(Received for publication, July 27, 1994; and in revised form, November 7, 1994)
From the
Brain sodium channels are a complex of (260 kDa),
1
(36 kDa), and
2 (33 kDa) subunits.
subunits are functional
as voltage-gated sodium channels by themselves. When expressed in Xenopus oocytes,
1 subunits accelerate the time course of
sodium channel activation and inactivation by shifting them to a fast
gating mode, but
subunits expressed alone in mammalian cells
activate and inactivate rapidly without co-expression of
1
subunits. In these experiments, we show that the Chinese hamster cell
lines CHO and 1610 do not express endogenous
1 subunits as
determined by Northern blotting, immunoblotting, and assay for
1
subunit function by expression of cellular mRNA in Xenopus oocytes.
subunits expressed alone in stable lines of these
cells activate and inactivate rapidly. Co-expression of
1 subunits
increases the level of sodium channels 2- to 4-fold as determined from
saxitoxin binding, but does not affect the K
for saxitoxin. Co-expression of
1 subunits also shifts
the voltage dependence of sodium channel inactivation to more negative
membrane potentials by 10 to 12 mV and shifts the voltage dependence of
channel activation to more negative membrane potentials by 2 to 11 mV.
These effects of
1 subunits on sodium channel function in
mammalian cells may be physiologically important determinants of sodium
channel function in vivo.
Voltage-sensitive Na channels are the membrane
proteins responsible for initiation of the action potential in most
excitable cells(1) . Na
channels isolated from
rat brain are heterotrimeric, composed of a 260-kDa
subunit, a
36-kDa
1 subunit, and a 33-kDa
2 subunit(2) .
Multiple cDNAs encoding Na
channel
subunit
isoforms have been cloned and sequenced (reviewed in (2, 3, 4) ). The primary structure of the rat
brain
1 subunit deduced from cDNA sequence predicts a membrane
glycoprotein with a type 1 transmembrane topology including a single
transmembrane segment(5) , in agreement with previous
biochemical analyses(2) . Rat brain
1 subunit mRNA is
expressed in brain, spinal cord, heart, and skeletal muscle
tissues(5, 6) . Although the Na
channel
subunit is sufficient for expression of functional
Na
channels(7, 8, 9, 10, 11, 12) ,
co-expression of rat brain
1 subunit and rat brain type IIA
subunit mRNAs in Xenopus oocytes results in acceleration of
the macroscopic rates of activation and inactivation, a hyperpolarizing
shift in the voltage dependence of inactivation, and an increase in
peak current amplitude(5, 13) . Furthermore, the
functional effects of
1 are not limited to the adult central
nervous system because similar functional effects of
1 subunits
have been reported on skeletal muscle µ1
subunits and on
embryonic brain type III
subunits(13, 14, 15) .
Na channel
subunits expressed alone in Xenopus oocytes function in two gating modes characterized by fast and
complete inactivation versus slow and incomplete
inactivation(16, 17, 18) . Expression of
1 subunits in Xenopus oocytes shifts the proportion of
Na
channels gating in the fast mode as compared to the
slow(13, 14) . This mode shift is responsible for the
acceleration of activation and inactivation. In contrast to Xenopus oocytes, Na
channel
subunits expressed
alone in transfected mammalian cells gate primarily in the fast
mode(12, 19, 20) , and no functional effect
of
1 subunits on Na
channels expressed in
mammalian cells has been defined. An essential step in understanding
the possible physiological significance of
1 subunits, therefore,
is to investigate the functional effects of
and
1
co-expression in transfected mammalian cells and to determine whether
the fast gating observed in these cells is caused by endogenous
1
subunit expression or by other factors contributed by the genetic
background of mammalian somatic cells. In the present study, we show
that functional co-expression of rat brain type IIA
and
1
subunits in Chinese hamster cells results in hyperpolarizing shifts in
both the voltage dependence of Na
channel activation
and inactivation and an increase in Na
channel
expression at the plasma membrane as compared to
alone.
Furthermore, we show that fast gating of Na
channel
subunits expressed alone in these cells is not due to the
presence of endogenous
1 subunits, but may be due instead to
differences in biosynthesis, post-translational modification, or
protein-protein interactions in mammalian somatic cells.
Synthetic Na channel peptides were synthesized using the solid phase method of
Merrifield(23) . The identities of the synthetic peptides were
confirmed by amino acid analysis and/or mass spectrometry. The
synthetic peptide, SP1, corresponds to residues 554-563 in the
primary sequence of the rat brain type IIA Na
channel
subunit(9) . The synthetic peptide,
1-1, corresponds
to amino acid residues 1-18 of the
1 subunit(5) .
Anti-peptide antibodies were prepared as described by Gordon et
al.(24) .
