Blockade by cAMP of native sodium channels of adult rat
skeletal muscle fibers
Jean-François
Desaphy,
Annamaria
De Luca, and
Diana Conte
Camerino
Unità di Farmacologia, Dipartimento Farmaco-Biologico,
Facoltà di Farmacia, Università degli Studi di Bari,
I-70125 Bari, Italy
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ABSTRACT |
Although the
skeletal muscle sodium channel is a good substrate for cAMP-dependent
protein kinase (PKA), no functional consequence was observed for this
channel expressed in heterologous systems. Therefore, we investigated
the effect of 8-(4-chlorophenylthio)adenosine 3',5'-cyclic
monophosphate (CPT-cAMP), a membrane-permeable cAMP analog, on the
native sodium channels of freshly dissociated rat skeletal muscle
fibers by means of the cell-attached patch-clamp technique. Externally
applied CPT-cAMP (0.5 mM) reduced peak ensemble average currents by
~75% with no change in kinetics. Single-channel conductance and
normalized activation curves were unchanged by CPT-cAMP. In contrast,
steady-state inactivation curves showed a reduction of the maximal
available current and a negative shift of the half-inactivation
potential. Similar effects were observed with dibutyryl adenosine
3',5'-cyclic monophosphate but not with cAMP, which does
not easily permeate the cell membrane. Incubation of fibers for 1 h
with 10 µM H-89, a PKA inhibitor, did not prevent the effect of
CPT-cAMP. Finally, the
-adrenoreceptor agonist isoproterenol
mimicked CPT-cAMP when applied at 0.5 mM but had no effect at 0.1 mM.
These results indicate that cAMP inhibits native skeletal muscle sodium
channels by acting within the fiber, independently of PKA activation.
sodium current; patch clamp; H-89; isoproterenol
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INTRODUCTION |
VOLTAGE-GATED SODIUM channels are responsible for the
initial rise and subsequent conduction of action potential in skeletal muscle. Because the action potential is an all-or-none response to the
rapid activation of sodium channels, it has been long thought that
sodium channels might be much less sensitive to acute physiological modulation than other voltage-gated channels. However, in the past
decade, numerous studies have shown that sodium channels can also be
modulated by second messengers, directly or indirectly through
phosphorylation (5, 9). On the other hand, a subtle change
in the gating properties of the skeletal muscle sodium channel (SkM1),
as occurs in some inherited muscular disorders, can induce severe
alterations of muscle function such as myotonia or paralysis (3). This
supports the hypothesis that fine regulation of sodium channels may
have a great importance for physiological function.
Accordingly, voltage-gated sodium channels have been shown to be
targets of cAMP in the brain and the heart. Phosphorylation of the rat
brain sodium channel II (rBII) by the cAMP-dependent protein kinase
(PKA) has been extensively studied (5, 9). Four serine residues within
the intracellular loop between domains I and II [interdomain I-II
(ID I-II)] of the channel protein can be phosphorylated by PKA
(20), and phosphorylation of Ser-573 is necessary and sufficient for
the inhibitory effect of PKA on this channel (26). Such a
phosphorylation is physiologically important for the neurons, occurring
in response to neurotransmitters such as dopamine (4, 22). The cardiac
sodium channel (H1) is also phosphorylated by PKA within the ID I-II of
the channel protein (10, 19, 29). This results in sodium current
inhibition or activation, depending on the protocol used (9, 21). In addition, cardiac sodium channels have been shown to be blocked directly by internal cAMP in neonatal rat heart, independently of PKA
activation (13), or by external cAMP through surface membrane receptors
in adult frog and mammalian myocytes (27). Compared with those of the
rBII and H1 channels, the ID I-II of the SkM1 channel is much shorter
and lacks the entire known consensus site for phosphorylation by PKA.
In spite of this, biochemical studies have shown that SkM1 is a good
substrate for phosphorylation by PKA both in vitro and in vivo and that
phosphorylation occurs at a single serine residue located in the ID
I-II (30, 33). Nevertheless, functional studies failed to find any
effect of cAMP on the biophysical properties of SkM1 channels when
expressed in oocytes (10, 24) or in a mammalian cell line (1).
