(Circulation. 2001;103:1303.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
Characterization of Sodium Channel 
- and ß-Subunits in Rat and Mouse Cardiac Myocytes
From the Departments of Pharmacology (J.D.M., C.C., L.N.M., L.L.I.) and Internal Medicine (R.M., F.C.B.), University of Michigan, Ann Arbor, and Department of Pharmacology (I.R., H.A., R.S.K.), Columbia University, College of Physicians and Surgeons, New York, NY.
Correspondence to Lori L. Isom, PhD, Department of Pharmacology, The University of Michigan, 1301 MSRB III, 1150 W Medical Center Dr, Ann Arbor, MI 48109-0632. E-mail lisom{at}umich.edu
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Abstract |
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Background—Sodium channels isolated from mammalian brain are composed of
-,
ß1-, and ß2-subunits.
The composition of sodium channels in cardiac muscle, however, has not
been defined, and disagreement exists over which ß-subunits are
expressed in the myocytes. Some investigators have demonstrated
ß1 expression in heart. Others have not
detected any auxiliary subunits. On the basis of Northern blot analysis
of total RNA, ß2 expression has been thought
to be exclusive to neurons and absent from cardiac
muscle.
Methods and Results—The
goal of this study was to define the subunit composition of cardiac
sodium channels in myocytes. We show that cardiac sodium channels are
composed of -, ß1-, and
ß2-subunits. Nav1.5 and Nav1.1 are expressed
in myocytes and are associated with ß1- and
ß2-subunits. Immunocytochemical localization
of Nav1.1, ß1, and ß2
in adult heart sections showed that these subunits are expressed at the
Z lines, as shown previously for Nav1.5. Coexpression of Nav1.5 with
ß2 in transfected cells resulted in no
detectable changes in sodium current.
Conclusions—Cardiac
sodium channels are composed of - (Nav1.1 or Nav1.5),
ß1-, and ß2-subunits.
Although ß1-subunits modulate cardiac sodium
channel current, ß2-subunit function in heart
may be limited to cell
adhesion.
Key Words: cells • signal transduction • genes • ion channels
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Voltage-gated sodium channels initiate action potentials in excitable cells.1 Brain sodium channels are composed of a single pore-forming
-subunit and 2 auxiliary
ß-subunits, with a stoichiometry of
1:1:1.1 ß-Subunits do not
form the ion-conducting pore but rather modulate channel gating and
cell surface expression levels and interact with extracellular matrix
and cell adhesion molecules.2
Recently, 2 additional members of the ß-subunit gene family have been
identified: ß1A and
ß3.3 4
The subunit structure of cardiac sodium channels has not
been well defined. At least 2 -subunit mRNAs are expressed in heart:
Nav1.5 and Nav1.1.5
ß1-Subunit mRNA is expressed in rat and human
heart6 7 but was
not detected in mouse heart.8
ß1-Subunit polypeptides have been demonstrated
in rat heart9 ; however,
purified preparations of cardiac sodium channels from chicken and rat
did not show detectable associated ß-subunits after
immunoprecipitation with -subunit
antibodies.10 11
Nav1.5+ß1 coexpression has been studied in
heterologous expression systems with variable and conflicting
results.7 12 13 14 15 16
ß1A polypeptides and
ß3 mRNA have also been detected in
heart.3 4
ß2 transcripts are not detectable in heart by
Northern blotting
techniques.17 Thus, it was
postulated that ß1,
ß1A, and ß3 may be
expressed in heart, whereas ß2 was most likely
absent. Recent studies, however, have compelled us to reexamine this
hypothesis. Nav1.5 sodium channels become permeable to
Ca2+ after activation of protein kinase
A.18 This mode of the
channel, called slip-mode conductance, requires
ß1 and ß2
coexpression with . Nav1.5 - and
ß2-subunits covalently associate in HEK293
cells18 ; it was not
determined, however, whether ß2-subunits are
expressed in cardiac muscle.
The purpose of this study was to define the sodium channel
- and ß-subunits expressed in cardiac myocytes. Using specific
antibodies, we identified Nav1.1, Nav1.5, ß1,
and ß2. The developmental time course of
ß2 expression in heart shows that it is
detectable by postnatal day 15. Nav1.5, ß1,
and ß2 associate in cardiac myocytes, as do
Nav1.1, ß1, and ß2.
