or SP6
(Nav 1.8) mMessage mMachine kit (Ambion, Texas, USA).
Expression and electrophysiology in Xenopus oocytes
Xenopus laevis females were anesthetized with 1.5 mg/ml tricaine
(Sigma, Oakville, Canada), and two or three ovarian lobes were surgically removed. Follicular cells surrounding the oocytes were removed
by incubation at 22 °C for 2.5 h in calcium-free oocyte medium
(82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl2 , and 5 mM Hepes, pH 7.6)
containing 2 mg/ml collagenase (Sigma, Oakville, Ontario, Canada).
The oocytes were first washed in calcium-free medium and then with a
50% Leibovitz’s L-15 medium (Life technologies, Burlington, Ontario,
Canada) enriched with 15 mM Hepes, 5 mM L -glutamine, and 10 mg/
ml gentamicin (pH 7.6). The oocytes were then stored in this medium
until further use. Fifty to hundred healthy stage VI–V oocytes were
selected and 50 nl of cRNA was microinjected into each oocyte. Equal
amounts of Nav 1.8 and b1 -, b2 -, or b3 -subunit cRNAs, at concentrations of 2 lg/ll, were microinjected into the oocytes. Oocytes were
stored at 18 °C and used for experiments 4–5 days post-injection. The
growth medium was changed every two days and unhealthy oocytes
were removed from the medium.
Whole-cell sodium current from cRNA-injected oocytes was measured using two-microelectrode voltage clamp at room temperature,
22–23 °C. The oocytes were impaled with <2 MX electrodes containing
3 M KCl and were voltage-clamped with an OC-725 oocyte clamp
(Warner Instruments, Hamden, CT, USA). The bath Ringer solution
contained: 90 mM NaCl, 2 mM KCl, 1.8 mM CaCl2 , 2 mM MgCl2 , and
5 mM Hepes (pH 7.6). Currents were filtered at 1.5 kHz with an 8-pole
Bessel filter and sampled at 10 kHz. Data were acquired and analyzed
with pCLAMP software v7 (Axon Instruments).
Current density for each oocyte was determined from the peak
current amplitude evoked in response to test potentials to +20 mV or
to +10 mV.
Time constant of inactivation was determined by fitting the current
decay to a single exponential function. The time constant was plotted
versus the test voltage: I ¼ AI expðt=sh Þ þ C, where I is the current,
AI is the percentage of channel inactivating with the time constant sh ; t
is time, and C is the steady-state asymptote.
Voltage-dependence of activation was determined from I/V curves
where inward Naþ currents were elicited using depolarizing pulses
from a holding potential of )100 mV to potentials ranging from )80 to
+60 mV in 5-mV increments. Current activation curves of the channels
were plotted using the following Boltzmann equation: GNa =GNa max ¼
1=ð1 þ expðV1=2 V Þ=kv Þ, where GNa (conductance) for each oocytes
clamped were determined by dividing the peak Naþ current by the
driving force (Vm ENa ). The reversal potential (ENa ) was estimated by
extrapolating the linear ascending segment of decay gradient between
+20 and +40 mV of an I–V curve to the voltage axis. V is the test
voltage, V1=2 is the voltage at which the channels are half-maximal
activated, and kv is the slope factor. Conductance versus voltage data
was fitted with a two-state Boltzmann equation.
Voltage-dependence of inactivation was determined by eliciting
500 ms conditioning pulses to voltages between )110 and +30 mV in 5mV increments followed by a standard test pulse to either +20 or
+10 mV. Test currents were normalized and plotted versus conditioning voltage. Inactivation curves were fitted to Boltzmann relation:
I=Imax ¼ 1=ð1 þ expðV V1=2 Þ=kv Þ where V is the test voltage, V1=2 is
the voltage at which the channels are half-maximal activated, and kv is
the slope factor.
K. Vijayaragavan et al. / Biochemical and Biophysical Research Communications 319 (2004) 531–540
Antibody generation
Site-directed antibodies against b1 - and b3 -subunits were generated
against synthetic peptides corresponding to the C-terminal cytoplasmic
tails of the human b1 and b3 proteins. Peptides CTGVQVAE (b1 subunit) and CSAVPVEE (b3 -subunit) were synthesized by standard
solid phase techniques (Severn BioTech) and conjugated to tubercullinPPD carrier using sulphoSMCC (Pierce) via the N-terminal cysteine residues. The resulting conjugates were used to raise antibody
responses in NZW and affinity purification against the immunogen
peptide used to isolate the polyclonal antibody from the bleed sera (as
described in [21]). b2 -subunit polyclonal antibody generation has been
described previously [21].