Expression of channels in heterologous systems provides a convenient
and controlled model for studying the biophysical properties and the modulation of these channels. However, compared with the native tissue,
such a system may express a channel protein with different posttranslational modifications or different folding within the membrane and may lack physiological receptors and intracellular signaling pathways. Therefore, we decided to investigate the effect of
the membrane-permeable PKA activator 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) on native SkM1
channels in freshly dissociated fibers of rat fast-twitch skeletal
muscle by means of the cell-attached patch-clamp technique. The results shown in this study strongly suggest that cAMP blocks skeletal muscle
sodium channels by acting within the cell, independently of PKA activation.
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MATERIALS AND METHODS |
Cell preparation.
Fibers from flexor digitorum brevis muscles of the hind feet were
obtained from adult rats as previously described (7). Briefly, animals
were killed either by an overdose of urethan (intraperitoneal
injection) or by decapitation. Flexor digitorum brevis muscles were
promptly removed and placed in Ringer solution supplemented with
2.5 mg/ml collagenase (3.3 IU/ml, type XI-S, Sigma, St.
Louis, MO). They were shaken at 70 min
1 for 1-2 h at
32°C under a 95% O2-5%
CO2 atmosphere. During this incubation, dissociated cells were sampled, rinsed several times with
bath solution, and transferred into the RC-11 recording chamber (Warner
Instrument, Hamden, CT).
Cell-attached recordings.
Sodium currents were recorded at room temperature (21 ± 2°C) in
the cell-attached configuration of the patch-clamp method (12) with an
AxoPatch 1D amplifier and a CV-4-0.1/100U headstage (Axon Instruments,
Foster City, CA). Pipettes were formed from Corning 7052 glass (Garner
Glass, Claremont, CA) with a vertical puller (PP-82, Narishighe, Tokyo,
Japan). They were coated with Sylgard 184 (Dow Corning) and heat
polished on a microforge (MF-83, Narishighe, Tokyo, Japan). Pipettes
had resistances ranging from 2 to 4 M
when filled with the recording
pipette solution. Voltage-clamp protocols and data acquisition were
performed with pCLAMP 6.0 software (Axon Instruments) through a 12-bit
analog-to-digital/digital-to-analog interface (Digidata 1200, Axon
Instruments). Currents were low-pass filtered at 2 kHz (
3 dB) by
the amplifier four-pole Bessel filter and digitized at 10-20 kHz.
Membrane passive responses were controlled during the experiments as an
index of the goodness of the seal; those patches in which eventual
change might have modified sodium current characteristics were
discarded. Capacitance currents were almost totally canceled by the
compensation circuit of the amplifier. For representation of ensemble
average current traces, we further eliminated residual capacitance
transients and leak by subtraction of the scaled passive current
response recorded on return to the holding potential. In some patches,
we also used the P/4 subtraction procedure of Clampex (pCLAMP 6.0 package). We found no quantitative differences between the methods in
the effect of compounds on peak current amplitude or current kinetics.
After 10 min of incubation in the CsCl-enriched bath solution, the
membrane potential
(Vm) of the
fibers was steadily depolarized to
7.6 ± 1.0 mV
(n = 45 fibers), as measured by means
of single intracellular microelectrodes. The values of potential given
in the manuscript are not corrected from
Vm. Sodium
currents were elicited by depolarizing pulses from a holding potential
of
100 mV to
20 mV, applied at a frequency of 1 or 0.5 Hz. Recordings were initiated at least 5 min after the gigaseal
formation, when current amplitude reached a steady level. We calculated
the rate of current rundown during the first 5-10 min of recording
(before application of the compounds), and we discarded from our
analysis all patches that showed a rundown corresponding to a peak
current reduction of >15% in 20 min. For the study of both
activation and steady-state inactivation, a single voltage-clamp
protocol was used: the holding potential was
100 mV; a first
pulse 450 ms in duration was applied from
120 to +80 mV in 10-mV
increments and allowed the construction of the current-voltage
relationship; a second pulse of 30 ms in duration was applied at
20 mV and allowed the measurement of steady-state inactivation
in function of the first-pulse potential. This protocol was repeated
five times, and peak current amplitude values were averaged at each potential and reported as means ± SE. The activation curve was constructed from the current-voltage relationship by converting current
to conductance: the current amplitude was divided by the driving force
(V
VNa), where
V is the potential applied to the patch and VNa is
the equilibrium electrochemical potential for sodium ions, estimated to
be approximately +70 mV. Activation curves were fitted with the
Boltzmann equation
G/Gmax = 1/{1 + exp[(V
Vx)/K]},
where G is conductance,
Gmax is the
maximal conductance, K is the slope
factor, and Vx is
the potential at which one-half of the channels are activated. The
steady-state inactivation curves were fitted with the Boltzmann
equation
I/Imax = 1/{1 + exp[(V
Vh)/K]},
where I is current,
Imax is the
maximal current, K is the slope
factor, and Vh is
the potential at which one-half of the channels are inactivated.
