(Received for publication, June 9, 1995)
From the
Voltage-gated Na channels consist of a large
subunit of 260 kDa associated with
1 and/or
2 subunits
of 36 and 33 kDa, respectively.
subunits of rat cardiac
Na
channels (rH1) are functional when expressed alone
in Xenopus oocytes or mammalian cells.
1 subunits are
present in the heart, and localization of
1 subunit mRNA by in
situ hybridization shows expression in the perinuclear cytoplasm
of cardiac myocytes. Coexpression of
1 subunits with rH1
subunits in Xenopus oocytes increases Na
currents up to 6-fold in a concentration-dependent manner.
However, no effects of
1 subunit coexpression on the kinetics or
voltage dependence of the rH1 Na
current were
detected. Increased expression of Na
currents is not
observed when an equivalent mRNA encoding a nonfunctional mutant
1
subunit is coexpressed. Our results show that
1 subunits are
expressed in cardiac muscle cells and that they interact with
subunits to increase the expression of cardiac Na
channels in Xenopus oocytes, suggesting that
1
subunits are important determinants of the level of excitability of
cardiac myocytes in vivo.
Cardiac Na channels are responsible for the
rapid, depolarizing upstroke in the cardiac action potential. Their
function is critical for the rapid spread of depolarization through the
heart and, ultimately, for cardiac contractility. Cardiac Na
channels have properties that distinguish them from other well
characterized voltage-dependent Na
channels. They are
less sensitive to tetrodotoxin (Baer et al., 1976), and their
kinetics of activation and inactivation are slower and more complex
(Brown et al., 1981). A Na
channel
subunit cDNA encoding this channel has been isolated from newborn rat
heart (rH1) (
)(Rogart et al., 1989) and denervated
rat skeletal muscle (Skm2, Kallen et al.(1990)) and a closely
related Na
channel has been isolated from human
cardiac muscle (Gellens et al., 1992). Expression of these
subunit cDNAs in Xenopus oocytes (Cribbs et
al., 1990; White et al., 1991; Gellens et al.,
1992) and mammalian cells (Qu et al., 1994; O'Leary and
Horn, 1994) yields Na
currents with functional
properties and tetradotoxin sensitivity characteristic of the native
cardiac Na
channel.
In electric eel electroplax,
Na channels are composed of a single large
subunit with a molecular mass of 230-270 kDa (Agnew et
al., 1980; Miller et al., 1983; Norman et al.,
1983). The major form of the Na
channel in rat brain
is a heterotrimeric complex of an
subunit (260 kDa), a
noncovalently bound
1 subunit (36 kDa), and a disulfide-linked
2 subunit (33 kDa) (Catterall, 1992). Na
channels
in rat skeletal muscle are heterodimeric, composed of an
subunit
and only one
subunit (Barchi, 1983; Kraner et al., 1985)
which is encoded by the same gene as the brain
1 subunit (Makita et al., 1994). Currents due to brain or skeletal muscle
Na
channel
subunits expressed alone by injection
of mRNA in Xenopus oocytes are small and have abnormally slow
kinetics (Auld et al., 1988; Krafte et al., 1988;
Joho et al., 1990; Krafte et al., 1990). Coexpression
of the
1 subunit increases channel expression, shifts the gating
mode from slow to fast, speeds activation and inactivation kinetics,
and causes a hyperpolarizing shift in the voltage dependence of
activation and inactivation (Isom et al., 1992; Cannon et
al., 1993; Makita et al., 1994; Patton et al.,
1994; Isom et al., 1995). Thus,
1 subunits both modify
the functional properties of brain and skeletal muscle Na
channels and increase the efficiency of their expression.
The
role of the 1 subunit in the heart has been less clear. Initial
studies using subunit-specific antibodies identified the
1 subunit
polypeptide in the heart (Sutkowski and Catterall, 1990).
1
subunit mRNA also has been identified in heart (Isom et al.,
1992; Tong et al., 1993; Yang et al., 1993; Makita et al., 1994) and cDNAs homologous to the rat brain
1
transcript have been cloned from rat cardiac muscle (Bennett et
al., 1993; McClatchey et al., 1993; Yang et al.,
1993). The human and rat cardiac
1 cDNAs are identical in sequence
to their brain counterparts (Makita et al., 1994). (
)However, purified preparations of cardiac Na
channels from chicken and rat do not have associated
1
subunits (Lombet and Lazdunski, 1984; Cohen and Levitt, 1993), and
there have been conflicting reports concerning the functional
significance of
1 subunits in the heart. Yang et
al.(1993) reported no effect of
1 expression, while Kyle et al.(1993) observed a depolarizing shift in the voltage
dependence of inactivation as a result of
1 coexpression.