Western blot
Membrane fractions of oocytes were isolated using a protocol
adapted from Shih et al. [23]. Briefly 50–100 oocytes injected with
Nav 1.8, b-subunits and different combinations of b-subunits were
gently rinsed with ice-cold 1 phosphate-buffered saline (PBS) and
each set of oocytes was then resuspended in 1 ml of ice-cold buffered
10% sucrose solution (10% sucrose, 150 mM NaCl, 5 mM KCl, and
20 mM Hepes, pH 7.5) containing protease inhibitors (0.5 lg/ml leupeptin, 0.7 lg/ml pepstatin, 2 lg/ml aprotinin, 0.1 mM EDTA, 0.5 mM
PMSF (phenylmethylsulfonyl fluoride), and 0.5 mM DTT). Oocytes
were homogenized (Dounce homogenizer) and overlaid on a 4 °C 10–
20–50% discontinuous sucrose gradient in the same buffer without
protease inhibitors. Sucrose gradients were spun in a swing bucket
rotor (Sorval AH-650) at 37,000 rpm for 35 min at 4 °C. Membrane
fraction was collected from 20 to 50% interface, diluted 1:3 with icecold water, and pelleted by centrifugation in an AM 50.14 Jouan rotor
(Table-top centrifuge, Jouan MR22i) at 11,000 rpm for 10 min at 4 °C.
Membrane proteins were homogenized further (Dounce homogenizer,
type A pestle) on ice, in 500 ll of solubilization buffer (75 mM KCl,
75 mM NaCl, 50 mM sodium phosphate (pH 7.2), 2 mg/ml soybean
lipids (Avanti, Alabaster), 1% Triton X-100, and protease inhibitor
cocktail). Insoluble materials were removed by centrifugation at
8000 rpm for 30 min at 4 °C. Protein concentration was determined
using the Bio-Rad protein assay (Bio-Rad laboratories) and BSA as a
standard. Equal amounts of protein (20–50 lg) were boiled in sample
buffer, separated by 8–12% SDS–PAGE gels (3.9% stacking: 8–12%
separating gel) according to the Laemmli method. Proteins were separated using the Mini-Protean 3 system (Bio-Rad) and transferred to
PVDF membranes (Millipore Immobilon-P transfer membrane) by the
Mini Trans-Blot electrophoretic transfer cell (Bio-Rad) overnight at
0.45 A. The membranes were blocked in 5% skimmed milk in 1 PBS
for 2 h at room temperature and then incubated overnight at 4 °C with
the primary antibody against Nav 1.8 and the b-subunits. The primary
antibodies against the b1 , b2 , and b3 -subunits were diluted to 1:200 in
blocking buffer (1 PBS/0.1% Tween/5% skimmed milk).
After the overnight incubation with the primary antibodies, the
membranes were rinsed 3 times for 10 min with wash buffer containing
1 PBS/0.1% Tween. Secondary goat anti-rabbit antibody conjugated
with peroxidase H + L (Jackson ImmunoResearch, 111-035-003) was
diluted 1:10,000 in the blocking buffer and incubated at room temperature for 1 h. Membranes were then rinsed 3 times for 10 min with
wash buffer. Detection was performed using the enhanced chemiluminescence (ECL) technique (Amersham–Pharmacia ECL kit, RPN
21029) according to manufacturer’s instructions and exposed on X-ray
Kodak Film (Scientific Imaging Film, Biomax MR) at different times
(20 s to 10 min).
Statistical analysis
Data were expressed using means SEM. A randomized block
design was involved to compare the different b-subunits and combination of b-subunits. Duncan’s comparison technique was used to
533
evaluate all b-subunits to Nav 1.8. The univariate normality assumptions were verified with the Shapiro–Wilk test. The Brown and
Forsythe’s variation of Levene’s test statistic was used to verify the
homogeneity of variances. The results were considered significant with
p values 60.05. All analyses were conducted using the statistical
package SAS v8.2 (SAS Institute, Cary, NC, USA).