Results are reported as means ± SE for
n patches. Student's
t-tests were performed for paired or
grouped data with Fig. P 6.0 software (Biosoft, Cambridge, UK).
Solutions and chemicals.
Ringer solution contained (in mM) 145 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 MOPS, and 5 glucose.
Bath solution contained (in mM) 145 CsCl, 5 EGTA, 1 MgCl2, 10 HEPES, and 5 glucose. Pipette solution contained (in mM) 150 NaCl, 1 MgCl2, 1 CaCl2, and 10 HEPES. All solutions
were buffered at pH 7.3. cAMP, CPT-cAMP, dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), and
(
)-isoproterenol hydrochloride were purchased from Sigma (Milan,
Italy). These compounds were dissolved in bath solution before addition
to the recording chamber at the final desired concentration. To
maintain the characteristics of the seal, compounds were applied away
from the patched cell. This procedure explains the delay needed to
observe initiation of effect and to reach the maximum effect. The PKA
inhibitor
N-[2-(p-bromocinnamylamino)-ethyl]-5-isoquinolinesulfonamide (H-89) (6), obtained from Calbiochem (La Jolla, CA), was first diluted
at 10 mM in DMSO and then diluted in Ringer or in bath solutions at the
final concentration.
 |
RESULTS |
The membrane-permeable cAMP analog CPT-cAMP decreases
macroscopic-current-like sodium currents.
Patches contained a large number of sodium channels, generally >20.
Thus depolarizations, applied from the holding potential of
100
mV to the potential of
20 mV, allowed recording of
macroscopic-current-like sodium currents with rapid onset and total
inactivation in <3 ms (Fig.
1A).
Bath application of 0.5 mM CPT-cAMP reduced current amplitude by
~75% (n = 13; Fig.
1A,
left and Table
1). The resulting scaled current generally
showed no change in current kinetics (Fig.
1A,
right), apart from a slight slowing
of inactivation observed in only two patches (not shown). The delay
between application of CPT-cAMP and initiation of the effect was 248 ± 30 s, and the maximum effect was reached in 987 ± 63 s
(n = 13; Fig.
1B). If no compound was applied,
sodium current amplitude remained quite stable during the same
interval, with only a light rundown corresponding to a peak current
reduction of ~10% in 30 min (Fig.
1B).

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Fig. 1.
Effect of 8-(4-chlorophenylthio)adenosine 3',5'-cyclic
monophosphate (CPT-cAMP) bath application on macroscopic-current-like
sodium currents in a cell-attached patch of rat skeletal muscle fiber.
A, left: ensemble average sodium
currents constructed from 50 consecutive traces elicited by
depolarizing patch membrane to 20 mV from holding potential of
100 mV, before (control) and after application of CPT-cAMP.
A, right: scaled current recorded
after effect of CPT-cAMP fairly superimposed control current.
B: time course of changes in peak
sodium current induced by CPT-cAMP. Sodium currents were elicited as in
A (same cell). Peak current amplitudes
were measured on each trace, averaged every 10 consecutive traces, and
reported as means ± SE as a function of recording time. Nucleotide
was added to bath solution at final concentration of 0.5 mM (box).
Dashed line, typical time course of change in peak sodium current
during same interval in absence of compound.
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Table 1.
Effect of nucleotides and isoproterenol on peak sodium current
amplitude in cell-attached patches of skeletal muscle fibers
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The control current-voltage relationship peaked between
40 and
10 mV (
25.3 ± 1.9 mV,
n = 19) and reached zero-current level near +60 mV (Fig.