In
these experiments, we have investigated whether 1 subunit mRNA is
expressed in cardiac muscle cells using in situ hybridization,
and we have examined the functional role of
1 subunits in
modulating cardiac Na
channel expression and kinetics
by coexpression of mRNA for
and
1 subunits in Xenopus oocytes. We report that
1 subunit mRNA is expressed in
cardiac muscle cells in vivo. When expressed in Xenopus oocytes in conjunction with the rH1
subunit,
1 subunits
substantially increase Na
channel expression, but have
no detectable effects on physiological properties. Thus,
1
subunits are likely to be important determinants of the level of
functional expression of Na
channels in cardiac cells,
but do not alter their physiological properties significantly.
The deletion mutant 1
Val138-Ser159
was constructed by first removing a portion of the 5`-untranslated
region of p
1.C1Aa (Isom et al., 1992) by deletion of
nucleotides 1 through 175. This region is predicted to contain
stem-loop structures which may decrease the efficiency of
1
expression (Patton et al., 1994). Single stranded DNA was
prepared by interference helper phage VCS-M13 infection of XL1-blue
transformants (Stratagene, La Jolla, CA), and served as template for
oligonucleotide-directed ``loop out'' deletion mutagenesis. A
36-base ``clamp'' oligonucleotide was designed to anneal to
two segments of the sense strand template which flanked the region to
be deleted. In vitro mutagenesis reactions were performed as
described for the Sculptor
IVM system (Amersham Corp.).
The resulting mutant phagemid contained a 66-base pair deletion of the
nucleotides encoding Val
through Ser
. The
deleted amino acids are located in the extracellular domain of the
1 polypeptide, adjacent to the proposed transmembrane segment, and
do not contain a putative glycosylation site.
For two-microelectrode voltage-clamp experiments, the
oocytes were continuously perfused at room temperature (23-25
°C) with Ringer's solution (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl, 10 mM HEPES, pH
7.2). Whole cell Na
currents were studied using the
conventional two-microelectrode voltage-clamp technique (Patton and
Goldin, 1991) with a CA-1 amplifier (DAGAN). For cell-attached
macropatch recordings, oocytes were first manually stripped with fine
forceps under a dissecting microscope after shrinking with a hypertonic
solution (200 mM potassium aspartate, 20 mM KCl, 10
mM EGTA, 1 mM MgCl
, 20 mM HEPES,
pH 7.4-7.5; Methfessel et al.(1986)). During recordings,
the oocytes were bathed in a high K
solution (110
mM KCl, 10 mM NaCl, 10 mM EGTA, 1 mM MgCl
, 10 mM HEPES, pH 7.2) to bring the
membrane potential to approximately 0 mV. The electrode tip was coated
with Sylgard (Dow Corning) and filled with 150 mM NaCl, 1.5
mM CaCl
, 2 mM MgCl
, 5 mM HEPES, pH 7.4 (tip resistance 0.5-1 m
). Macropatch
current was recorded in the cell-attached configuration (Hamill et
al., 1981) using an AxoPatch-1C amplifier (Axon Instruments). The
voltage-clamp protocols are described in figure legends or
corresponding text. Conductance-voltage (g-V) relationships were
calculated from current-voltage (I-V) relationships according to g
= I/(V-V
), where I is the peak current measured at
voltage V and V
is the measured reversal potential.
Normalized conductance-voltage relationships and inactivation curves
were fit with a Boltzmann distribution, 1/(1 +
exp[(V-V
)/k]), where V
is the voltage of half-activation or half-inactivation and k is a slope factor. Pooled data are reported as means ± S.E.
Statistical comparisons were made using Student's t test, with p < 0.05 taken as the critierion of
significance.