Results
To study the functional properties of the Nav 1.8
channel in the presence of individual and various combinations of b-subunits, we cloned the genes encoding
the Nav 1.8 a-subunit [13], b2 , and b3 -subunits from the
Sprague–Dawley rat DRG using RT-PCR (see Methods
and materials). Fidelities of the cloned cDNAs were
confirmed by sequencing and comparison to the published sequences (Acc U53833 for Nav 1.8, U37026 for
b2 , and AJ243396 for b3 ).
Western blot analysis of Nav 1.8 and b-subunit interaction
To examine the interaction between Nav 1.8 and the
b-subunits, we used antibodies raised against the individual b-subunits. Western blot analysis of the oocyte
membrane fractions for the individual b-subunits revealed that the specific b-subunit antibodies detected the
b-subunit against which they were directed and that
the antibodies had little cross-reactivity for the other
b-subunits. All three b-subunits, whether injected alone
or in combinations, were detected in the membrane
fraction, suggesting co-localization with the Nav 1.8
subunit (Figs. 1A–C).
Modification of Nav 1.8 levels of expression by the
different b-subunits
Since all the subunits were able to co-locate at the
membrane, we investigated the effect of individual and
combined interactions of b1 , b2 , and b3 -subunits on
Nav 1.8. To do so we microinjected individual and
combinations of the rat b-subunit cRNAs into stage IV/
V Xenopus oocytes along with the Nav 1.8 channel
cRNA. Equal amounts of each cRNA were microinjected into the oocytes. Fig. 2 shows typical macroscopic
currents of oocytes expressing Nav 1.8 alone, with the
individual and different combinations of b1 -, b2 -, and b3 subunits. Whole-cell sodium currents were evoked by
applying a series of depolarizing voltage steps between
)80 and +65 mV in 5-mV increments from a holding
potential of )100 mV. For Nav 1.8 channels co-expressed
with b1 alone or in combination with one or both of the
other b-subunits, levels of current expression were larger
and inactivation was rapid compared to the Nav 1.8
a-subunit alone or with b2 b3 .
Fig. 3 shows the peak current amplitude of Nav 1.8
in the absence or presence of the various b-subunits.
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K. Vijayaragavan et al. / Biochemical and Biophysical Research Communications 319 (2004) 531–540
Fig. 1. Association of Nav 1.8 with individual and different combinations of b-subunits. Solubilized membrane proteins from oocytes injected with
Nav 1.8 alone, with individual and different combinations of b-subunits. Proteins were resolved by SDS–PAGE under reducing conditions in the
presence of 1% b-mercaptoethanol and immunoblotted using an anti-b1 antibody, anti-b2 antibody, and anti-b3 antibody.
Fig. 2. Typical whole-cell Naþ currents of oocytes expressing Nav 1.8 alone, with individual and various combinations of b1 -, b2 -, and/or b3 -subunits.
The inward currents were elicited be applying depolarizing steps between )80 and +65 mV in 5-mV increments from a holding potential of )100 mV
(see insets). Dashed lines represent zero current levels.
Oocytes injected with Nav 1.8 alone expressed small currents ()620.6 66.2 nA, n ¼ 16). Co-expression of b1 increased levels of Nav 1.8 current expression 3.3-fold
()2059.3 222.3 nA, n ¼ 19) (p ¼ 0:00005), in contrast
b2 ()527.9 76.6 nA, n ¼ 23) (p > 0:05) and b3 -subunits
()777.3 90.0 nA, n ¼ 18) (p > 0:05) failed to increase
expression of Nav 1.8 channels. Our data suggest that only
the b1 -subunit modulates current amplitudes of the
Nav 1.8 channel. When we co-expressed combinations of
b-subunits with Nav 1.8, combinations that included the
b1 -subunit expressed larger currents than those oocytes
that expressed Nav 1.8 alone or b2 b3 -subunits ()476 118.0, n ¼ 12) (Fig. 2). Our data further suggest that the
b1 -subunit modulates Nav 1.8 peak current amplitudes.
