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Role of Chloride Conductance
Correspondence to Thomas Holm Pedersen: thp{at}fi.au.dk
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: lactic acid • muscle fatigue • action potentials • Na+ channels • Cl– channels
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Moreover, myotonia congenita is often treated with Na+ channel blockers (Lehmann-Horn and Jurkat-Rott, 1999), again indicating that the initiation of action potentials in skeletal muscles depend on the interplay between excitatory Na+ currents and shunting Cl– currents.
During exercise, active muscle lose K+ and extracellular K+ ([K+]o) has been reported to increase from around 4 to 10 mM in plasma, and even higher levels of [K+]o have been reported in the interstitium (Sréter, 1962; Hnik et al., 1976; Hermansen et al., 1984; Hallen et al., 1994; Green et al., 2000; Juel et al., 2000; Sejersted and Sjøgaard, 2000). Exposure of isolated rat muscles to elevated [K+]o corresponding to the levels measured in vivo starts a chain of events that primarily affects the excitatory Na+ currents. At elevated [K+]o, fibers become depolarized, Na+ channels become slow inactivated (Ruff, 1996), the amplitude of single action potentials becomes reduced (Rich and Pinter, 2001), and the amplitude and integrated area of compound action potentials (M-waves) decrease. Based on such findings, elevated [K+]o leading to reduced muscle excitability through loss of excitatory Na+ currents is believed to be a component in muscle fatigue in working muscle (Sejersted and Sjøgaard, 2000).
Intensive exercise, however, is usually associated with an intracellular accumulation of lactic acid and consequently a drop in muscle pH. Interestingly, Lehmann-Horn et al. (1987) have shown that the contractile force of isolated fiber bundles from intercostal muscles of a patient suffering from hyperkalaemic periodic paralysis show partial recovery from depression caused by increased [K+]o when exposed to lowered pH. Likewise, in studies on isolated rat muscles depressed by increased [K+]o, it was found that within the physiological range, reductions in muscle pH produce a substantial recovery of excitability and force (Nielsen et al., 2001). Later, a study on mechanically skinned fibers of the rat showed a similar enhancement of t-system excitability in depolarized fibers with intracellular acidosis (Pedersen et al., 2004). Importantly, this latter study showed that the effect of acidosis was caused by a down-regulation of t-tubular Cl– permeability. These findings suggest that the effect of pH on excitability of skeletal muscle is related to a change in GCl–. This notion tallies with previous studies on both amphibian and mammalian skeletal muscle tissue, which indicate that reduced muscle pH can lower GCl– (Hutter and Warner, 1967; Palade and Barchi, 1977). Based on these observations and the importance of GCl– in action potential generation and propagation, we hypothesize that the increase in excitability in K+-depressed muscles induced by muscle acidification is caused by a reduction in GCl–. To evaluate this hypothesis, the aim of the present study was to examine the effects of muscle pH on GCl– and, further, to examine whether the observed changes in GCl– can explain the recovery of excitability and force observed when pH is lowered in muscles depressed by elevated [K+]o (Nielsen et al., 2001). Part of the results has been presented in a preliminary form (Pedersen, 2004).
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MATERIALS AND METHODS |
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10 mm of intact nerve attached. All handling of animals was in accordance with Danish Animal welfare regulations.
Muscles were incubated in standard Krebs-Ringer bicarbonate buffer containing (in mM) 122 NaCl, 25 NaHCO3, 2.8 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.3 CaCl2, and 5.0 D-glucose. Total buffer [Cl–] was 127.4 mM. In Cl–-free buffer, methanesulphonate salts replaced NaCl and KCl, and Ca(NO3)2 replaced CaCl2. In buffers with reduced [Cl–], NaCl was partly replaced by sodium methanesulphonate. In buffers with high K+, KCl replaced part of the NaCl whereby [Cl–], osmolarity, and ionic strength were kept constant. The pH of all standard buffers equilibrated with 5% CO2 in O2 was 7.4 (referred to as normal pH). To acidify muscles, buffers were equilibrated with 24% CO2 in O2 (low pH). In a previous study, we showed that when CO2 was increased to 23%, the pH of the standard Krebs-Ringer buffer decreased by 0.56 units and produced a maintained reduction in the intracellular pH of the muscles of 0.39 units (Nielsen et al., 2001). All experiments were carried at 30°C.