Immunocytochemistry revealed Nav1.1, ß1, and
ß2 expression in adult cardiac muscle along
the Z lines. Coexpression of Nav1.5 and
ß2-subunits in tsA201 cells did not result in
any detectable changes in sodium current over alone. We conclude
that cardiac sodium channels contain ß1- and
ß2-subunits and that either Nav1.1 or Nav1.5
-subunits can form the ion-conducting
pore.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Antibodies
Antibodies to ß1 or ß2 used for Western blots were described previously.19 20 An extracellular epitope ß2 antibody was obtained from Dr W.A. Catterall, University of Washington, and used for immunolocalization. Anti–SP11-I21 and anti-rH1 were obtained from Alomone Laboratories. The anti-rH1 antibody was raised in rabbit against the peptide D492RLPKSDSEDGPRALNQLS510(C) with an additional C-terminal cysteine and affinity-purified. Anti–cardiac
-sarcomeric actin and anti–-actinin were from
Sigma. Fluorescent secondary antibody conjugates were from Vector
Laboratories.
Human Fetal Heart ß2
cDNA Clone
A cDNA clone for a human fetal heart sodium channel
ß2-subunit was obtained as previously
described (accession number
AF107028).18
RT-PCR Analysis of Rat and Mouse mRNA
Poly-A mRNA was obtained from Clonetech. Reverse
transcription–polymerase chain reaction (RT-PCR) was performed by use
of the Titan One Tube PCR system (Roche Molecular Biochemicals).
Primers CTFNS (5'-CTGTACCTTCAACTCCTGCTATACC-3') and E3'
(5'-ATGACTGCCACCGTGGAGTCCCGCTCTG-3') were used. Each reaction contained
50 ng of mRNA. First-strand cDNA was synthesized at 55°C for 30
minutes, and then PCR was performed as follows: 94°C for 2 minutes;
10 cycles of 94°C for 15 seconds, 65°C for 30 seconds, and 68°C
for 30 seconds; and 25 cycles of 94°C for 15 seconds, 65°C for 30
seconds, and 68°C for 30 seconds, plus 5 additional seconds for each
cycle. A final step for 7 minutes at 68°C was performed. This PCR
amplifies a 335-bp product from mRNA and a 1061-bp product from genomic
DNA that includes introns 1 and 2 of SCN2B.
Northern Blot Analysis of Total Mouse
RNA
Samples of total mouse mRNA were purified with Trizol
reagent (Life Technologies). Northern blot analysis was performed as
previously
described.17
Preparation and Culture of Cardiac
Myocytes
Primary cultures of neonatal rat cardiac myocytes
were prepared as previously
described.22 Myocytic purity
was monitored by immunofluorescence using anti–cardiac -sarcomeric
actin and averaged 96±3% 48 hours after
plating.
Expression of Nav1.5 Sodium Channels
in HEK Cells
Nav1.5 cDNA was subcloned into pcDNA3.1/Zeo (+)
(Invitrogen), and HEK 293 cells were transfected with lipofectamine
(Life Technologies). Clones were selected with 400 µg/mL zeocin
(Invitrogen) and tested electrophysiologically for the presence of
sodium current. Clone 21, used in this study, showed current amplitude
of 
500 pA/pF and was maintained in culture with 200 µg/mL
zeocin.
Immunoprecipitation and Western
Blot
Immunoprecipitations were performed from heart
membranes, prepared as described for
brain,23 or solubilized
cardiac myocytes. Western blot analysis of cardiac -subunits was
performed as
described.20
Immunocytochemical Analysis of Nav1.1
, ß1, and ß2
Expression in Heart
Mice were anesthetized by intraperitoneal injection
of Beuthanasia-D (Schering-Plough Animal Health Corp). Hearts were
washed by injection of 50 mL prewashing buffer (g/L: NaCl 8, dextrose
4, sucrose 8, calcium chloride 0.23, sodium cacodylate 0.34) and
perfused with 50 mL perfusion solution (g/L: sucrose 40,
paraformaldehyde 40, sodium cacodylate 14.34). Hearts were incubated in
perfusion solution at 4°C overnight with constant rotation. The
solution was changed to 30% sucrose, and the incubation was continued
overnight at 4°C with constant rotation. Cryostat sections (0.4 µm
thick) were cut and postfixed in 2% paraformaldehyde, treated for 10
minutes with 0.5% Triton X-100 in TBS buffer (10 mmol/L Tris-HCl [pH
7.5], 150 mmol/L NaCl), and then blocked at room temperature in the
same solution containing 5% newborn calf serum. Primary antibodies
(anti–SP11-I, 1:100; anti-ß1, 1:100;
anti-ß2, extracellular epitope 1:50; anti-
actinin, 1:400) or primary antibodies preadsorbed with peptides (40
µg/mL for ß1 or ß2,
50 µg/mL for Nav1.1) were then added to the above solution, which
also contained 0.1% Tween-20, and incubated for 1.5 hours at room
temperature. The sections were washed with TBS-T. Secondary antibodies
(fluorescein isothiocyanate–conjugated goat anti-rabbit IgG for ,
ß1, and ß2 or Texas
red–conjugated goat anti-mouse IgG for -actinin) were then added,
and the incubation was continued for 1.5 hours at room temperature.