The
Chinese hamster ovary (CHO) cell line CNaIIA, which stably expresses
rat brain type IIA subunits(20) , was transfected with
pCDM8.
1 using DOTAP reagent as described above. pSV2*Hyg was
co-transfected with pCDM8.
1 to confer resistance to hygromycin B.
Northern blot analysis of
10 µg of each RNA sample was carried out as described previously (13) except that Boehringer Mannheim Nylon membrane was
substituted for nitrocellulose. An antisense cRNA probe was transcribed
from the plasmid p1.C1Aa, incorporating digoxigenin-11-UTP
(Boehringer Mannheim) and hybridized to the blot as described in the
product literature. Hybridization and washing of the blot were
performed at 65 °C. Wash solutions contained 0.5% SSC and 0.5% SDS.
Chemiluminescent detection of the digoxigenin-labeled probe was
accomplished using LumiPhos (Boehringer Mannheim) according to the
manufacturer's instructions. The blot was then exposed to Kodak
X-Omat AR film at room temperature for 30 min.
Whole cell voltage clamp
experiments on mammalian cells were performed as described
previously(20) . The extracellular solution contained: 130
mM NaCl, 4 mM KCl, 1.5 mM CaCl,
1.0 mM MgCl
, 10 mM HEPES, and 5 mM glucose, adjusted to pH 7.4 with NaOH. For one series of
experiments comparing CNaIIA cells to CNaIIA
1-B6 cells, a low
Na
solution containing 13 mM NaCl and 117
mM choline Cl was used. Intracellular solutions contained
either fluoride or aspartate as the dominant intracellular anion. The
fluoride-based solution contained: 105 mM CsF, 40 mM CsCl, 10 mM NaCl, 10 mM CsEGTA, and 10 mM HEPES. The aspartate-based intracellular solution contained: 140
mM cesium aspartate, 5 mM NaCl, 3 mM MgCl
, 10 mM HEPES, and 5 mM CsEGTA.
When the fluoride-based solution was included in the pipette, the
voltage-dependent parameters underwent a slow shift in the
hyperpolarizing direction after achieving the whole cell configuration
that was largely complete within 10 min. Therefore, at least 10 min
were allowed before recording voltage-dependent properties when using
this solution. Such shifts were not observed with the aspartate-based
intracellular solution.
The voltage dependence of channel activation
was determined from the peak current recorded during 16-ms-long
prepulses to a range of potentials. Chord conductance (G) was
calculated from peak current (I) according to G = I/(V - V)
where V is the test pulse potential and V
is the measured reversal potential. Inactivation curves were
measured using a 200-ms-long prepulse to a range of potentials followed
by a test pulse to 0 mV. Conductance-voltage curves and inactivation
curves were fit with a Boltzmann relationship, G = 1/(1
+ exp((V - V
)/k)))
where V
is the midpoint of the curve, and k is a slope factor.
Figure 1:
Northern blot analysis of RNA from
parental and transfected cell lines. Total cellular RNA was isolated
from the parent cell lines 1610 and SNaIIA and the 1
subunit-transfected cell lines. 10 µg of each RNA sample was
electrophoresed on a 7.4% formaldehyde gel as described under
``Experimental Procedures.'' Following electrophoresis, the
RNA was transferred to Boehringer Mannheim Nylon membrane, UV
cross-linked, hybridized with a cRNA probe synthesized from
p
1.C1Aa, and detected by chemiluminescence as described under
``Experimental Procedures.'' Lane 1, SNaIIA; lane 2, SNaIIA
1-16; lane 3,
SNaIIA
1-D1; lane 4, SNaIIA
1-15; lane
5, SNaIIA
1-19; lane 6,
1-B3; lane
7, SNaIIA
1-D3; lane 8,
1610.
Figure 2:
Detection of 1 and
subunit
polypeptides in membrane fractions or cell extracts isolated from
parent and transfected cell lines. A, Na
channel
1 subunit polypeptide was identified in membrane
preparations of SNaIIA
1-16 (indicated as 16) or
1610 cells. Membrane proteins (125 µg/lane) were separated by
SDS-PAGE, transferred to nitrocellulose, and probed with
anti-
1-1 antibody (lanes 1 and 2) or
anti-
1-1 antibody that had been preincubated with
1-1 peptide (lanes 3 and 4) as described
under ``Experimental Procedures.'' Visualization of the blot
was performed with the Enhanced Chemiluminescence Detection System
(Amersham) using Kodak X-Omat AR film. B, Na
channel
subunit polypeptide was identified in cell extracts
prepared from 1610 or SNaIIA
1-16 (indicated as 16)
cells using a combination of immunoprecipitation, phosphorylation with
cAMP-dependent protein kinase and [
-
P]ATP,
and SDS-PAGE as described under ``Experimental Procedures.'' Lane 1, 1610 cell extract immunoprecipitated with anti-SP1
antibody followed by phosphorylation; lane 2,
SNaIIA
1-16 cell extract immunoprecipitated with anti-SP1
antibody followed by phosphorylation; lane 3,
SNaIIA
1-16 cell extract immunoprecipitated with nonimmune
serum followed by phosphorylation. Gels were subjected to
autoradiography against Kodak X-Omat AR film at -70 °C with a
DuPont CRONEX Lightning Plus intensifying screen for 2
h.
