The effects of 15-mer phosphorothioate antisense
oligodeoxynucleotides (aODNs) specifically designed against the
RNAs of either of two closely related Na+ channel isoforms,
hSkM1 or hH1, were tested in human myotubes. Fluorescence
(3'-fluorescein isothiocyanate) labeling showed that mere
incubation of cultures with aODNs did not result in aODN uptake, but
liposome-mediated transfer was successful and resulted in cytoplasmic
and nuclear localization of ODNs. Intracellular fluorescence was stable
for at least 3 days. At 5 µM, the hH1-specific aODN was effective in
suppressing ion channel function, but the hSkM1-specific aODN was not.
Reverse transcription-polymerase chain reaction gave corresponding
results on the mRNA level. However, in HEK-293 cells stably expressing
hSkM1, the same hSkM1-specific aODN was able to reduce Na+
currents (2.4 ± 0.5 nA, n = 11; controls:
6.5 ± 1.0 nA, n = 14). We conclude that
cellular uptake of aODNs and intracellular access to the RNA target are
necessary, but not always sufficient conditions for an effective block
of mRNA translation in intact cells.
 |
Introduction |
A
modern strategy in pharmacology for interfering with receptor or
channel proteins is to manipulate lifetime and translation efficacy of
their coding mRNAs using antisense oligodeoxynucleotides (aODNs)
(Wagner, 1995
; Li et al., 1997; Branch, 1998). Such aODNs can be very
specific because they exert their action by binding to a unique part
(with a length of 12-20 bases) of their target mRNA. Their specificity
lets them distinguish between closely related isoforms of RNAs
(Hescheler, 1994; Brinkmeier et al., 1997) and even between wild-type
and point-mutant RNAs (Duroux et al., 1995).
For an effective inhibition of protein translation in vivo or in
cultured cells, the aODNs have to be protected against extracellular degradation, they must also be able to pass through the lipophilic plasma membrane, and finally they must be made to escape intracellular degradation and sequestration into organelles (Nakai et al., 1996; Crooke, 1998). Most importantly, when aODNs have reached their RNA,
they must find access to their specific target site within the
three-dimensional folding of this molecule (Branch, 1998).
Some of these tasks can now easily be solved, e.g., degradation is
substantially slowed when phosphorothioate-protected ODNs are used
instead of phosphodiester ODNs (Crooke, 1998). The accessibility of a
specific target site in a given RNA can be tested in advance in vitro
by means of binding assays or translation arrest assays (Schu and
Brinkmeier, 1999). As for the specificity for closely related isoforms
of voltage-gated Na+ channels, we have earlier
developed highly specific 15-mer aODNs against the RNAs of the
Na+ channels in human heart (hH1, Gellens et al.,
1992) and skeletal muscle (hSkM1, Chahine et al., 1994). Using an in
vitro assay and Xenopus oocytes as a cellular model, we
showed that these aODNs are clearly able to discriminate between the
two isoforms (Brinkmeier et al., 1997). The aim of the present
study was to achieve uptake of these aODNs into intact cultured cells
and to test whether they are able to selectively prevent translation of
the respective RNAs.
| |
Materials and Methods |
Cells and Application of Oligonucleotides.
Human myotubes
expressing both the tetrodotoxin (TTX)-sensitive skeletal
muscle-specific hSkM1 and the TTX-insensitive heart-specific hH1
Na+ channel (Ruppersberg and Rüdel, 1988;
Yang et al., 1993; Kaspar et al., 1994) were cultivated from satellite
cells isolated from muscle biopsies (Brinkmeier et al., 1993).
Proliferation of myogenic stem cells was supported in a medium composed
of a 1:1 (v/v) mixture of Ham's F-12 medium (Biochrom, Berlin,
Germany) and Dulbecco's modified Eagle's medium (PAA
Laboratories, Linz, Austria) with 5% fetal calf serum (FCS), 5% horse
serum (both Life Technologies, Inc., Karlsruhe, Germany), and
2.5 mg/ml glucose, 0.3 mg/ml glutamine, and 1.2 g/l
NaHCO3. Two days after seeding, the serum content of the medium was reduced to 2% FCS and 2% horse serum, allowing the
cells to differentiate into multinucleated myotubes within 2 weeks
(Brinkmeier et al., 1993). HEK-293 cells stably expressing the
-subunit of hSkM1 (Mitrovic et al., 1994) were used in ancillary experiments. The cells were grown in a medium composed of 90% minimum
essential medium (MEM) and 10% FCS. To select for high expression of
hSkM1 the medium contained in addition 800 µg/ml of the antibiotic
geneticin (G418; Boehringer Mannheim, Mannheim, Germany).