Sections were washed, mounted with Dabco (Sigma), and visualized with a
BioRad MRC 600 confocal laser scanning microscope in the Microscopy and
Image Analysis Laboratory Core Facility at the University of
Michigan.
Electrophysiology
Cell Culture
TsA201 cells (a gift from Dr Mohamed Chahine,
University of Laval) were grown under standard conditions and
transfected with Nav1.5 alone or Nav1.5+hß1-,
Nav1.5+hß2-, or
Nav1.5+hß1+hß2-subunits
with lipofectamine as
described.24
hß1 cDNA was a gift from Dr A. George,
Vanderbilt University.
mRNA Extraction and RT-PCR
mRNA extraction and RT-PCR were performed with the
mRNA Captur(e) Kit and the Titan One Tube RT-PCR kit (Roche Molecular
Biochemicals). The primers used were, for the detection of
hß1:
SCN1B-F(5'-GACCAACGCTGAGACCTTCAC-3')/SCN1B-R(5'-CACGAGCCATATGGTCAACAC-3');
for the detection of hß2:
hß2n90
(5'-GGAGGTCACAGTACCTGCCACCCTC-3')/hß2n480
(5'-CACGGCCACCGTGAAGTCCC-3'); and for the detection of human ß-actin:
hß-ACTIN556F
(5'-CACTGTGCCCATCTAC-GAGG-3')/hß-ACTIN1169R(5'-CGGACTCGTCATACTCC-TGCTT-3').
First-strand cDNA was synthesized at 60°C for 30 minutes, and then
PCR was performed as follows: 94°C for 2 minutes, then 10 cycles of
94°C for 30 seconds, 55°C (for hß1 and
hß2) or 45°C (for hß-actin) for 30
seconds, and 68°C for 45 seconds, followed by 25 cycles of 94°C for
30 seconds, 55°C or 45°C as described above for 30 seconds, and
68°C for 45 seconds, plus 10 additional seconds for each cycle. A
final step for 7 minutes at 68°C was then
performed.
Whole-Cell Voltage Clamp
Currents were measured at room temperature by
whole-cell patch-clamp procedures with Axopatch 200B amplifiers (Axon
Instruments) with previously described recording solutions and voltage
protocols.24 Data were
collected and analyzed with pClamp8 and Origin software (Axon
Instruments and Microcal Software). The voltage-dependence of
inactivation was determined by measuring current in response to pulses
to -20 mV that had been preceded by conditioning pulses (500 ms) to a
series of voltages. Holding potentials were -100 mV, and
[Na+]o was 130
mmol/L. For the voltage-dependence of activation, current was measured
in response to pulses from -80 to +60 mV, and
[Na+]o was 10
mmol/L (with N-methyl-glucamine
used as an equimolar Na+ substitute). Data
are presented as mean±SEM. Two-tailed Student’s
t test was used to compare
means; a value of P<0.05 was
considered statistically significant. Data were filtered with a
Boltzmann relationship, where V1/2 is the
voltage where half of the channels are available (or activated) and k
is the slope
factor.
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Results |
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Antibody Specificity
To determine the specificity of the rH1 antibody for Nav1.5, we performed Western blots of heart and brain membranes using anti-rH1 versus anti–SP11-I antibodies. Using anti–SP11-I, we observed that Nav1.1 is expressed both in brain and in cardiac myocytes, whereas Nav1.5 could be detected in myocytes but not in brain membranes with anti-rH1 (Figure 1A
).
Figure 1B shows that anti-rH1, but not anti–SP11-I,
recognizes Nav1.5 sodium channels expressed in HEK 293 cells. These
results indicated that the 2 antibodies are selective for the 2 channel
subtypes and that cross-reactivity would not complicate our
experiments.
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Expression of
ß2 mRNA in Heart
Our previous studies suggested that
ß2 expression is limited to neuronal
tissues.17 Thus, we decided
to retest cardiac RNA for ß2 expression by
more sensitive methods. RT-PCR showed that ß2
mRNA is expressed in mouse heart
(Figure 2A). This experiment was designed with
oligonucleotide primers flanking introns 1 and 2 of the
ß2
gene17
(Figure 2B) such that contaminating genomic DNA could be
clearly separated from the lower-molecular-weight mRNA (cDNA) band
(Figure 2A, arrows). Northern blot experiments confirmed our
previous results that ß2 transcript is not
detectable in total heart RNA (data not shown). We conclude that
ß2 mRNA is present in cardiac muscle tissue,
but at significantly lower levels than in brain.