2A).
CPT-cAMP reduced sodium current at all voltages and did not modify the
voltage at which current amplitude was maximal (Fig.
2A). The voltage dependence of the
activation curve was not modified by the nucleotide (Fig.
2B), suggesting that no change in
fiber Vm occurred
in response to the cyclic nucleotide.
Vx was
42.9 ± 2.2 mV (n = 10) in
control, and the maximal shift observed in response to CPT-cAMP was
2.8 mV (mean ± SE =
1.0 ± 0.8 mV,
n = 10). In contrast, the maximal
sodium conductance, measured on nonnormalized activation curves, was reduced by 59.7 ± 3.5%, 10 min (603 ± 26 s) after the
beginning of the CPT-cAMP effect.

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Fig. 2.
Effect of CPT-cAMP (0.5 mM) bath application on current-voltage
relationship and activation curve of sodium currents in a cell-attached
patch of rat skeletal muscle fiber. A:
current-voltage relationships were constructed before (control) and
during application of CPT-cAMP from protocol shown in
inset. Same cell as in Fig. 1.
B: activation curves were constructed
from current-voltage relationships by converting current to
conductance. Data were fitted to Boltzmann equation with following
parameters: half-activation potential
(Vx) = 40.7 mV and slope factor (K) = 5.4 mV in control;
Vx = 41.8
mV and K = 5.6 mV with CPT-cAMP. Same
cell as in A.
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Although cell-attached patches always contained numerous channels,
measurement of single-channel current amplitude was possible on late
single-sodium channel openings that occurred within the depolarization.
CPT-cAMP did not modify single-channel current amplitude, which
supports a lack of membrane potential change in the presence of the
nucleotide (Fig. 3). Single-channel
conductance was 19.6 ± 1.6 pS in control and 19.3 ± 1.1 pS in
the presence of CPT-cAMP (n = 3, P = 0.853, Student's
t-test for paired samples).

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Fig. 3.
Effect of CPT-cAMP bath application on single-sodium channel current
and conductance in cell-attached patches of rat skeletal muscle fibers.
Inset: single-channel currents
measured at 20 mV before and during application of 0.5 mM
CPT-cAMP. Sodium channel current amplitudes measured on square-shaped
single-channel events, as illustrated in
inset, are reported as a function of
command pulse potential. Data are means ± SE of current amplitudes
measured on 3 patches before ( ) and after ( ) application of
CPT-cAMP. They are well fitted by linear correlation
(P < 0.001 for both), the slope of
which gives values of single-channel conductance as 19.6 and 19.3 pS
before and after application of nucleotide, respectively.
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cAMP acts within the cell independently of PKA activation.
To test whether the action of CPT-cAMP was within the cell or resulted
from activation (specific or nonspecific) of a sarcolemmal receptor, we
applied cAMP, which does not easily permeate the cell membrane, and
recorded sodium currents within a period of at least 1,200 s. Such a
duration is greater than the mean time needed to observe the maximal
effect of the membrane-permeable analog CPT-cAMP. Only a slight
reduction of sodium current was measured in the external presence of
0.5 mM cAMP, which ranged from
4.1 to
18.0% of control
current (Fig. 4 and Table 1). In contrast,
0.5 mM DBcAMP, another membrane-permeable derivative of cAMP, reduced
peak sodium current in the same extent as CPT-cAMP (Fig. 4 and Table
1).

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Fig. 4.
Effects of nucleotides and isoproterenol bath applications on sodium
currents in cell-attached patches of rat skeletal muscle fibers.
Ensemble average sodium currents were constructed from 50 consecutive
traces elicited as in Fig. 1A,
before (control) and after application of compounds. Representative
examples are shown for 0.5 mM dibutyryl adenosine
3',5'-cyclic monophosphate (DBcAMP;
A), 0.5 mM cAMP
(B), 0.5 mM CPT-cAMP in presence of
10 µM H-89 (C), 0.1 mM
isoproterenol (ISO; D), and 0.5 mM
isoproterenol (E). Averaged values
of peak current reduction are reported in Table 1.