Figure 3:
Functional effects of mRNA from parental
and transfected cell lines on Na channels expressed in Xenopus oocytes. Na
currents were recorded
from Xenopus oocytes injected with type IIA
subunit cRNA
(5 ng/µl) alone or with p
1.C1Aa cRNA (5 ng/µl),
SNaIIA
1-16 cell poly(A)-selected mRNA, or 1610 cell
poly(A)-selected mRNA. Currents were evoked by applying a depolarizing
pulse to 0 mV from a holding potential of -90 mV. The records
were normalized with respect to the peak current
amplitudes.
Figure 4:
Analysis of [H]STX
binding to parental and transfected cell lines. A, saturation
binding. SNaIIA and SNaIIA
1-16 cells were grown past
confluency, suspended, and incubated with increasing concentrations of
[
H]STX (0.1, 0.2, 0.5, 1, 2, 5, or 10
nM, 10 nM point not shown) (20 Ci/mmol) in the
presence and absence of 10 µM TTX for 1 h at 4 °C as
described under ``Experimental Procedures.'' Specific binding
data were normalized to protein using Peterson analysis(18) .
Scatchard analysis of the saturation binding data was performed using
LIGAND/EBDA to calculate K
(SNaIIA
= 0.55 ± 0.12 nM, SNaIIA
1-16 =
0.50 ± 0.16 nM) and B
(SNaIIA
= 3.2 ± 0.4 fmol/mg of protein, SNaIIA
1-16
= 14.5 ± 1.3 fmol/mg protein). These data are
representative of three separate experiments. Closed squares,
SNaIIA
1-16; open diamonds, SNaIIA. B,
comparison of [
H]STX binding at saturation. Each
indicated cell line was grown past confluence, suspended, and incubated
with 5 or 10 nM [
H]STX in the presence
and absence of 10 µM TTX as described under
``Experimental Procedures.'' Specific binding data were
normalized to protein using Peterson analysis(18) . The mean
and standard deviation values for each cell line were analyzed using
Sigma Plot (Jandel Scientific). The error bars shown reflect n independent culture experiments as follows: SNaIIA, n = 6; SNaIIA
1-16, n = 6;
SNaIIA
1-15, n = 5; SNaIIA
1-D1, n = 5; SNaIIA
1-D3, n = 5;
SNaIIA
1-19, n =
4.
Figure 5:
Na currents in cells
expressing
subunits or
+
1 subunits. A,
currents in response to a 16-ms pulse from a holding potential of
-100 mV to -20 mV in an SNaIIA cell and in an
SNaIIA
1-16 cell. B, voltage dependence of
Na
channel activation and inactivation in cells
expressing
subunits alone and in four independent cell lines
expressing
+
1 subunits. Mean conductance-voltage
curves (open symbols) and inactivation curves (filled
symbols) for control cells (
), SNaIIA
1-16 cells
(
), SNaIIA
1-D1 cells (
), SNaIIA
1-15 cells
(
), and SNaIIA
1-D3 cells (
). Conductance-voltage and
inactivation curves for individual experiments were fit with Boltzmann
functions by a least squares technique as described under
``Experimental Procedures.'' Mean curves were calculated from
the mean values of V
and k obtained
from the fits. Mean values for fits to inactivation curves for each
cell line are reported in Table 2. Mean values of V
and k for fits to conductance-voltage
curves for each cell line are reported in Table 3.
The voltage dependence of Na channel inactivation was determined using 200-ms-long prepulses
in 4 cell lines co-expressing
and
1 subunits and compared to
the parent cell line expressing
alone. Measurements were obtained
after 10 min of recording to minimize effects of the spontaneous
negative shift in the voltage dependence of activation and inactivation
in fluoride-containing intracellular solution that had largely
stabilized by this time. In each cell line expressing
1 subunits,
the voltage dependence of inactivation was shifted 10 to 13 mV negative
to the parent cell line (Fig. 5B and Table 2).