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ß1 and
ß2 Associate With Cardiac Sodium
Channels
Analysis of heart membranes in the presence and absence
of ß-mercaptoethanol showed the presence of an immunoreactive
ß2 band that shifted on reduction from >200
kDa to 33 kDa, indicating -ß2 covalent
interactions
(Figure 3A). Western blot analysis of primary cardiac
myocytes with anti-ß2 antibody revealed an
immunoreactive band at 40 kDa
(Figure 3B).
Figure 3C shows the developmental time course of
ß2 expression in heart from early embryo to
adulthood. ß2-Subunits are expressed only
after birth, becoming detectable at postnatal day 15.
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Coimmunoprecipitation experiments showed that in heart
membranes
(Figure 4) as well as in myocytes
(Figure 5A), Nav1.5 associates with
ß1 and ß2.
Figure 5B demonstrates that ß1 and
ß2 are also associated with Nav1.1 in cardiac
myocytes. Thus, sodium channels in cardiac myocytes are composed of
-, ß1-, and
ß2-subunits, and either Nav1.1 or Nav1.5 can
form the ion-conducting pore.
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Immunolocalization of Nav1.1 ,
ß1, and ß2 in
Heart
Previous immunolocalization of Nav1.5 revealed labeling
of surface and T-tubular membrane systems of atrial and ventricular
myocytes when viewed in cross section. In longitudinal sections,
labeling was also observed at terminal intercalated disks in
ventricular muscle in accordance with Z-line
appearance.25 We used
Nav1.1, ß1, and ß2
antibodies to investigate the localization of these subunits in
longitudinal sections of cardiac muscle. As shown in
Figure 6, A and D, both ß1 and
ß2 colocalized with -actinin
(Figure 6, B and E), a marker for cardiac-muscle Z lines. As
shown in
Figure 7, A and B, Nav1.1 also showed a labeling pattern
similar to that of -actinin. Because anti–SP11-I antibody does not
recognize Nav1.5
(Figure 1B
), we could be confident that our results were not
complicated by antibody cross-reactivity and that Nav1.1 is indeed
expressed in the myocytes. Antibodies preadsorbed with peptides showed
no specific signals
(Figure 6, C and F;
Figure 7C). The Nav1.1 -, ß1-,
and ß2-labeling results are similar to
previous results for
Nav1.5.25 Thus, Nav 1.1,
Nav1.5, and ß-subunits are colocalized in heart muscle.
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Electrophysiological Analysis
We used RT-PCR to investigate the presence of
endogenous sodium channel auxiliary subunits in tsA201 cells.
Figure 8 indicates that endogenous expression of
ß2 is not detected in our assays. In contrast,
tsA201 cells do express ß1 mRNA. Transfection
with ß1 strongly increased the level of
ß1 mRNA, however, suggesting that it might be
possible to detect functional consequences of coexpression of Nav1.5
with both ß-subunits in this cell line. We expressed Nav1.5 with and
without hß1 and hß2
in tsA201 cells and studied the properties of the expressed channels
(Table).
We found no differences in peak current density due to
hß1 or hß2, but did
detect an effect of hß1 on the
voltage-dependence of inactivation. Coexpression of Nav1.5 with
hß1 caused a significant +5-mV shift in the
half-maximal voltage-dependence of inactivation
(V1/2), similar to previous
reports.16 These data
indicate that the endogenous levels of ß1
expression in tsA201 cells are not sufficient to saturate the effect of
ß1 on inactivation. Because of the endogenous
ß1, the 5-mV shift we measure is likely to be
an underestimate of the influence of ß1 on
inactivation. In contrast to ß1, we did not
detect any functional effects of coexpression of Nav1.5 with
ß2 on the voltage-dependence of inactivation
or activation.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Sodium channels isolated from mammalian brain are composed of 1
- and 2
ß-subunits.1 The subunit
structure of cardiac sodium channels has not been as well defined. At
least 2 -subunit mRNA transcripts, Nav1.1 and Nav1.5, have been
identified in adult
heart.5 26 High-
and low-affinity populations of STX receptors, presumably corresponding
to Nav1.1 and Nav1.5, respectively, have also been identified in adult
rat heart, with high-affinity receptors estimated to make up 25% to
50% of the total population of sodium
channels.5
ß1 mRNA and protein are expressed in heart
tissue at high
levels6 9 ; however,
its association with -subunits has not been demonstrated.