Isometric Force and Compound Action Potentials (M-waves)
Muscles were stimulated to contract either by field stimulation or by stimulating the nerve using a suction electrode. In experiments using field stimulation, muscles were mounted on force transducers at optimal length and stimulated by applying an electrical field across the central part of the muscle through platinum electrodes. Tetani were elicited every 10 min using 2-s trains of 0.2-ms pulses at 30 Hz. An electrical field of 24 V/cm was used, which was supramaximal stimulation for muscles incubated in standard Krebs-Ringer buffer at both pH 7.4 and at 6.8. In some experiments with 4-wk-old animals, force and M-waves were recorded simultaneously. In these experiments, tetanic contractions were evoked via the nerve using 5 mA constant current pulses with 0.15-ms duration delivered at 30 Hz. M-waves were recorded from a circular silver electrode with a recording area of 0.79 mm2 (Overgaard and Nielsen, 2001).
Intact muscles were used in experiments with 4-wk-old animals. To minimize problems with diffusion of oxygen and other compounds between the core of the muscles and the buffer, muscles from adult animals used for experiments involving contractions were split in two equal halves from tendon to tendon with only one of them being used for experiments. In pilot experiments, intact muscles from adult animals showed the same contractile properties and the same loss of force at elevated [K+]o as did the split muscles but the time to reach a steady force at elevated [K+]o was considerably longer for the whole muscles.
Recordings of Cable Parameters and Single Action Potentials
Recordings of cable parameters and action potentials were done using a two-electrode constant current technique. Both electrodes were connected to an Axoclamp-2a amplifier. One electrode was used for passing current into the cells (current electrode), the other for measuring the intracellular potential (voltage electrode). Resistances of microelectrodes were between 5 and 20 M
. The current electrode was filled with 2 M K-citrate and the voltage electrode was filled with 3 M KCl. The membrane potential recorded by the microelectrodes and the current pulses injected in the fiber were displayed on an oscilloscope and recorded by a computer. Pilot experiments showed that when two electrodes were placed in the same fiber in muscles from 4-wk-old animals, the fiber depolarized rapidly, and the use of large holding currents would be necessary for maintaining a constant resting membrane potential. To avoid this problem, all experiments in which fibers were penetrated with two electrodes were done on muscles from adult animals.
The cable parameters were measured by injecting hyperpolarizing constant current pulses of 75 ms duration. The steady displacement of the membrane potential (
Vm) during a constant current pulse (I) was recorded at three to five locations in each fiber. For each fiber investigated, the three to five Vm/I ratios were plotted on a log scale against the inter-electrode distance (x) on a linear scale and fitted to a two parameter exponentially decaying function (y(x) = y0e–bx), giving a straight line (see Fig. 2). From fibers that showed an accurate fit (r2 
0.99), the ordinate intercept (y0) was taken as the input resistance (Rin), and the length constant (
) was calculated from the slope of the fitted line (b = 1/). Assuming an internal resistivity (Ri) of 180 cm (Albuquerque and Thesleff, 1968), Rin and were used to calculate the membrane conductance in accordance with the methods of Boyd and Martin (1959) based on the theory derived by Hodgkin and Rushton (1946). The values of the longitudinal resistance of the fiber (ri) and the membrane resistance (rm) were calculated as ri = 2Rin/ and rm = 2Rin. Fiber diameter (d) was calculated as d = (4Ri/(
ri))1/2 and specific membrane resistance (Rm) as Rm = drm. Membrane conductance (Gm) was then calculated as Gm = 1/Rm. The component conductance of K+ (GK+) was taken as the membrane conductance in Cl–-free buffer, disregarding the contribution of other ion conductances. GCl– was calculated by subtracting GK+ from Gm in Cl–-containing buffer. All fibers in which the resting potential depolarized more than 7 mV, while the two electrodes were inserted in the fiber, were disregarded. Likewise, fibers with a resting membrane potential more depolarized than –60 mV at a [K+]o of 4 mM or more than –50 mV at a [K+]o of 11 mM were disregarded.