In situ hybridization of free-floating sections
was carried out using modifications of methods described previously
(Miller et al., 1989; Black et al., 1994). Briefly,
adult Sprague-Dawley rats were anesthetized using sodium pentabarbitol,
the heart was removed, immediately frozen in powdered dry ice, and
stored at -70 °C. Sagittal sections (40 µm) through the
long axis of the heart were cut on a sliding microtome and then placed
into 4% paraformaldehyde fixative for 45 min. Tissue sections were then
rinsed in 0.05 M phosphate-buffered saline for 20 min, water
for 2 min, 0.1 M triethanolamine, pH 8.0, for 2 min and then
in 0.25% acetic anhydride in 0.1 M triethanolamine for 10 min
to reduce nonspecific binding. The tissue sections were then rinsed in
2 SSC for 10 min, 70% ethanol for 2 min, 95% ethanol for 2 min,
100% ethanol for 2 min, 70% ethanol for 2 min, and finally in water for
2 min. Sections were prehybridized for 3 h at 42 °C in a buffer
containing 44.6% formamide, 8.9% dextran sulfate, 0.27 M NaCl,
7 mM Tris, pH 8.0, 0.7 mM EDTA, 0.9
Denhardt's, 16 mM dithiothreitol, 0.45 mg/ml yeast tRNA,
and 4.6
Genius Northern blocking solution (Boehringer
Mannheim). The tissue was then transferred to hybridization solution
containing 41% formamide, 8.2% dextran sulfate, 0.25 M NaCl,
6.5 mM Tris, pH 8.0, 0.66 mM EDTA. 0.82
Denhardts, 14.8 mM dithiothreitol, 1.2 µg/ml yeast tRNA,
4.1
Genius Northern blocking solution, and 7.5 µg/ml of the
digoxigenin-labeled probe and incubated overnight at 42 °C. Tissue
sections were rinsed in 1
standard sodium chloride/sodium
citrate (SSC; Miller et al. 1989) for 30 min, treated with 25
µg/ml RNase A in RNase buffer (10 mM Tris, 0.5 M NaCl, and 1 mM EDTA) for 30 min, rinsed in 1
SSC
for 30 min, rinsed in 0.1
SSC at 45 °C for 40 min, rinsed
in 0.1
SSC at room temperature for 15 min, and then in 0.1 M Tris-buffered saline, pH 7.4, for 30 min. The tissue was
then blocked using 10% normal sheep serum in 0.1 M Tris-buffered saline for 1 h at room temperature before being
placed in alkaline phosphatase-conjugated anti-digoxigenin F(ab)
antibody (diluted 1:400 in 0.1 M Tris-buffered saline
containing 10% normal sheep serum and 0.1% Triton X-100) for 24 h at
room temperature. The tissue was then washed in 0.1 M Tris-buffered saline for 1 h followed by 1 h of rinsing in a
solution containing 100 mM Tris, 50 mM
MgCl
, and 100 mM NaCl, pH 9.5. The tissue was then
reacted with 4-nitro blue tetrazolium chloride (0.45 mg/ml),
5-bromo-4-chloro-3-indolyl phosphate (0.175 mg/ml), and levamisole
(0.24 mg/ml) in the reaction buffer, rinsed in stop buffer (10 mM Tris and 1 mM EDTA, pH 8.0) for 30 min and the free
floating sections were finally mounted on gelatin-subbed slides, air
dried, coverslipped using Biomeda gel mount (Fischer), and viewed using
a Leitz Dialux microscope.
Control experiments included omitting
probes from hybridization solution and substituting sense probes for
antisense probes. In addition, 1 antisense probe was hybridized to
liver tissue to test for nonspecific labeling with the probe. No
specific labeling was observed in the controls.
Figure 1:
In situ hybridization of 1 mRNA in heart. Adult rat heart sections
were processed for in situ hybridization as described under
``Experimental Procedures.'' A, ventricular
papillary heart tissue hybridized with
1 antisense probe
demonstrating the presence of
1 mRNA in muscle cells. Arrowheads outline the nuclei. B and C,
higher magnifications of heart tissue hybridized with
1 antisense
probe illustrating that labeling is present in the cytoplasm
surrounding the nucleus. D, tissue section hybridized with
1 sense probe to illustrate the lack of hybridization and the
specificity of the
1 antisense probe. Scale bars: A equals 20 µm; B-D equal 10
µm.
Figure 2:
Effect of rat brain Na channel
1 subunit mRNA co-injection with rH1
subunit
mRNA on expressed Na
currents in Xenopus oocytes. Currents were recorded in oocytes injected with
alone (25 ng/µl, left) or
(25 ng/µl) +
1 (50 ng/µl, right) mRNA during pulses from a holding
potential of -100 mV to potentials ranging from -45 to 0 mV
in 5-mV increments using two-microelectrode voltage
clamp.