The effects of aODNs on the translation of Na+
channel RNAs were tested with several chemically different types of
15-mer aODNs (Interactiva, Ulm, Germany), i.e., standard,
phosphorothioate-capped at positions 1 and 15, completely
phosphorothioate-modified ODNs and the latter additionally labeled at
the 3' end with fluorescein isothiocyanate (FITC). In some experiments
aODNs were chemically coupled to a carrier peptide (penetratin; Oncor
Appligene, Heidelberg, Germany), originally derived from a
Drosophila homeobox peptide (Brugidou et al., 1995) to
facilitate their uptake into cells.
As for the designation of our ODN constructs, the number corresponds to
the start nucleotide of the target sequence of the published cDNAs
(Gellens et al., 1992; Chahine et al., 1994), and the additions
"asen" and "sen" indicate whether the ODN is an antisense or a
sense construct, respectively. The used aODNs were those that had been
found most effective in in vitro tests (Brinkmeier et al., 1997), i.e.,
TTCACCTCGTACTGC (3866asen) against hSkM1 RNA, GCAGTACGAGGTGAA (3866sen)
for control, and CTCTTCATACCCCCT (4444asen) against hH1 RNA (sequences
from 5' to 3' end). As an independent control we used in addition a
scrambled ODN containing the same bases as 3866asen in randomized
order: CTCTCAGCTTCCTAG.
Oligonucleotides were dissolved in water and stored as stock solutions
(1 mM) at 
70°C. For incubation they were diluted with serum-free
medium to final concentrations between 1 and 15 µM. In most
experiments liposomes (LipofectAMINE; Life Technologies, Inc.) were
used to improve cellular uptake of ODNs. For this, 5 µl of the
liposome mixture and 5 µl of the ODN stock solution were diluted with
200 µl of serum-free MEM. This mixture was incubated for 45 min at
room temperature, added to 800 µl of MEM, and the so obtained
solution was applied to nearly confluent myotube or HEK-293 cell
cultures for 5 h. Then the cultures were washed and supplied with
their standard serum-enriched medium. During incubation, the culture
medium was changed every day. The cells were used for electrophysiology
on day 2 or 3. For this, myotubes were converted into myoballs
(Pröbstle et al., 1988).
Electrophysiology.
Whole-cell Na+
currents were recorded at room temperature using an EPC-7 patch-clamp
amplifier (List, Darmstadt, Germany). The standard external solution
contained 140 mM NaCl, 3.5 mM KCl, 1.0 mM CaCl2,
1.0 mM MgCl2, and 2 mM HEPES, pH 7.4 (all
ingredients from Merck, Darmstadt, Germany). The pipettes were filled
with standard internal solution containing 140 mM CsCl, 1.4 mM
MgCl2, 10 mM EGTA, and 10 mM HEPES. Pipette
resistances were 0.5 M
for myotubes and 1 to 1.5 M for HEK-293
cells. Seal resistance, cell capacity, and access resistance were
determined as described (Pröbstle et al., 1988; Hamm et al.,
1996). The voltage error due to series resistance was estimated to be
lower than 4 mV for all experiments.
For the determination of current-voltage curves, a cyclic pulse program
was applied, each cycle consisting of a constant 100-ms prepulse to
135 mV and an 8-ms test pulse that was varied from 65 to +31 mV in
4-mV steps. The peak currents, INa, were
plotted against the test potential and the voltage dependence of
steady-state activation of the Na+ channels
(m3
curve) derived from these
plots (Hamm et al., 1996). Boltzmann equations were fitted to the data
points for the determination of the position of the point of
inflection, V1/2, and steepness, k, of the curves at V1/2. To
investigate the voltage dependence of steady-state inactivation of the
Na+ channels (h curve),
a cyclic pulse program was used with each cycle consisting of a 100-ms
prepulse that was varied between 135 and 19 mV in 4-mV steps, and a
test pulse to 20 mV. The current peaks recorded during the test
pulses were normalized and plotted against the prepulse potential and
the Boltzmann parameters registered. In some experiments TTX (Sigma,
Deisenhofen, Germany) was applied during the measurements.