For the measurement of action potentials, the current and the recording electrode were placed 0.3 mm apart in the same fiber, and depolarizing constant current pulses were injected through the current electrode while the response of the membrane was recorded by the voltage electrode. Pilot experiments showed that in muscles at 4 mM K+, action potentials always occurred within the first 25 ms of the current pulses, so to obtain the highest possible sampling frequency for data acquisition (81 KHz) the pulse duration was set to 25 ms. To determine the rheobase current in muscles at 4 mM K+, single depolarizing current pulses with increasing strength (steps of 5 nA starting at 10 nA) were injected until an action potential was elicited. If fibers did not produce an action potential in response to a 100 nA current, they were considered to be unexcitable. In the excitable fibers, the rate of rise of the action potential was calculated from the slope of the action potential upstroke between 10 and 90% of the action potential peak.
Intracellular Water Content
For determination of intracellular water content, muscles from adult rats at 11 mM K+ were equilibrated for 100 min in buffer containing 14C-sucrose (0.1 µCi/ml and 1 mM nonlabeled sucrose). After incubation, the tendons were cut off and the muscles were blotted, weighed for determination of wet weight, and subsequently dried overnight at 60°C. After determination of dry weight, the muscles were soaked for 20 h in 0.12 M trichloroacetic acid (TCA), and the content of 14C-sucrose in the supernatant was determined by scintillation counting. Intracellular water content, expressed per gram dry weight, was calculated from the total water content by subtracting the distribution volume of 14C-sucrose.
Chemicals and Isotope
All chemicals were of analytical grade. 9-Anthracene-carboxylic acid (9-AC) and methanesulphonate salts were from Sigma-Aldrich and 14C-sucrose was from Amersham Biosciences.
Statistics
All data are expressed as means ± SEM. The statistical significance of any difference between groups was ascertained using Student's two-tailed t test for nonpaired observations between two groups and one-way Anova between more than two groups.
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RESULTS |
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Vm/I ratios were calculated and related to the inter-electrode distance as illustrated in Fig. 2 B, which shows measurements from single fibers from each of the four groups of muscles reflecting each group average. In Cl–-containing buffer, low muscle pH led to larger Vm/I ratios than normal pH, indicating that the membrane conductance had been reduced by acidification. In marked contrast, fibers in Cl–-free buffer appeared unaffected by acidification, indicating that the effect of pH on membrane conductance only was related to a change in GCl–. To investigate the effect of acidosis on the component conductance of K+ and Cl– in more qualitative terms, , Rin, and Gm were determined in each of the four groups in accordance with the methods of Boyd and Martin (1959). Table I shows the calculated values of , Rin, and Gm for fibers at 4 mM K+ (normal pH with Cl–) and from the four groups of fibers at 11 mM K+. When pH was reduced from 7.4 to 6.8 in muscles incubated in Cl–-containing buffer at 11 mM K+, increased significantly by 18% (P < 0.01) and Rin by 40% (P < 0.01). Consequently, muscles incubated in Cl–-containing buffer showed a reduction in membrane conductance from 2136 ± 150 µS/cm2 at pH 7.4 to 1393 ± 56 µS/cm2 at pH 6.8 (P < 0.01). In muscles incubated in Cl–-free buffer, there were no significant changes in or Rin with acidification, which was also reflected by a small and insignificant change in GK+ from 405 ± 20 µS/cm2 at normal pH to 455 ± 30 µS/cm2 at low pH (P < 0.16). Based on these results, acidification from 7.4 to 6.8 by increasing CO2 from 5 to 24% caused a reduction of GCl– from 1731 ± 151 µS/cm2 at normal pH to 938 ± 64 µS/cm2 at low pH corresponding to a 46% decrease (P < 0.01).
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). Neither incubation in Cl–-containing buffer at pH 6.8 or in Cl–-free buffer at pH 7.4 or 6.8 caused any change in the intracellular water content of the muscles, the contents being 2.78 ± 0.02, 2.72 ± 0.01, and 2.79 ± 0.02 g H2O/g dry weight, respectively (P < 0.46, n = 3 in all groups).