K. Vijayaragavan et al. / Biochemical and Biophysical Research Communications 319 (2004) 531–540
535
Fig. 3. Effects of co-expression of individual and different combinations of b-subunits on the expression levels of Nav 1.8. Co-expression of the
b1 -subunit alone and in combination with other b-subunits caused the largest increase in Nav 1.8 peak current amplitudes compared to b2 and b3 . The
data are means SEM of n values indicated on the histogram. *p < 0:05 compared with Nav 1.8.
Alterations of Nav 1.8 decay kinetics and gating by the
b-subunits
voltage-dependence at test potentials from 0 to +40 mV
(Fig. 4B). Furthermore, we observed that at the voltage of
peak current (+20 mV), Nav 1.8 expressing oocytes coinjected with b1 b3 , b1 b2 , and b1 b2 b3 displayed similar time
constants of current decay as Nav 1.8 co-expressed with
b1 , which was 3 ms (Table 1). b2 b3 , however, did not
affect the time constant of channel inactivation (Table 1).
Fig. 4D shows typical current trace, at +20 mV, for oocytes expressing Nav 1.8 and b1 , b1 b3 , b1 b2 , b1 b2 b3 or b2 b3 .
The reduction in time constant for the rapidly inactivating component, by b1 b3 , b1 b2 , and b1 b2 b3 was voltagedependent at test potentials from 0 to +40 mV (Fig. 4D).
Thus, the results suggest that the b1 -subunit significantly
accelerates Nav 1.8 inactivation kinetics.
We also assessed the effects of the b-subunits on
Nav 1.8 steady-state activation and inactivation. Relative
In addition to changes in current amplitude, the different b-subunits also altered the decay kinetics and gating of Nav 1.8 channel differentially. The current decay
was best fitted with a single exponential function. Coexpression of Nav 1.8 with b1 caused the most significant
(p ¼ 0:006) acceleration of the time constant current decay (sh ); in contrast co-expression of b2 or b3 did not
appreciably alter the inactivation time course (p > 0:05)
(Table 1). Fig. 4B shows typical current traces, at +20 mV,
for oocytes expressing Nav 1.8 alone and with the b1 -, b2 or b3 -subunits. The reduction of Nav 1.8 time constant of
current decay observed with b1 co-expression was consistent for a wide range of voltages and showed strong
Table 1
Effects of individual and combined b-subunits expression on Nav 1.8 Naþ channels
Voltage-dependence of
Time constant of
Activation
Nav 1.8
Nav 1.8 + b1
Nav 1.8 + b2
Nav 1.8 + b3
Nav 1.8 + b1 =b2
Nav 1.8 + b1 =b3
Nav 1.8 + b2 =b3
Nav 1.8 + b1 =b2 =b3
Inactivation
Current decay
V1=2
kv
n
V1=2
kv
n
ms
n
10.3 1.2
1.6 07
9.5 0.8
11.5 1.1
1.7 0.4
2.9 0.9
7.2 1.0
1.7 1.1
)6.6 0.4
)6.3 0.1
)5.7 0.7
)6.0 0.5
)6.7 0.2
)6.6 0.2
)8.0 0.6
)6.6 0.3
9
24
24
24
3
21
17
19
)52.9 0.8
)58.4 1.5
)49.0 0.2
)47.9 0.6
)46.6 0.9
)55.1 0.9
)51.3 1.2
)54.4 1.0
8.9 0.2
7.1 0.4
9.9 0.5
9.0 0.5
10.3 0.5
8.7 0.4
10.0 0.5
8.9 0.4
6
15
26
17
9
20
16
18
3.8 0.2
2.9 0.1
3.6 0.1
3.6 0.1
3.3 0.2
2.6 0.1
3.6 0.2
3.0 0.2
15
24
22
24
8
14
12
20
V1=2 and kv values for voltage-dependence of activation and inactivation were obtained by fitting the activation and inactivation curves with a
Boltzmann equation as described in the Methods and materials. The time constant of current was obtained by fitting the decay phase of maximum
Naþ current (+20 mV for Nav 1.8, with b2 and b3 and +10 mV for Nav 1.8 with b1 ) with a single exponential function. Significant statistical differences
with respect to Nav 1.8 channel alone are denoted by , where the p values <0.05 are the criterion for significance.