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To further evaluate whether the effect of CPT-cAMP on sodium currents
was dependent on activation of PKA, the fibers were incubated with 10 µM H-89 for at least 45 min before application of 0.5 mM CPT-cAMP. In
three cells from three independent experiments, H-89 by itself had no
effect on sodium currents. Despite the presence of the PKA inhibitor,
CPT-cAMP still reduced peak current (Fig. 4 and Table 1) with kinetics
(maximal effect in 907 ± 83 s) similar to that observed in the
absence of H-89.
The
-adrenoreceptor agonist isoproterenol was also tested because of
its ability to increase intracellular cAMP levels in a physiological
manner (Fig. 4). Reduction of sodium currents was observed in response
to application of 0.5 mM isoproterenol, ranging from 17.8 to 79.3% of
control current (Table 1). In contrast, 0.1 mM isoproterenol had no
significant effect on sodium current, the reduction of which was
limited to ~10% within 1,200 s of application (Table 1).
CPT-cAMP modifies the voltage dependence of sodium current
steady-state inactivation.
Many sodium channel blockers exert their action by shifting the channel
inactivation toward more negative potentials. Therefore, we examined
the effect of CPT-cAMP on the steady-state inactivation curves (Fig.
5). In control conditions, the different
patches showed steady-state inactivation curves with similar slopes
(mean slope factor ± SE = 5.6 ± 0.1 mV,
n = 19). However, half-inactivation potentials (Vh)
were quite variable, ranging from
63.7 to
98.1 mV (mean ± SE =
81.9 ± 2.6 mV,
n = 19). After application of 0.5 mM
CPT-cAMP, the slope factor was not significantly modified (mean ± SE = 5.8 ± 0.2 mV, n = 10, P = 0.272 with paired control data),
whereas Vh was
shifted toward more negative potentials by 8.5 ± 1.8 mV
(n = 10). However, this shift was more
or less pronounced, ranging from
2.0 to
18.6 mV (Fig.
5), depending on the initial
control value (see below). The
Imax was reduced by 51.7 ± 3.0% (n = 10), but, in
contrast to the
Vh shift, this effect developed similarly in all patches.

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Fig. 5.
Effect of CPT-cAMP (0.5 mM) bath application on steady-state
inactivation curve of sodium currents in cell-attached patches of rat
skeletal muscle fibers. Steady-state inactivation curves were
constructed from currents elicited by protocol shown in
inset. Two examples are illustrated,
which showed minimal ( , ) and maximal ( , ) shift in
half-inactivation potential
(Vh) between
control conditions ( , ) and CPT-cAMP effect ( , ). Data were
fitted to Boltzmann equation with following parameters: ,
Vh = 64.9
mV and K = 6.4 mV; ,
Vh = 83.5
mV and K = 7.0 mV; ,
Vh = 98.1
mV and K = 6.1 mV; ,
Vh = 100.1
mV and K = 6.0 mV.
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Fig. 6.
Effects of cAMP and CPT-cAMP on voltage dependence of sodium current
steady-state inactivation in cell-attached patches of rat skeletal
muscle fibers. Shifts of
Vh are reported
as a function of value of
Vh measured in
same cell before application of nucleotide. Data points obtained with
CPT-cAMP ( ) are linearly correlated
(r = 0.79,
P < 0.01). In contrast, shift of
Vh observed with
cAMP ( ) is not correlated to control value of
Vh
(r = 0.30,
P = 0.43).
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A spontaneous negative shift of the steady-state inactivation of sodium
currents has been reported to occur during patch-clamp recording (14,
18, 21). To test such a possibility, we compared the shifts of
Vh in response to
the active CPT-cAMP and to the inactive cAMP. Because of the
variability in the control
Vh observed between patches, we reported the shifts in
Vh as a function
of the control values of
Vh (Fig. 6). The
data points obtained for CPT-cAMP were correlated by a linear
regression (r =
0.79,
P < 0.01, n = 10).Therefore the
Vh shift obtained
in response to the membrane-permeable cAMP derivative was more
pronounced for the less negative control values of
Vh. In contrast,
the linear regression of the data points obtained in the experiments
performed with cAMP did not give a satisfactory correlation
(r =
0.30, P = 0.43, n = 9). In these conditions, it
appears that Vh
shift in the presence of cAMP was independent of the control value of Vh. This shift,
which reached a mean value of
3.9 ± 0.3 mV in 1,255 ± 103 s (n = 9), was significantly lower
than that measured in response to CPT-cAMP
(P < 0.05) and likely corresponds to
a spontaneous shift of steady-state inactivation, as has been reported in other studies (14, 18, 21).