ß2 transcripts are not detectable in total
cardiac RNA by Northern blotting
techniques.17 In purified
preparations of chicken and rat cardiac sodium channels, ß-subunits
could not be detected at
all.10 11 Thus,
although a number of investigators presumed that cardiac sodium
channels were most likely composed of - and
ß1-subunits, this has remained controversial.
The purpose of the present study was to define the subunit composition
of cardiac sodium channels. Cardiac myocytes express
ß1 and ß2
polypeptides, and Nav1.5 physically associates with both ß-subunits.
ß2-Subunit polypeptides are expressed
postnatally in heart. -, ß1-, and
ß2-subunits are localized to the Z lines in
heart sections. We also identified Nav1.1 in cardiac myocytes and
showed that it associates with ß1 and
ß2. We conclude that sodium channels expressed
in cardiac myocytes are composed of either Nav1.1 or Nav1.5 and that
both associate with ß1 and
ß2. Although ß1 has
modulatory effects on Nav1.5, ß2 has no
detectable effects in our system, suggesting that the effects of
ß2 in heart in vivo may involve cell adhesion
and cytoskeletal communication as opposed to channel
gating.
What is the physiological role of ß-subunits in heart?
Brain and skeletal muscle sodium channels expressed in oocytes exhibit
slow inactivation kinetics. Coexpression of
ß1-subunits produces a significant increase in
the rate of inactivation of these
channels.27 28 In
contrast, expression of Nav1.5 in oocytes produces channels that
inactivate rapidly in the absence of
ß-subunits.13 Some groups
have reported that ß1 has no observable
effects on Nav1.5 functional
expression.7 12
Others reported that coexpression of ß1 and
Nav1.5 results in increased current density with no detectable effects
on channel kinetics or
voltage-dependence.13 14
Some groups have found modulation of channel sensitivity to lidocaine
block and subtle changes in channel kinetics and gating properties in
response to ß1
expression,15 whereas others
have reported significant shifts in the voltage-dependence of
steady-state inactivation, similar to the present
results.16 A Nav1.5 mutation
associated with long-QT syndrome affects the voltage-dependence of
channel inactivation by altering the interaction of Nav1.5 and
ß1.16
Finally, Nav1.1 -subunits are modulated by
ß1- and ß2-subunits
when expressed in oocytes.28
Thus, ß-subunits may modulate cardiac sodium channels and play a role
in cardiac physiology.
ß1 and ß2 are cell adhesion molecules of the immunoglobulin superfamily.27 Both interact with extracellular matrix molecules and participate in homophilic cell adhesion, resulting in cellular aggregation and recruitment of ankyrin to the plasma membrane at points of cell-cell contact.2 We have proposed that a major function of ß-subunits is cell adhesion, contributing to channel localization, clustering, and nodal formation in brain and peripheral nerve.2 Cardiac sodium channels reside at specific locations as well.25 In the present study, we observed Nav1.1, ß1, and ß2 labeling along Z lines in longitudinal sections. We have also observed ß1A staining of surface membranes of cardiac myocytes when viewed in cross section.3 It has been proposed that cardiac sodium channels may be targeted and clustered to specific locations in a manner similar to that observed for sodium channels in brain.25 The presence of ß-subunits in cardiac myocytes may facilitate sodium channel localization and clustering to discrete functional domains via cell-adhesive interactions. Treatment of inside-out patches of ventricular cells with cytochalasin-D induced sodium channels to enter a mode characterized by lower peak open probability with a greater persistent activity, consistent with a decrease in the rate of inactivation.29 Sodium channels in ankyrinB-knockout mice display reduced current density and abnormal kinetics that contribute to prolonged action potential duration and abnormal QT-rate adaptation.30 Thus, cytoskeletal interactions may be critical to sodium channel localization and gating in the heart as well as in the brain. We propose that the presence of ß1- and ß2-subunits in cardiac myocytes may facilitate channel-cytoskeletal interactions and play a key role in the regulation of the cardiac action potential.
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Acknowledgments |
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This study was supported by grant IBN-9734462 from the National Science Foundation to Dr Isom and in part by NIH-5P60-DK-20572 from the National Institute of Diabetes and Digestive and Kidney Diseases to the Michigan Diabetes Research and Training Center, University of Michigan. We thank Seth Beebe, Sajida Jackson, and Christy Avery for expert technical assistance.
Received June 6, 2000; revision received September 8, 2000; accepted September 15, 2000.
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