Reduction in GCl– Recovers Excitability and Force Production in Muscles at Elevated [K+]o
To test if the improved excitability and force production that was induced by increasing CO2 to 24% in muscles from 4-wk-old animals at elevated [K+]o (Fig. 1) could be explained by a reduction in GCl–, as observed in muscles from adult animals (Table I), we examined the effect of reducing GCl– in muscles from young rats by lowering [Cl–]o to 80 mM while keeping pH at 7.4. Measurements of membrane conductance showed that lowering [Cl–] in the buffer from the normal 127.4 mM to 80 mM reduced GCl– of muscles from adult rats at 11 mM K+ to 761 ± 102 µS/cm2 (n = 15), which was similar to the reduction seen when these muscles were acidified with 24% CO2 (Table I, P < 0.1). Fig. 3 A shows that in addition to the reduction in GCl–, the lowering of [Cl–]o to 80 mM led to a recovery of M-wave area and force production of similar magnitude as the recoveries induced by increasing CO2 to 24%. In accordance with the data shown in Table I, the reduction in [Cl–]o had no effect on the membrane potential, the potentials being –55 ± 1 mV at both 127.4 mM Cl– and 80 mM Cl–. Fig. 3 A further shows that recovery of excitability and force in muscles at 11 mM K+ also could be induced by addition of 100 µM of the specific muscle Cl– channel (ClC-1) inhibitor 9-AC.
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), led to a recovery of force that was similar to the recovery of force induced by increasing CO2 to 24%. Other experiments showed that similar results were obtained in muscles pretreated with 10–5 tubocurarine (unpublished data).
Acidosis Decreases Rheobase Current and Increases the Number of Excitable Fibers in Muscles at Elevated [K+]o
The recovery of M-waves induced by acidification in muscles at elevated [K+]o (Figs. 1 and 3) strongly suggests that acidification improves muscle excitability. To further investigate the effects of acidification on the excitability of muscles at normal and elevated [K+]o, intracellular recordings of action potentials were done in muscles at normal pH (5% CO2) and in acidified muscles (24% CO2). In these experiments, action potentials were elicited in single fibers by injecting depolarizing constant current pulses of 25 ms duration and recording the membrane response by another intracellular electrode. Since this method required the penetration of fibers with two electrodes, the experiments were done on muscles from adult rats. Table II shows that in muscles at 4 mM K+, acidification produced a significant reduction in the rheobase current of 26%, corresponding to a 15 nA decrease. When muscles were examined at 11 mM K+ (as used in Table I and Figs. 1 and 3), the examination of rheobase current and action potentials was encumbered by a complete loss of excitability in most of the fibers, as judged from their inability to respond to a 100 nA current injection with an action potential. For that reason, [K+]o was only increased to 9 mM, and all fibers were excited by injection of a single 100-nA depolarizing constant current pulse. Fig. 4 A shows that when the muscles were incubated at normal pH, this procedure only elicited action potentials in 55% of the examined fibers (n = 40). In muscles at low pH, however, the same stimulation elicited an action potential in 95% of the tested fibers (n = 40). This indicates that upon acidification, an additional 40% of the fibers responded with an action potential to the 100-nA stimulus. Examining the action potentials of those fibers that were excitable under the different conditions showed that acidification also had effects on action potential shape. Fig. 4 shows representative action potentials from fibers at 4 mM K+ and normal pH (Fig. 4 B), at 9 mM K+ and normal pH (Fig. 4 C), and at 9 mM K+ and low pH (Fig. 4 D). Clearly, elevated [K+]o caused a depression of the action potential, which was partly recovered by acidification. Table II shows that the potentiation of the action potentials by low pH was present both in muscles at 4 and 9 mM K+, producing a 25 and 43% increase in the rate of rise of the action potential and a 8 and 7 mV increase in the action potential peak in fibers at 4 and 9 mM K+, respectively.