Quantification of RNA Concentration.
Myotube cultures were
harvested by trypsin treatment, once washed with serum-free medium,
centrifuged and the pellets stored at 70°C. RNAs were isolated with
the RNeasy kit (Qiagen, Hilden, Germany), the integrity of the
preparations checked by agarose gel electrophoresis, and the RNA
concentration adjusted to 100 ng/ml for all samples. To quantify the
concentrations of hH1 and hSkM1 mRNAs, 1 µl of each RNA solution was
added to 19 µl of a one-tube RT-PCR master mix (Roche Molecular
Biochemicals, Mannheim, Germany) containing 20 picomoles of upstream
and downstream primers. The reverse transcription-polymerase chain
reaction (RT-PCR) was then carried out in a real time PCR analysis
system (Lightcycler; Roche Molecular Biochemicals) in the
presence of the fluorescent indicator SYBER green (contained in the
master mix). Two primers were used each for hH1 and hSkM1 RNA. For hH1
the oligonucleotide (from 5' to 3') GAG CAG CCT CAG TGG GAA TA served
as upstream primer and GCC TGC TTG GTC ACA ATG T as downstream primer.
The hSkM1 RNA primers were CGC TGG CTC AAT GTC A (upstream) and AGC CAA
AGA TGA TGA AGA TG (downstream). As standards to quantify the mRNA
levels, different dilutions of plasmid hSkM1 or hH1 cDNA were added to
the master mix instead of RNA samples and amplified parallely. The
Lightcycler system is a device to measure the raising concentration of
double-stranded PCR products with a fluorimetric technique during a
PCR. We used SYBER green as a fluorophore. To increase the specificity
of the SYBER green-based detection we performed the measurements in an
additional 81°C step (hH1) or at 84°C (hSkM1) during each
amplification cycle.
| |
Results |
Uptake of the ODNs into Myotubes.
In preliminary experiments
we exposed myotubes to standard, phosphorothioate-capped, and
completely phosphorothioate-modified aODNs (1 to 10 µM) or aODNs (1 µM) coupled to the carrier peptide penetratin (Brugidou et al., 1995)
and investigated the amplitudes of the Na+
currents conducted by these cells. The results did not suggest that the
aODNs inhibited RNA translation. Labeling of phosphorothioate-capped or
completely phosphorothioate-modified aODNs with FITC showed that these
aODNs were not incorporated into cells.
Coincubation of FITC-labeled, completely phosphorothioate-modified
aODNs with cationic liposomes caused fluorescence in nearly all
myotubes of all 20 investigated cultures. The stain was predominantly found in the nuclei, but also the cytoplasm was homogeneously stained.
Fluorescence appeared to be stable for at least 3 days of observation.
Effects of aODNs on Sodium Currents Conducted by Myotubes.
Because myotubes regularly express both hH1 and hSkM1, we applied aODNs
against either RNA to test for selective suppression of the translation
of the RNA for either channel. Because hSkM1 is more sensitive to TTX
than hH1, the simplest test was to compare the TTX sensitivities of the
channels in treated and untreated cells.
The Na+ currents conducted by the cells that had
incorporated the anti-hH1 aODN 4444asen were of about the same
amplitude as the currents recorded from untreated controls (average
current density for aODN cells: 106.1 nA/nF; controls: 106.0 nA/nF) but clearly, a higher than normal fraction of the current was sensitive to
TTX (Fig. 1A). On average, the fraction
of TTX-sensitive channels was increased by a factor of 2 (Fig. 1B;
Table 1).
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Fig. 1.
A, suppression of hH1-conducted
Na+ currents in myoballs treated with anti-hh1
ODN 4444asen (top) and absence of suppression of hSkM1-conducted
Na+ currents in myoballs treated with anti-hSkM1
ODN 3866asen (bottom). Three days before current recordings, test cells
had been incubated with phosphorothioate ODNs (5 µM) and cationic
lipids (5 µl/ml), whereas control cells, tested in parallel, were
incubated with cationic lipids only. The illustrated transient
Na+ currents were all elicited by 8-ms square
voltage pulses going from 85 mV for 100 ms to 135 mV and then to
21 mV, first with the cells in standard extracellular fluid, and then
in extracellular fluid with 100 nM TTX added. For each of the four
cells, the amplitudes of the currents in standard solution (ranging
between 7 and 10 nA) were normalized to unity. Addition of the
TTX-containing solution inhibits the current more than control in the
case of blockade of hH1 RNA, indicating high efficacy of aODN 4444asen.
