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

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 beta -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

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 MOmega 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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

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.

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 (open circle ) and after (bullet ) 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.

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.

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 beta -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 (open circle , bullet ) and maximal (, ) shift in half-inactivation potential (Vh) between control conditions (bullet , ) and CPT-cAMP effect (open circle , ). 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; open circle , Vh = -98.1 mV and K = 6.1 mV; bullet , 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 (bullet ) are linearly correlated (r = -0.79, P < 0.01). In contrast, shift of Vh observed with cAMP (open circle ) is not correlated to control value of Vh (r = -0.30, P = 0.43).

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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 beta -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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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