To examine the concentration
dependence of this increase in Na current amplitude,
1 RNA was injected at concentrations ranging from 10 to 500
ng/µl with a constant 25 ng/µl of
subunit RNA. The
amplitude of the expressed Na
current increased as the
amount of
1 RNA injected was increased (Fig. 3A).
No current was observed when
1 subunits were injected in the
absence of the
subunit. These results were obtained in one series
of oocytes injected simultaneously and studied at times ranging from 62
to 78 h after injection. Similar results were obtained in two other
experimental series of this kind.
Figure 3:
Effect of 1 subunit mRNA coinjection
on rH1 Na
current expression. Mean current levels were
measured 72 h after injection in a single batch of oocytes (n = 6 for each point). A, effects of
1 mRNA
concentration. Each point was significantly different from the adjacent
one (p < 0.05) except for the last two right-hand bars with the highest
1 mRNA concentrations. B,
comparison of effects of wild type and mutant
1 subunit on
Na
channel expression. rH1
mRNAs was injected
into oocytes with or without
1 mRNA or with an inactive mutant
1 mRNA (
Val
-Ser
) with those amino
acids deleted. The amounts of
and
1 mRNAs injected are
indicated beneath the histogram bars.
Significant increases in
Na channel current amplitude were observed at a
1:
RNA molar ratio of 5:3. The observed increase in current
amplitude saturated at a
1:
RNA molar ratio of 50:1, with a
half-maximal increase at a
1:
molar ratio of 10:1 (equivalent
to a weight ratio of 2:1). The maximal increase in current induced by
1 subunit RNA coinjection was 3-6-fold, depending on the
batch of oocytes (mean = 5.2 ± 1.4-fold, n = 22 oocytes). This is comparable to the increase observed
when rat brain type IIA Na
channel
subunits were
coinjected with
1 subunit mRNA in Xenopus oocytes (Isom et al., 1992) and to the increase in
[
H]saxitoxin binding observed as a result of type
IIA
subunit and
1 subunit coexpression in transfected
mammalian cells (Isom et al., 1994).
An alternative
explanation for the observed increase in Na current
amplitude is that
1 subunit mRNA may exert nonspecific effects on
Na
channel expression or mRNA expression in general.
To examine that possibility, a deletion mutant of rat brain
1
(
1
Val
-Ser
) was constructed as
described under ``Experimental Procedures.'' The functional
effects of this mutant
1 subunit were tested by coinjecting it
with the rat brain type IIA
subunit (
1:
molar ratio of
100:1). The macroscopic Na
current time courses from
oocytes injected with transcripts encoding the rat brain type IIA
subunit alone and in conjunction with
1
Val
-Ser
were very similar.
Current-voltage relationships, steady-state inactivation curves, and
the time course of recovery from inactivation also were unaffected
(data not shown). This indicates that
1
Val
-Ser
is inactive, probably
because it does not associate with the
subunit. Coinjection of
1
Val
-Ser
with rH1
subunit
mRNA (10:1 molar ratio) caused no increase in current amplitude (Fig. 3B). Wild-type
1 subunits caused the normal
increase in current amplitude in the same batch of oocytes (Fig. 3B). Similar results were observed in two other
experiments. Thus, an active
1 subunit is necessary to observe the
increase in current with
1 subunit RNA coinjection.
Figure 4:
Effects of coinjection of 1 subunit
mRNA on the time course of rH1 Na
current. A,
current traces recorded in cell-attached macropatch configuration from
oocytes injected with
subunit mRNA alone (500 ng/µl, left) and in oocytes coinjected with
1 subunit mRNA
(
100 ng/µl +
1 200 ng/µl, right)
during depolarizations to -80, -70, -60, -50,
-40, -30, -20, -10, 0, 20, 60, and 80 mV from a
holding potential of -120 mV. B, averages of macropatch
current traces during pulses to the indicated voltages from oocytes
injected with rH1
subunit mRNA alone (solid lines, n = 18) or coinjected with
1 subunit mRNA (dotted
lines, n = 15). The average current traces shown
were constructed by normalizing the amplitudes of current traces from
individual experiments and then averaging
them.