Addition of TTX-containing solution inhibits the current less than
control in the case of blockade of hSkM1 RNA, indicating low efficacy
of aODN 3866asen. B, summary of results from similar experiments with a
number of cells ascribed to each column in parentheses. Fraction of
hH1-conducted current (±S.E.M.) of total Na+
current recorded from myoballs treated with anti-hH1 aODN 4444asen
(left) and anti-hSkM1 aODN 3866asen (right). Plotted are the quotients
of the maximum values of current-voltage relationships recorded in
presence/absence of 100 nM TTX. ***, significantly different from
control 2, P < .001, Mann-Whitney U
test.
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TABLE 1
Effect of antisense oligonucleotides on amplitude and voltage
dependence of activation and inactivation of moyball Na+
currents
Sodium currents (INa) were recorded from
myoballs in external solution (ext.) and after application of 100 nM
TTX. Mean values ± S.E.M. are given for all data, the number of
tested cells is given in parentheses.
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Fluorescent cells that supposedly had incorporated anti-hSkM1 aODN
3866asen also conducted Na+ currents of the same
amplitude as controls, but this aODN did not seem to have an effect on
the translation of its target RNA because, unlike with anti-hH1 aODN
4444asen, the TTX sensitivity of the conducted
Na+ currents was unchanged (Fig. 1).
The relative fractions of hH1 and hSkM1 channels in a myotube can also
be determined from the voltage dependence of the steady-state inactivation (h curve) because the points of
inflection of the respective individual curves differ by about 20 mV
(Pröbstle et al., 1988). Incubation of myotubes with anti-hH1
aODN 4444asen resulted in h curves having an
average inflection point at 78.3 mV (Fig.
2), which is close to the 73 mV of a
pure population of hSkM1 channels in HEK-293 cells (Table
2, column 4) and in tsA201 cells
(transformed human embryonic kidney cells of the HEK-293 cell line,
expressing the simian virus 40 T-antigen) (Wang et al., 1996).
Untreated control cultures and myotubes having incorporated the
anti-hSkM1 aODN 3866asen had their points of inflection at about 90
mV (Fig. 2), a value close to that for a pure population of hH1
channels (99 mV, Wang et al., 1996). An even more negative value,
about 100 mV, was noted with all myotubes when currents were
investigated in the presence of TTX (Table 1, column 6) Thus, also
investigation of the inflection point of the h
curve suggested that anti-hH1 aODN 4444asen was effective, whereas
anti-hSkM1 aODN 3866asen was not.
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Fig. 2.
Suppression of hH1 currents (top) and absence of
suppression of hSkM1 currents (bottom) demonstrated by the size of the
shift of the h curve (voltage dependence of
steady-state inactivation) of Na+ channels on
addition of 100 nM TTX (see text). Myoballs treated with aODNs 4444asen
(top left) or 3866asen (bottom left) in absence (filled symbols) or
presence (open symbols) of TTX. Control cells incubated with liposomes
only. The big left-shift seen in the presence of TTX with cells treated
with aODN 4444asen indicates an effective suppression of the current
conducted by hH1 channels. Same concentrations and incubation
conditions as in Fig. 1.
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TABLE 2
Effects of antisense oligonucleotides on magnitude and
electrophysiological characteristics of hSkM1 Na+ currents
recorded from HEK-293 cells
The number of tested cells is given in parentheses.
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Effects of aODNs on mRNA Levels of hH1 and hSkM1.
To test
whether the low efficacy of the anti-hSkM1 aODN 3866asen is also seen
on the mRNA level, we determined mRNA concentrations of both hSkM1 and
hH1 mRNAs in myotubes with a quantitative PCR technique. Myotube
cultures having been treated with 4444asen for 3 days showed about a
60% decrease of hH1 RNA compared with control. A scrambled ODN and the
3866asen against hSkM1 showed, as expected, nearly no effects (Fig.
3A). In contrast, hSkM1 RNA was resistant
against the application of its specific 3866asen oligonucleotide.