Effects on the voltage dependence of activation were more variable with
activation being shifted 2 to 11 mV negative relative to the parent
cell line in
1-expressing lines (Fig. 5B and Table 3). Voltage dependences of activation and inactivation were
also studied using an aspartate-based intracellular solution. With this
intracellular solution, as opposed to the fluoride-based intracellular
solution, shifts in voltage-dependent parameters during intracellular
dialysis in the whole cell voltage clamp are minimal. However, it was
much more difficult to obtain and maintain high resistance seals on
these transfected cells without fluoride, preventing collection of a
large set of recordings under these conditions. In the aspartate-based
intracellular solution, voltage dependences of activation and
inactivation were more positive than in the fluoride-based solution ( Fig. 6and Table 4). However, the half-activation and
inactivation voltages were shifted -6 mV and -11 mV,
respectively, in the SNaIIa
1-16 cell line expressing the
1 subunit. Thus, in this solution, as in the fluoride-based
solution, the voltage dependences of activation and inactivation for
SNaIIA
1-16 were more negative than for the parent SNaIIA
cell line. These results confirm the more complete data in the
fluoride-containing intracellular solution ( Table 2and Table 3), demonstrating a significant negative shift in
activation and inactivation in this cell line due to co-expression of
the
1 subunit of the Na
channel.
Figure 6:
Voltage dependence of Na
channel activation and inactivation in cells expressing
subunits
alone or
+
1 subunits measured using aspartate
intracellular solution. Mean conductance-voltage curves (open
symbols) and inactivation curves (filled symbols) for
control cells (circles) and SNaIIA
1-16 cells (squares). Conductance-voltage and inactivation curves for
individual experiments were fit with Boltzmann functions by a least
squares technique as described under ``Experimental
Procedures.'' Mean curves were calculated from the mean values of V
and k obtained from the fits. Mean
values of V
and k for fits to
conductance-voltage curves and mean values for fits to inactivation
curves for each cell line are reported in Table 4.
Co-expression of Na channel
and
1
subunits in Xenopus oocytes demonstrated that
1 subunits
play a modulatory role in Na
channel function in that
cell type (5) .
1 subunits increased peak current
amplitude, shifted the voltage dependence of Na
channel inactivation in the hyperpolarizing direction, and
accelerated the rates of Na
channel activation and
inactivation through a mechanism involving an increase in the
proportion of channels gating in the fast
mode(5, 13) . In the present study, we observed
similar modulatory effects for
and
1 co-expression in
mammalian somatic cell lines.
1 subunits cause a hyperpolarizing
shift in the voltage dependence of inactivation and increase the
functional expression of Na
channels at the plasma
membrane. In addition,
1 subunits cause a variable shift in the
voltage dependence of Na
channel activation in the
hyperpolarizing direction. Our results provide the first evidence for
modulation of the functional properties of sodium channels expressed in
mammalian somatic cells by
1 subunits and support the hypothesis
that
1 subunits have important modulatory effects on sodium
channels in vivo.
Previous reports have demonstrated the
efficient expression of functional Na channels in
various mammalian cell lines by transfection of
subunits of rat
brain sodium channels alone(12, 20) . Similar results
have been obtained with transient expression of skeletal muscle sodium
channels (19) and stable expression of cardiac sodium channels (33) in mammalian cell lines. In each case, transfection of
subunits alone resulted in expression of Na
channels which activated and inactivated with a rapid time
course. It was possible that this fast gating could have resulted from
endogenous expression of
1 subunits by each of the several cell
lines used in these previous experiments. Alternatively, the rapid time
course of gating could be explained by differences in protein synthesis
and processing between mammalian cells and Xenopus oocytes. In
the present study, we have demonstrated through Northern blot and
immunoblot analysis and through expression of poly(A)-selected mRNA in Xenopus oocytes that the parent 1610 cell line does not
express endogenous
1 subunits or
1 subunit-like activity.
Thus, our results support the conclusion that
1 subunit expression
has direct physiological effects on Na
channel
activation, inactivation, and expression levels in mammalian cells. The
fast gating mode observed for Na
channel
subunits expressed in mammalian cells is likely to be caused by factors
in addition to
1 subunit expression such as post-translational
glycosylation, acylation, or processing, association of Na
channel
subunits with other proteins such as cytoskeletal
components, or the higher temperature at which these processes take
place in a mammalian cell.
In Xenopus oocytes, where some
of the post-translational events observed in mammalian cells are
altered or absent, 1 subunits appear required to stabilize the
subunit in a conformation that gates in the fast
mode(13) . While
1 subunits most likely also contribute to
stabilization of the functional state of sodium channels in mammalian
cells, other elements are evidently present which exert a sufficient
stabilizing effect to ensure Na
channel gating in the
predominant fast mode in the absence of
1 subunits. Efficient,
stable co-expression of Na
channel
and
1
subunits now provides a system in which the role of the auxiliary
1 subunits in Na
channel biosynthesis and
processing can be assessed, and other cellular factors which influence
Na
channel functions such as gating mode can be
identified.