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Fig. 4. Effects of co-expression of individual and different combinations of b-subunits on time constant of Nav 1.8 current decay. Sodium currents in
oocytes expressing Nav 1.8 alone, with b1 , b2 or b3 or b-subunit combinations were elicited by test pulses to +20 mV from a holding potential of
)100 mV. (A) Representative current traces from Nav 1.8 in the absence and presence of each b-subunit. Currents were normalized to facilitate the
comparison of the kinetics. The dashed line represents the zero current level. Co-expression of the b1 -subunit with Nav 1.8 caused the most significant
acceleration of current decay kinetics at +20 mV (p ¼ 0:006). (B) Mean voltage-dependence of current decay of Nav 1.8 in the absence and presence of
each b-subunit. Co-expression of Nav 1.8 with b1 also caused the most significant reduction in time constants of current decay, at a wide range
of voltage from 0 to +40 mV compared to the other b-subunits. (C) Representative current traces of Nav 1.8 co-expressed with various combinations
of b-subunits. (D) Mean time constants for Nav 1.8 in the presence of different combinations of b-subunits, determined over a range of test potentials.
Only the combination of auxiliary subunits with b1 caused acceleration of Nav 1.8 current decay and showed voltage-dependence.
conductance was determined from similar families of
Naþ currents shown in Fig. 2. We compared the normalized conductance of Nav 1.8 in the presence of each
b-subunit and plotted the values versus the test voltage
(Fig. 5A). Co-expression of b1 with Nav 1.8 caused an
8.3 mV hyperpolarized shift (p < 0:0001) in the activation curve (Table 1). For Nav 1.8 co-expressed with either b2 or b3 , we observed no shift in the steady-state
activation curve (p > 0:05) (Table 1).
To investigate the effects of b-subunits on voltagedependence of Nav 1.8 inactivation, we applied 500 ms
conditioning pulses to voltages between )110 and
+35 mV and then determined the fraction of available
channels with test pulses to +10 or +20 mV. We normalized the fraction of available currents and plotted
the data versus the conditioning pulses (Fig. 5B). The
smooth curves are Boltzmann fits and midpoints (V1=2 )
and slope factor (kv ) are shown in Table 1. Co-expression of b1 caused a 5.5 mV hyperpolarized shift in the
midpoint of Nav 1.8 steady-state inactivation (p ¼ 0:02).
The b2 -subunit on the other hand shifted the midpoint
of Nav 1.8 steady-state inactivation by 3.9 mV in the
depolarized direction (Table 1, p ¼ 0:01). The b3 -sub-
unit also caused a 5 mV shift of steady-state inactivation
in the depolarized direction (Table 1, p ¼ 0:02).
When we co-expressed different combinations of the
b-subunits with Nav 1.8, we observed that combinations
including b1 caused similar negative shifts in steady-state
activation as when the channel was co-expressed with b1
alone (Table 1). Fig. 5C illustrates the steady-state activation of Nav 1.8 with different combinations of
b-subunits. For the steady-state inactivation of Nav 1.8,
we observed depolarized shifts by b1 =b2 . However in the
presence of b1 =b3 , b2 =b3 , and b1 =b2 b3 we observed little
modulation in the voltage-dependence of inactivation in
comparison with Nav 1.8 (Fig. 5D).
Fig. 5E is a scatter plot of the V1=2 of steady-state
activation and inactivation of Nav 1.8 in the presence
of different b-subunits, combinations including b1 ,
consistently gave a hyperpolarized shift in the V1=2 of
steady-state activation. Our data suggest that only the
b1 -subunit modulates the steady-state activation of
Nav 1.8 because, the channel showed similar shifts in the
voltage-dependence of activation when co-expressed
with b1 alone or in combination with other b-subunits.