 |
DISCUSSION |
The phosphorylation of voltage-gated sodium channels by PKA has been
demonstrated biochemically for the major sodium channel isoforms of
brain (rBII) (20), heart (H1) (19), and skeletal muscle (SkM1) (30,
33). When sodium channels are expressed in heterologous systems,
PKA-mediated phosphorylation results in functional inhibition of rBII
channels (11, 17, 25, 26) and activation of H1 channels (10) but lacks
effect on SkM1 channels (1, 10, 24, 25). Accordingly, native sodium channels have been shown to be modulated by activation of the cAMP-PKA
pathway in neurons (4, 22) and cardiac myocytes (18, 21, 29). In
contrast, nothing is known about a possible effect of this
intracellular pathway on native skeletal muscle sodium channels.
We recorded macroscopic-current-like sodium currents in cell-attached
patches of freshly dissociated skeletal muscle fibers. These currents
peaked between
40 and
10 mV and reversed near +60 mV.
They showed a slight rundown, limited to 10-15% of peak current
reduction in 30 min. During the same interval, activation curves
remained rigorously unchanged, while steady-state inactivation curves
slightly shifted by ~4 mV toward more negative potentials. Moreover,
with 100 nM tetrodotoxin in the pipette, these currents were reduced by
>80%, confirming that they transit through the adult isoform of
skeletal muscle sodium channels (7). Thus our experimental conditions
allow study of acute modulation of native sodium channels in adult rat
skeletal muscle fibers.
In these conditions, application of the cell membrane-permeable
derivatives of cAMP, CPT-cAMP and DBcAMP, strongly reduced sodium
currents, with no change in activation and inactivation rates. This
effect resulted principally from the reduction of the
Imax, in other
words from the reduction of the open channel probability. Furthermore,
the cyclic nucleotides shifted the steady-state inactivation curve
toward more negative potentials in a manner dependent on the initial
condition (see below). Both effects were likely independent, since we
did not find any correlation between the percentage of reduction of
Imax and the
negative shift of Vh. In contrast,
single-channel conductance, single-channel current amplitude, and
voltage dependence of activation curves were not modified, therefore
indicating that the cAMP analogs did not affect the
Vm. The effect of
cAMP derivatives on the skeletal muscle sodium channels differs from
those observed on brain and cardiac sodium channels. In fact, brain
sodium currents are reduced through the cAMP pathway with no change in
kinetics and no effect on the voltage dependence of steady-state
inactivation (4, 11, 17, 22). The situation is less clear for the
cardiac sodium channel, for which some investigators described sodium
current inhibition (18, 29) whereas others reported sodium current
enhancement (10, 23). Discrepancies also appeared regarding the
steady-state inactivation of cardiac sodium currents with no change of
voltage dependence (23) or negative shift of
Vh (21) in
response to the cAMP pathway activation. In the present study, we also
found some variability in the effect of CPT-cAMP on the voltage
dependence of the steady-state inactivation of skeletal muscle sodium
channels. Considering the spontaneous shift observed in control
conditions, the shift observed in the presence of CPT-cAMP appeared to
be significant only in those patches for which the control inactivation curve was initially less negative (Fig. 6). This phenomenon may reflect
fiber-specific differences in the initial state of the channels. For
example, higher basal levels of cAMP in some fibers may have already
shifted the voltage dependence of sodium current inactivation curves
toward the left and, in consequence, may have prevented a further
effect of exogenously applied nucleotides on this parameter. In this
case, the amplitude of control sodium currents should have been minor
in the fibers that showed less negative sodium current inactivation
curves. We failed to find such a correlation by comparing the control
values of Imax
and Vh. However,
because sodium channels are heterogeneously distributed on the
sarcolemma, the control
Imax value
calculated in a small sample of cell-attached patches
(n = 10) may not really reflect sodium
current density. Thus different cell-specific basal states of the
sodium channel might explain the variability in the cAMP effect on the
voltage dependence of channel inactivation observed in the present study.