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) clearly shows that muscles from adult animals have less tolerance to elevated [K+]o than muscles from 4-wk-old animals. Thus, at normal pH, the force production of muscles from 4-wk-old animals incubated at 11 mM K+ roughly corresponded to the force production of muscles from adult rats incubated at 9 mM K+.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The force-depressing effect of elevated [K+]o is most likely related to the depolarization of the muscle fibers, which has been shown to cause a loss of Na+ current due to slow inactivation of Na+ channels (Ruff, 1996), leading to a loss of the ability of the fibers to initiate and propagate action potentials (Cairns et al., 1995, 1997; Overgaard et al., 1999; Sejersted and Sjøgaard, 2000; Rich and Pinter, 2003). This reduction in excitability can be seen in Fig. 1, which in agreement with other studies (Overgaard et al., 1999; Overgaard and Nielsen, 2001) shows that the reduction in force at 11 mM K+ was well correlated with a concomitant reduction in M-wave area. Interestingly, at 11 mM K+, the membrane potential of muscles from adult animals was the same as in muscles from 4-wk-old animals. Thus, the reduced tolerance to elevated [K+]o in muscles from adult rats could not be related to a larger effect of increased [K+]o on the membrane potential but must result from a reduced tolerance to depolarization per se.
During intensive exercise, the accumulation of extracellular K+ occurs simultaneously with an accumulation of lactic acid and ensuing reduction in muscle pH (Fitts, 1994). Thus, to determine the effect of high [K+]o on muscle function under circumstances that approach the conditions during exercise, muscles were acidified by elevated CO2 pressure, which acidifies both the intra- and extracellular compartment of the muscles (Nielsen et al., 2001). Similar to previously reported effects of lactic acid (Nielsen et al., 2001), lowering pH by increasing the CO2 tension clearly increased the tolerance of the muscles to elevated [K+]o. This effect was present in muscles from both 4-wk-old (Fig. 1) and adult animals (Fig. 5).
Judged from the increase in M-wave area (Fig. 1), the recovery of force induced by low pH was related to an increase in the number of fibers producing an action potential in response to electrical stimulation of the preparation. Since similar increase in the tolerance to elevated [K+]o was found in currarized muscles activated by field stimulation, the mechanism for the protective effect of acidosis had to reside distally to the neuromuscular junction. Together this indicates that the increase in force production in muscle at elevated [K+]o upon acidification is caused by an increase in the ability of the muscle fibers to initiate and propagate action potentials. This conclusion is supported by the finding that in muscles from adult rats that were incubated at 9 mM K+, the relative force (Fig. 5) and the proportion of fibers that produced an action potential in response to injection of a 100-nA current (Fig. 4) were closely related before and after acidification. Interestingly, in muscles from adult animals incubated at 4 mM K+, acidification reduced the rheobase current and caused a greater rate of rise and higher peak of the action potentials (Table II), indicating that acidification also causes an increase in the excitability of muscles incubated at normal [K+]o.
Effect of Acidosis on Membrane Conductance
Table I shows that within the physiological range, a reduction in pH, obtained by raising CO2 from 5 to 24%, in muscles at 11 mM K+ led to a large reduction in Gm. Since GCl– is known to account for the largest fraction of the total membrane conductance in resting muscle fibers (Bretag, 1987), the reduction in Gm at low pH was most likely related to a reduced GCl–. Indeed, low pH was without effect on Gm in muscles incubated in Cl–-free buffer, showing that the effect of low pH on Gm was the result of a 46% reduction in GCl–. This effect of pH is in accordance with other reports on the influence of acidosis on GCl– in native tissue (Hutter and Warner, 1967; Palade and Barchi, 1977). The finding that low pH was without effect on Gm in muscles incubated in Cl–-free buffer also indicates that reduced pH did not change GK+ significantly. This conclusion is further supported by the finding that the reduction in pH had no effect on Vm. In muscles at 11 mM K+ and normal pH, GCl– accounted for around 81% of the total membrane conductance, which agrees convincingly with previous investigations conducted at 5 mM K+ under otherwise similar conditions (Albuquerque and Thesleff, 1968; McArdle and Albuquerque, 1973). The large increase in total membrane conductance at 11 mM K+ compared with 4 mM K+ roughly agrees with the constant field theory (Hodgkin and Katz, 1949) and with the results of Kwiecinski et al. (1984).