However, all the b-subunits are able to modulate the
K. Vijayaragavan et al. / Biochemical and Biophysical Research Communications 319 (2004) 531–540
537
Fig. 5. Voltage-dependence of activation and inactivation of Nav 1.8 when co-expressed with the different b-subunits. The voltage-dependence of
activation in oocytes was determined by eliciting depolarizing pulses from a holding potential of )100 mV to potentials ranging from )80 to +60 mV
in 5-mV increments. Current activation smooth curves were plotted using the Boltzmann equation (see Methods and materials). (A,C) Mean steadystate activation (filled symbols) of Nav 1.8 in the presence of (A) individual and (C) various combinations of b-subunits. (B,D) Mean steady-state
inactivation (open symbols) was measured using a 500 ms conditioning pulse to voltages between )110 and +35 mV. The fraction of available current
was determined using test pulses to +20 or +10 mV and the normalized currents were plotted versus the conditioning voltage. The V1=2 values of
steady-state activation and inactivation of Nav 1.8 alone is represented by circles (d; s), b1 by squares (j; ), b2 by triangles (m; n), and b3 by
diamonds (r; }). For the various combinations of b1 b2 by inverted triangles (.; ,), b1 b3 by hexagons ( , ), b2 b3 by dotted square ( , ), and
b1 b2 b3 by dotted circles ( , ).
voltage-dependence of Nav 1.8 inactivation either alone
or in combination with other b-subunits.
Discussion
Modulation of Nav 1.8 by b-subunits
Nav 1.8 is likely to be co-expressed with the b1 , b2 , and
b3 -subunits in C-fiber neurons from the DRG. In this
study, we show that each of these b-subunits is able to
interact and modulate the channel when co-expressed in
Xenopus oocytes and that co-expression of multiple
b-subunits caused a different effect from that of individual subunits on the Nav 1.8 Naþ channel. Qu et al.
[26] report that the b3 -subunit induces persistent currents when expressed with Nav 1.2. In contrast, our results show that b3 either alone, or in combination with
b1 or b2 , does not induce Nav 1.8 persistent currents.
Furthermore, unlike the results observed for the Nav 1.3
channel [24], we observed that the functional effects of
b1 on the Nav 1.8 channel are dissimilar to those of the
b3 -subunits and thus are not interchangeable for Nav 1.8
channel complex. John et al. [25] demonstrate that b3
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K. Vijayaragavan et al. / Biochemical and Biophysical Research Communications 319 (2004) 531–540
co-expression with Nav 1.8 in mammalian cell lines gives
an increase in peak current amplitudes. In contrast, our
results demonstrated that b1 and not b3 increased the
Nav 1.8 mediated current. Clearly b1 and b3 are both able
to modulate channel activity, however the ability of
these subunits to increase current amplitudes may rely
on cellular components that interact differently in
Xenopus oocytes to mammalian cells.
Modulation of the b-subunits gating properties is
variable for different channel isoforms. For instance, coexpression of b2 with the brain Nav 1.2 channel causes a
positive shift in the steady-state inactivation but little
effect is observed for the Nav 1.3 channel [24,26,27]. Also
Zimmer and Benndorf [28] recently showed, using chimeras between b1 and b2 -subunits, that different regions
of the b1 -subunits are involved in modulating different
Naþ channels. The transmembrane segment of b1 is
more important for cardiac Nav 1.5 modulation of expression and gating, while for Nav 1.2, the extracellular
segment of the b1 -subunit is the crucial molecular
determinant [28].
In our study, the b1 -subunit alone, or in combination
with other b-subunits, accelerated the time constant of
inactivation, shifted the voltage-dependence of activation to more hyperpolarized potentials, and increased
levels of Nav 1.8 channel functional expression, either by
increasing channel density at the oocyte membrane or
by increasing individual channel conductance. These
negative shifts in the steady-state activation of Nav 1.8
by b1 could imply that the channel is more sensitive to
activation by smaller depolarizing potentials. On the
other hand, the b3 -subunit shifted the voltage-dependence of inactivation to more positive potentials and did
not influence the time constant of current decay, steadystate activation or expression of Nav 1.8. Positive shifts
in the steady-state inactivation of Nav 1.8 by b3 would be
expected to increase the fraction of channels available
for opening prior to depolarization. Co-expression of b1
and b3 -subunits with Nav 1.8 resulted in little change in
availability compared to channel alone. This could be
because the combined currents mediated by a–b1 (with a
5.5 mV hyperpolarized shift) and a–b3 (with a 5 mV
depolarized shift) channels expressed in the same oocyte
cancel each other out. The b2 -subunit did not alter the
kinetic properties or current amplitude of Nav 1.8 currents and only gave a small depolarizing shift in the
voltage-dependence of steady-state inactivation. Interestingly, however, the co-expression of b1 and b2 shifted
the Nav 1.8 channel availability to more depolarized
potentials in comparison to when channels were expressed with either b1 or b2 alone. This demonstrates
that the b2 -subunit in an a–b1 –b2 complex overrides the
hyperpolarizing shift in inactivation observed when b1
alone is co-expressed with Nav 1.8. However, b2 does not
seem to overcome the hyperpolarizing effect of b1 on the
voltage-dependence of activation or on the rate of fast
inactivation. More studies are required to elucidate the
reasons for the effect on steady-state inactivation. One
plausible explanation for this effect is that the binding of
the b1 -subunit on the channel may cause a conformational change that could reveal important site(s) for the
b2 -subunit modulation. The combined hyperpolarized
shift in steady-state activation and depolarized shift in
steady-state inactivation would be expected to increase
the window current, thus will cause the channel to be
more active.