To our knowledge, this report is the first one that describes an effect
of cAMP on the adult skeletal muscle sodium channel. In fact, previous
studies reported no effect of isoproterenol (1, 24, 25), external
DBcAMP (24), external 8-bromoadenosine 3',5'-cyclic
monophosphate (8-BrcAMP) (1), or cAMP intracellular injection (10) on
heterologously expressed SkM1 channels. These results are somewhat
surprising, since SkM1 channels are good substrate for phosphorylation
by PKA, both in vivo and in vitro (30, 33). We hypothesized that the
lack of cAMP-PKA effect on SkM1 channels may result from some
limitation of the heterologous expression of these channels. However,
the effects of cAMP on native SkM1 channels described in the present
study are likely to be independent of PKA activation, and thus of
phosphorylation of the channels, because incubation of muscle fibers
with the highly specific PKA inhibitor H-89 (6) did not prevent the inhibitory effect of cAMP analogs. Therefore, this study agrees with
the previous reports in that phosphorylation of SkM1 channels by PKA
may have no functional effect on these channels. On the other hand, a
difference remains in that cAMP inhibits sodium channels in the present
study, whereas it was without effect in previous studies. The
difference did not reside in the cAMP concentration used, since
Bendahhou and colleagues (1) used 8-BrcAMP at concentrations as high as
2 mM. One explanation may be the lack of one intermediate factor
between cAMP and the channels in the heterologous expression system.
Because cAMP itself, which has low membrane permeability, had no effect
on skeletal muscle sodium currents when externally applied, we excluded
an effect of the nucleotide on possible surface receptors of the
sarcolemma, as has been reported in cardiac myocytes (27). Therefore,
if such an intermediate factor exists, it must be intracellular. One
other possibility is that the heterologously expressed SkM1 channel
does not perfectly match the native SkM1 channel because of different
posttranslational maturation and/or different folding within
the membrane. In this case, SkM1 channels may also be sensitive to
direct action of cAMP in a manner specific to their expression in
native tissue. Direct action of cyclic nucleotides has been described
already for different voltage-gated channels (8, 13, 28). Among them,
the cardiac sodium channel has been shown to be blocked by cAMP and
other 6-aminopurines from the cytoplasmic side in inside-out excised
patches of neonatal rat heart myocytes (13). This block was observed in
the absence of a phosphate donor, therefore indicating a
PKA-independent effect of the cyclic nucleotide, and resembled in part
the blocking effect of local anesthetics, leading the authors to
hypothesize a channel-associated binding site accessible from the
cytoplasmic side (13).
It is difficult at the moment to imagine the physiological impact of
SkM1 channel modulation by cAMP. Our results suggest that only high
concentrations of the
-adrenoceptor agonist isoproterenol may
decrease sodium currents in rat skeletal muscle fibers. Therefore, epinephrine is likely unable to reduce sodium channel activity in adult
rat fast-twitch muscle, in agreement with the classical description of
this hormone as a positive inotropic agent in skeletal muscle (31). On
the other hand, the reduction of sodium current by cAMP might take on
importance in the case of the highest myoplasmic levels of the cyclic
nucleotide, as in slow-twitch muscles compared with fast-twitch muscles
(32), in unweighted slow-twitch muscles (15), in aged skeletal muscle
(16), or during endurance training (2, 32). Finally, because the
therapeutic interest of sodium current modulation generally resides in
sodium channel block, the description of new blocking agents acting
through cAMP might open new perspectives in pharmacology.
In conclusion, this study strongly suggests that the native skeletal
muscle sodium channels are blocked by internal cAMP independently of
PKA activation. This is in agreement with previous studies, which
showed that PKA-dependent phosphorylation of these channels has no
functional consequence for their biophysical properties. In contrast,
this is the first description of the inhibitory effect of cAMP on these channels.
 |
ACKNOWLEDGEMENTS |
This research was supported by the Italian Telethon (Project 901).
Association Française contre les Myopathies provided support for
J.-F. Desaphy.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: D. Conte Camerino, Dipartimento
Farmaco-Biologico, Via Orabona 4, I-70125 Bari, Italy.
Received 22 June 1998; accepted in final form 24 August 1998.
 |
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