The Role of Reduced GCl– and ClC-1 Channels in the Protective Effect of Acidosis
The finding that acidification of muscles at 11 mM K+ caused a reduction in GCl– (Table I) suggests that the concomitant improvement in the ability of the muscle fibers to respond to electrical stimulation can be explained by a reduction in Cl– currents, which changes the balance between excitatory Na+ currents and the shunting Cl– currents in favor of the excitatory currents. This role for GCl– is supported by the observation that in K+-depressed muscles at normal pH, substantial recovery of excitability and force could be induced by reducing buffer Cl– or by adding 9-AC. Importantly, in these experiments, large recoveries were seen even when 80 mM Cl– or 30 µM 9-AC were used to obtain graded reductions in GCl–, thereby mimicking the reduction in GCl– measured in acidified muscles. These results indicate that a reduction in GCl– plays a major role in the recovery of muscle function induced by acidification at elevated [K+]o. At variance with this, Cairns et al. (2004) recently reported a synergistic effect of increasing [K+]o and decreasing [Cl–]o on loss of force in soleus and extensor digitorum longus muscles from mice. This loss of force was probably due to the depolarization they observed when [Cl–]o was lowered at elevated [K+]o, perhaps indicating some active transport mechanism of Cl– at elevated [K+]o not seen in our results or in human skeletal muscle where it has been shown that Cl– is passively distributed over a range of [K+]o from 1 to 7 mM (Kwiecinski et al., 1984). In accordance with our results, van Emst et al. (2004) recently reported a recovery of twitch force in K+-depressed rat soleus muscles upon reduction of extracellular Cl–.
It should be noted that despite the tendency for GCl– to be lower in muscles at 80 mM Cl– and normal pH compared with acidified muscles (761 ± 102 vs. 938 ± 64 µS/cm2), the recovery of excitability and force induced by 80 mM Cl– was actually lover than the recovery induced by acidification (Fig. 3). One possible reason for this discrepancy could be that the cable parameters were measured in muscles from adult rats, whereas measurements of force and M-waves were done in muscles from young rats. Another possible explanation for the discrepancy could be that acidification, in contrast to reduced extracellular Cl–, leads to an increased screening of fixed negative charges on the outer membrane surface, effectively making the surface potential of the membrane more positive and consequently elevating the electric field within the membrane. According to Woodhull (1973), such a screening effect of extracellular H+ ions could shift the gating of inactivation of voltage-gated Na+ channels in the depolarizing direction and thereby increase the sodium currents during an action potential in depolarized fibers. If so, such an effect of low extracellular pH would contribute to the recovery of excitability seen in K+-depressed muscles upon acidification. At variance with this, a reduction of extracellular pH has also been found to block voltage-gated Na+ channels, which would counteract restoration of excitability upon muscle acidification (Hille, 1968; Woodhull, 1973; Kuzmenkin et al., 2002). In our results, however, this direct blocking effect of low extracellular pH must be minor since muscle acidification caused a pronounced increase in rate of rise of the action potentials and a reduced rheobase current in muscles at 4 mM K+, where a screening effect of low extracellular pH on membrane excitability would be of less significance compared with K+-depressed muscles. In contrast, intracellular acidification seems to have only minor effects on the steady-state activation and inactivation of voltage-gated Na+ channels in skeletal muscles, (Kuzmenkin et al., 2002).
Since the weakly voltage-dependent ClC-1 channel is believed to account for the largest part of GCl– in skeletal muscle (Jentsch et al., 2002), the above findings suggest the involvement of ClC-1 channels in the protective effect of acidosis. This is further indicated by the protective effect of 9-AC on excitability and force in K+-depressed muscles (Fig. 3). The activation of muscles requires a maintained excitability of both the sarcolemma and the t-tubular system. The measurements of M-waves and action potentials in the present study mainly reflect the function of the sarcolemma. Studies on skinned fibers (Coonan and Lamb, 1998; Pedersen et al., 2004), however, demonstrate that Cl– channels play a similar role for the function of the t-tubular system, which is further supported by the close correlation between force and sarcolemma excitability in the present study. When ClC-1 channels are expressed in heterologue expressions systems, however, the observed effects of acidosis on ClC-1 channels are unable to explain the reduced GCl– at low pH observed in native tissue (Rychkov et al., 1996). The apparent discrepancy between these observations from native tissue and expression systems is not clear. Irrespectively of this, the present study demonstrates that muscle excitability strongly depends on the balance between the number of Na+ channels that can be activated and the prevailing GCl–. Furthermore, it demonstrates that the mechanism underlying the protective effect of muscle acidification in K+-depressed muscles (Nielsen et al., 2001) is largely attributable to a down-regulation of GCl–, possibly conveyed by an inhibition of ClC-1 channels. These results fully agree with the recent study of Pedersen et al. (2004) on mechanically skinned muscle fibers showing enhanced t-system excitability in depolarized fibers through a reduction in t-system Cl– permeability. Together, these two studies underline the importance of Cl– channel regulation for maintenance of excitability and contractile function in working muscle.