Concentrations of messenger RNA coding for hSkM1 were all in the same
range regardless of the applied ODN (Fig. 3B). To discriminate between
two possible mechanisms, that of an early suppression followed by an
up-regulation of hSkM1 during days 2 and 3 and that of a compete lack
of inhibition we quantified the hSkM1 mRNA after 24 h (instead of
3 days) after treatment with 3866asen. Also at that time the aODN had
no influence on the hSkM1 mRNA level (data not shown).
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Fig. 3.
Messenger RNA levels of hH1 (A) and hSkM1 (B)
isolated from myotubes after a 3 days treatment with the hH1 antisense
oligonucleotide 4444asen, hSkM1 antisense oligonucleotide 3866asen, or
scrambled oligonucleotide. RNA concentrations were determined using a
series of dilutions of plasmid DNA and normalized to control (cells
without treatment). Mean values ± S.D. given from three
independent experiments (A) and two experiments (B), respectively.
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Effects of aODN 3866asen on Sodium Currents in HEK-293 Cells.
Because aODN 3866asen had been very effective in vitro, we decided to
test its efficacy in another cell system, i.e., HEK-293 cells stably
expressing the -subunit of hSkM1. As with myotubes, also with HEK
cells we found substantial uptake of FITC-labeled aODNs when the latter
had been mixed with liposomes. In contrast to the results with
myotubes, we found in all tested cell cultures only 30 to 40% of the
cells fluorescent. As with myotubes, the fluorescence remained stable
for at least 3 days in the cells that had taken up the aODNs. As
another difference to the observations with the myotubes where the
fluorescence was mainly contained in the nuclei, in the HEK cells it
was more or less homogeneously distributed in nuclei and cytoplasm.
The amplitudes of the Na+ currents conducted by
fluorescent cells were significantly smaller than those conducted by
nonfluorescent cells, indicating that aODN 3866asen had been effective
(Fig. 4; Table 2). The current conducted
by fluorescent cells was also much smaller than currents conducted in
control cells treated with the corresponding sense ODN 3866sen or with
no ODNs at all (Fig. 4; Table 2).
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Fig. 4.
Na+ currents recorded from
HEK-293 cells expressing hSkM1 that had been incubated with 5 µM aODN
3866asen. Controls from cells treated with liposomes only. A and B,
original recordings of families of Na+ currents
elicited by square voltage pulses from 85 to 135 mV and then to
various test potentials between 73 and +31 mV. Smaller amplitudes of
currents conducted by cells treated with the 3866asen aODN indicate
efficacy of this oligo in HEK cells. C, summarized results of
experiments as shown in A and B. The maximum values attained in
current-voltage curves for cells treated with antisense (asen) or sense
(sen) aODNs are compared with maximum values seen in controls treated
with liposomes only. Cells showing bright fluorescence during
microscopic inspection (columns 2 and 3) were selected for recordings:
asen: aODN 3866asen (5 µM); sen: corresponding 3866sen ODN (5 µM).
Another group of cells from the same culture showing no or little
fluorescence was studied accordingly (columns 4 and 5). Means ± S.E.M. Number of tested cells given in parentheses. *, difference
significant compared with groups (from left to right) 1, 3, and 4;
P < .05, Kruskall-Wallis nonparametric ANOVA test.
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Using HEK-293 cells, we were also able to investigate whether the aODN
treatment had any side effects on the activation and inactivation
parameters of those Na+ channels that had been
incorporated in the membrane in spite of suppression of RNA
translation. No difference was found in the positions of
m3 and
h curves between aODN (3866asen)-treated cells and the two controls, sense ODN-treated and only liposome-treated cells
(Fig. 5; Table 2, columns 3 and 4).
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Fig. 5.
Absence of influence of incorporation of aODN
3866asen on the gating characteristics of HEK-293 cells. Voltage
dependence of inactivation (A) and activation (B) of
Na+ channels in cells that were incubated with
the oligo ( ) or with solution containing liposomes only ( ). The
same concentrations and incubation conditions were used as described in
Fig. 1. Boltzmann curves (continuous lines) were fitted to each set of
data points.
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| |
Discussion |
The positive result of this study is that specific aODNs can be
incorporated into human muscle cells. The negative result is that one
of our aONDs tested was not effective even when clearly taken up by the
cells. For unknown reasons, the anti-hSkM1 aODN that can effectively
prevent RNA translation, as shown previously in vitro (Brinkmeier et
al., 1997) and now also in cultured HEK-293 cells, did not seem to be
effective in myotubes. We conclude from this latter finding that
cellular uptake of aODNs and intracellular access to the RNA target are
necessary, but not always sufficient conditions for an effective block
of RNA translation in intact cells.