Physiopathological significance
The b1 -, b2 -, and b3 -subunits have been identified in
the small sensory neurons where the Nav 1.8 channel is
predominantly expressed [7,21]. Nav 1.8 has been proposed to play an important role in action potential
generation in DRG neurons [29]. Thus, factors that
modulate the expression levels or localization of the
b-subunits will have important impact on the action
potential electrogenesis in C-fibers of the DRG neurons.
Since our Western blot analysis and electrophysiological
studies indicate that all three subunits co-locate in the
cell membrane and each either individually or in combination modulates the function of the channel, the
behavior of the channel will depend on the subunit and/
or subunits that are predominantly expressed at any
time. Changes in the sensitivity of the channel to voltage, due to shifts in the voltage-dependence of activation
and inactivation caused by an individual or combination
of b-subunits, are important determinants of action
potential threshold in these C-fibers. Even small shifts in
voltage-dependence of activation and inactivation
caused by the b-subunit(s) can be critical to the response
of a neuron to its synaptic input, since excitatory potentials depolarize neurons only by a few millivolts.
Furthermore, the other modulator effects on the Nav 1.8
channel caused by the b-subunits, including increased
number of active channels on the cell surface and rapid
inactivation, are some additional factors that can contribute to the hyperexcitability of the sensory neurons
during the pathophysiology of neuropathic pain.
Different effects of the auxiliary subunits on the
Nav 1.8 channel have been observed under different
neuropathic pain models. For instance, in human patients suffering neuropathic pain following spinal root
avulsion injury, an upregulation of the b1 - and b2 -subunits was observed [21], while in a chronic constriction
injury (CCI) model in rats an elevation of the b3 -subunits was observed [30]. Similarly axotomy has been
shown to increase TTX-R currents not only in the small
C-fibers but also in the large fibers [31]. Interestingly,
previous immunohistochemical studies by Novakovic
et al. [14] showed that there are key changes in the
subcellular distribution of Nav 1.8 following either CCI
or total transection of the sciatic nerve. Furthermore,
K. Vijayaragavan et al. / Biochemical and Biophysical Research Communications 319 (2004) 531–540
Gold et al. [32] recently showed that the redistribution
of Nav 1.8 in uninjured axons of the C-fibers may be
necessary for neuropathic pain. Both models induced
the translocation of presynthesized channels from the
neuronal somata to peripheral axons with consequent
clustering at the site of injury. The specific subcellular
redistribution of Nav 1.8 together with the type of
b-subunits that are predominantly expressed after peripheral nerve injury may be important determinants in
establishing peripheral nerve hyperexcitability and the
resultant neuropathic pain. It is also important to note
the presence of other Naþ channel subtypes in the sensory DRG that are also modulated by the b-subunits,
such as the Nav 1.2 and Nav 1.3 Naþ channels [24,26].
The diversity in Naþ channel isoforms combined with
various potential associations with auxiliary b-subunits
may influence the diverse electrophysiological responses
observed in the sensory neurons. It is possible that those
DRG cells that vary in their expression of membrane
properties may represent sensory neurons that transmit
different types of sensory information.
Acknowledgments
This study was supported by grants from the Heart and Stroke
Foundation of Quebec (HSFQ) and the Canadian Institute of Health
Research (CIHR) MOP-49502 awarded to Dr. M. Chahine. Dr. M.
Chahine is an Edwards Senior Investigator (Joseph C. Edwards
Foundation).
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