Effects of 1 subunits on the
voltage dependence of Na
channel activation and
inactivation were determined using both two-microelectrode and
cell-attached patch recording configurations. Mean voltage dependence
of Na
current activation was unaffected by
coexpression of the
1 subunit with the
subunit in
cell-attached patches or in two-microelectrode recordings (Fig. 5, A and B). The voltage dependence of
Na
channel inactivation was determined using
conditioning prepulses (98 ms long in cell-attached experiments, 500 ms
long in two-microelectrode experiments) followed by a test
depolarization. The mean voltage dependences of Na
channel activation and inactivation were not significantly
affected in either recording configuration due to coinjection of
1
subunit RNA (Fig. 5, A and B). The voltage
dependences of activation and inactivation determined in the
cell-attached configuration with or without
1 subunits were
shifted negatively compared to two-microelectrode recordings as has
been previously observed for the native cardiac channel (Kimitsuki et al., 1990).
Figure 5:
Effects of 1 subunit coinjection on
the voltage dependence of rH1 Na
current activation
and inactivation and on its time course of recovery from inactivation.
Determinations were either from cell-attached macropatches (A and C) or from two-microelectrode recordings (B and D). A and B, mean voltage
dependences of activation and inactivation with (
) and without
(
) coinjection of
1 subunits. The data plotted
correspond to mean values derived from Boltzmann fits to individual
experiments. In the cell-attached configuration the mean values for
alone (
) were: V
act = -50.4
± 5.9 mV with slope factor, k = -7.4
± 1.0 mV (n = 18), V
inact =
-99.8 ± 9.4 mV, k = 9.1 ± 4.0 (n = 17);
+
1 (
):
V
act = -53.0 ± 6.2 mV, k = -7.3 ± 0.6 (n = 15),
V
inact = -97.3 ± 9.4 mV, k = 9.4 ± 1.8 (n = 13). For
two-microelectrode recordings,
alone (
): V
act
= -27.8 ± 2.3 mV, k = -6.2
± 0.6 mV (n = 12), V
inact =
-52.0 ± 4.2 mV, k = 6.5 ± 1.0 (n = 6);
+
1 (
):
V
act = -28.7 ± 1.6 mV, k = -6.0 ± 0.8 (n = 10),
V
inact = -56.1 ± 3.5 mV, k = 6.5 ± 1.0 (n = 14). C and D, recovery from inactivation in oocytes injected with rH1
subunit mRNA alone (
) and in conjunction with
1
subunit mRNA (
). C, recovery at -120 mV following
a 16-ms pulse to -30 mV. Normalized peak currents from each data
set were fit with single exponentials,
alone:
= 7.81
ms;
+
:
= 6.19 ms. D,
recovery at -100 mV after a 5-s conditioning pulse to -10
mV. Both data sets were fitted with two exponentials,
alone:
= 6.23 ms,
= 1120.06
ms, A
= 0.45, A
= 0.55;
+
:
= 6.50 ms,
= 854.28 ms, A
= 0.44,
A
= 0.56.
Recovery from inactivation was also studied
in both the cell-attached and two-microelectrode recording
configurations using double-pulse protocols. In the cell-attached
configuration, Na channels were inactivated using a
16-ms conditioning pulse to -30 mV. The membrane potential was
then returned to -120 mV to allow channels to recover from
inactivation. The degree of recovery was assessed at various recovery
times with a test pulse to -30 mV. Recovery time courses at
-120 mV determined by plotting normalized peak test pulse current versus recovery time were well fit with single exponentials (Fig. 5C). In oocytes injected with
subunit RNA
alone,
= 6.9 ± 1.8 ms, and in oocytes coinjected
with
1 subunit mRNA,
= 7.3 ± 1.4 ms (n = 4). For recovery experiments in the two-microelectrode
configuration, the conditioning pulse was 5 s long and recovery was
studied at -100 mV. Such a long prepulse is expected to generate
both fast and slow inactivation of the Na
channel.
After such prepulses, fits of the recovery time course required two
exponential components (Fig. 5D). In oocytes injected
with
alone, the time constant of the fast component (
1) was
5.5 ± 3.1 ms, and the time constant of the slow component
(
2) was 1010 ± 174 ms, with initial amplitudes, A1 and A2,
of 52 and 48% respectively (n = 3). In oocytes
coinjected with
1 subunit mRNA,
1 was 6.0 ± 0.9,
2 was 868 ± 163, with A1 and A2 each equaling 50% (n = 3). Differences between results with
alone and with
1 coinjection were insignificant (unpaired Student's t test, p > 0.05). Consistent with the lack of effect on
recovery from inactivation, after a 10 Hz train of 20 15-ms long pulses
to -10 mV from a holding potential of -100 mV, current was
reduced to 92.4 ± 1.8% of its initial value with rH1
alone
and to 91.9 ± 1.9% with coinjection of
1 (n = 7). Again, these values were not significantly different
from each other (p > 0.05). Thus, in oocytes where effects
of
1 subunit RNA coinjection have been verified by recordings of
significantly increased Na
current levels, there was
no significant difference in Na
current time course,
voltage dependence of activation, voltage dependence of inactivation,
or recovery from inactivation when comparing Na
currents due to injection of rH1 Na
channel
subunit RNA alone or when coinjected with
1 subunit RNA.