The question of cellular uptake is still of particular concern in the
use of antisense oligonucleotides. Only in a few experimental settings
without special delivery system did aODNs reach intracellular concentrations that were sufficient for a specific block of translation (Melone et al., 1998). With our two human cell systems, i.e., primary
muscle cells and HEK cells, the difficult barrier of the plasma
membrane was only overcome when the aODNs were coincubated with
cationic lipids (Bennett et al., 1992). Visible fluorescence then
indicated that the intracellular aODN concentration was sufficient. Nonfluorescent cells, when tested with simple current measurements did
not show an antisense effect. For our cell and delivery systems, fluorescence-labeled aODNs resulted in homogeneous nuclear and cytoplasmic staining. This is in agreement with recent observations made with monocytes and lymphocytes using the cationic lipid-mediated uptake technique (Hartmann et al., 1998). Fortunately, we did not
observe signs of cytotoxicity, such as formation of intracellular granules, surface blebs, or detachment of cells as a consequence of the
use of our phosphorothioate-protected aODNs.
Studies on the kinetics of aODN uptake and efflux have given evidence
for a high initial uptake during the first hours followed by a slower
uptake process (24 h and after), the latter giving way to a dynamic
balance of efflux and influx (Li et al., 1997). A high initial uptake
rate might explain why we found 5 h of exposure time enough for
sufficient uptake yield with both cell systems. Our observation of a
fluorescence remaining fairly constant for 3 to 4 days can be explained
by a long half-time for the aODN efflux (4-5 days according to Li et
al., 1997).
The anti-hSkM1 aODN 3688asen was shown to have good access to its RNA
target sequence because it was very effective in translation arrest
assays and after coinjection with RNAs into X. laevis
oocytes (Brinkmeier et al., 1997). The expectation that an aODN, once it was taken up by a myotube as demonstrated by fluorescence, would
block RNA translation was not fulfilled. Several mechanisms may be
involved. In myotubes, hSkM1 could be more stable than hH1 and it could
be more stable in myotubes than in HEK-293 cells (Spiller et al.,
1998). For the TTX-sensitive Na+ channel in rat
myotubes, however, a half-life of 18 h was determined (Sherman et
al., 1985). A similar half-life for hSkM1 in human myotubes would be
short enough for a marked antisense effect under our experimental
conditions. A second possible mechanism, the compensatory up-regulation
due to post-transcriptional feedback mechanisms (Rothenberger et al.,
1990; Mosner et al., 1995), which has been shown to exist for the
skeletal muscle chloride channel (Chen et al., 1997), is also unlikely
because we have shown a normal hSkM1 RNA concentration at days 1 and 3 in the presence of the oligonucleotide. A third possibility seems to be
the most likely, a structural difference between the natural mRNA and
the coinjected and transfected cRNA. The structures of such huge RNAs (6.5 to 7.5 kb) are difficult to predict or determine (for a review of
RNA structure and structural transitions, see Klaff et al., 1996).
Considering the current knowledge of aODN action, our finding that
hSkM1 mRNA is stable in myotubes is best explained by a poor access of
3866asen ODN to its mRNA target sequence. Thus, in spite of its good
access to the hSkM1 cRNA in vitro, in X. laevis oocytes (Brinkmeier et al., 1997), and HEK-293 cells the OND 3866asen is
probably prevented by an unknown mechanism from reaching its mRNA
target sequence in myotubes, or else it cannot fulfill its degradative
antisense action.
We thank Dr. N. Mitrovic for supplying us with HEK-293 cells
stably expressing hSkM1 and E. Fuchs, K. Kiote, and M. Dürr for
expert technical assistance.
aODN, antisense oligodeoxynucleotide;
hH1, human heart voltage-dependent sodium channel;
hSkM1, human adult
skeletal muscle voltage-dependent sodium channel;
TTX, tetrodotoxin;
FCS, fetal calf serum;
MEM, minimum essential medium;
FITC, fluorescein
isothiocyanate;
asen, antisense;
sen, sense;
m3 curve and h curve, voltage dependence of steady-state
activation and inactivation, respectively;
HEK, human embryonic
kidney.
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Abstract
Introduction
channel 1 gene dosage.
J Physiol (Lond)
504:
75-81.