Our results show that 1 subunit mRNA is expressed in
cardiac muscle cells as assessed by high resolution in situ hybridization. Thus,
1 subunits are available to modulate rH1
Na
channels in vivo. These studies complement
previous work showing that
1 subunit mRNA and protein are present
in the heart without defining the cell-type expressing them (Sutkowski
and Catterall, 1990; Isom et al., 1992; Tong et al.,
1993; Yang et al., 1993; Makita et al., 1994).
Coexpression of rH1 subunits and
1 subunits in Xenopus oocytes substantially increases the level of
Na
currents. Since both
1 subunits and
subunits are present in cardiac myocytes, it is likely that
1
subunits associate with
subunits and increase the expression of
functional Na
channels in cardiac cells as well.
Purified Na
channels from chicken and rat heart do not
have associated
1 subunits (Lombet and Lazdunski, 1984; Cohen and
Levitt, 1993). However,
1 subunits dissociate easily from
detergent-solubilized Na
channels (Messner and
Catterall, 1986) and therefore may have been lost in purification.
Since the purified preparations of Na
channels from
heart have not been functionally reconstituted, it remains to be
determined whether these preparations which appear to lack
1
subunits can function in voltage-activated ion conductance.
Little
effect of 1 subunits was observed on the kinetics and voltage
dependence of expressed current. The lack of kinetic effects was
consistent with effects of human heart
1 subunits on human heart
Na
channel
subunit (Makita et al.,
1994). It differs from reports that coinjection of the rat brain
1
subunit RNA with the rat heart Na
channel
subunit RNA causes a 3 or 6 mV depolarizing shift in the voltage
dependence of inactivation (Kyle et al., 1993; Makielski et al., 1995) and from a report that
1 subunits cause a
3-mV hyperpolarizing shift in the voltage dependence of inactivation
(Nuss et al., 1995). These small differences in results may
reflect differences in the details of the experimental procedures used
in the different studies.
Injection of cardiac (rH1) subunit
mRNA into oocytes gave currents with comparable kinetics and voltage
dependence to Na
currents in native cardiac myocytes
(Satin et al., 1992). Recordings from our laboratory in
neonatal rat ventricular myocytes (Qu et al., 1994) gave
half-activation values of -34 mV and half-inactivation values of
-58 mV. Currents expressed in oocytes after injection of rH1
subunit RNA were half-activated at -28 mV and
half-inactivated at -52 mV (see Fig. 5B), only 6
mV more positive than Na
currents in ventricular
myocytes. These small differences contrast sharply with injection of
brain or muscle Na
channel
subunit RNA alone
into Xenopus oocytes. Those currents exhibit abnormally slow
kinetics and positively shifted voltage dependences of activation and
inactivation which were normalized by coinjection of
1 subunits
(Isom et al., 1992; Makita et al., 1994). Thus,
1 subunits have much more striking functional effects on brain and
skeletal muscle Na
channels than on cardiac
Na
channels.
During development of both rat retinal
ganglion cells and rat forebrain neurons in vivo, expression
of subunits and their assembly with
subunits is concurrent
with a 5- to 10-fold increase in the number of Na
channels (Wollner et al., 1988; Scheinman et
al., 1989; Sutkowski and Catterall, 1990). Thus, expression and
assembly of
subunits may be a rate-limiting step in Na
channel expression. In addition,
1 subunits stabilize
purified and reconstituted brain Na
channels. Channel
function was completely lost upon selective removal of
1 subunits
(Messner and Catterall, 1986; Messner et al., 1986). Our
results showing that coinjection of
1 subunit mRNA significantly
increases heart Na
channel expression in Xenopus oocytes are consistent with the hypothesis that
1 subunits
are important for the biosynthesis, assembly, and stabilization of the
cardiac Na
channel in vivo.