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ACKNOWLEDGMENTS |
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This study was supported by Helga and Peter Kornings Fond, The Lunbeck Foundation, The Danish Medical Research Council (22-02-0188), and a PhD grant for Thomas Holm Pedersen from the Faculty of Medical Sciences, University of Aarhus.
Olaf S. Andersen served as editor.
Submitted: 20 August 2004
Accepted: 10 January 2005
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S. Pierno, J.-F. Desaphy, A. Liantonio, A. De Luca, A. Zarrilli, L. Mastrofrancesco, G. Procino, G. Valenti, and D. Conte Camerino Disuse of rat muscle in vivo reduces protein kinase C activity controlling the sarcolemma chloride conductance J. Physiol., November 1, 2007; 584(3): 983 - 995. [Abstract] [Full Text] [PDF]
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P.-Y. Tseng, B. Bennetts, and T.-Y. Chen Cytoplasmic ATP Inhibition of CLC-1 Is Enhanced by Low pH J. Gen. Physiol., July 30, 2007; 130(2): 217 - 221. [Abstract] [Full Text] [PDF]
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 U. Rose, C. Derst, M. Wanischeck, C. Marinc, and C. Walther Properties and possible function of a hyperpolarisation-activated chloride current in Drosophila J. Exp. Biol., July 15, 2007; 210(14): 2489 - 2500. [Abstract] [Full Text] [PDF]
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 V. Shushakov, C. Stubbe, A. Peuckert, V. Endeward, and N. Maassen The relationships between plasma potassium, muscle excitability and fatigue during voluntary exercise in humans Exp Physiol, July 1, 2007; 92(4): 705 - 715. [Abstract] [Full Text] [PDF]
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 W. A. Macdonald, N. Ortenblad, and O. B. Nielsen Energy conservation attenuates the loss of skeletal muscle excitability during intense contractions Am J Physiol Endocrinol Metab, March 1, 2007; 292(3): E771 - E778. [Abstract] [Full Text] [PDF]
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O. B. Nielsen, K. Overgaard, K. Sahlin, and J. M. Renaud Lactic acid accumulation is an advantage/disadvantage during muscle activity J Appl Physiol, July 1, 2006; 101(1): 367 - 368. [Full Text] [PDF]
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S.-J. Zhang, J. D. Bruton, A. Katz, and H. Westerblad Limited oxygen diffusion accelerates fatigue development in mouse skeletal muscle J. Physiol., April 15, 2006; 572(2): 551 - 559. [Abstract] [Full Text] [PDF]
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G. D. Lamb, D. G. Stephenson, J. Bangsbo, and C. Juel Point:Counterpoint: Lactic acid accumulation is an advantage/disadvantage during muscle activity J Appl Physiol, April 1, 2006; 100(4): 1410 - 1412. [Full Text] [PDF]
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S. M. Sostaric, t. l. S. L. Skinner, M. J. Brown, T. Sangkabutra, I. Medved, T. Medley, S. E. Selig, I. Fairweather, D. Rutar, and M. J. McKenna Alkalosis increases muscle K+ release, but lowers plasma [K+] and delays fatigue during dynamic forearm exercise J. Physiol., January 1, 2006; 570(1): 185 - 205. [Abstract] [Full Text] [PDF]
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W. A Macdonald, T. H Pedersen, T Clausen, and O. B Nielsen N-Benzyl-p-toluene sulphonamide allows the recording of trains of intracellular action potentials from nervestimulated intact fast-twitch skeletal muscle of the rat Exp Physiol, November 1, 2005; 90(6): 815 - 825. [Abstract] [Full Text] [PDF]
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B. Bennetts, G. Y. Rychkov, H.-L. Ng, C. J. Morton, D. Stapleton, M. W. Parker, and B. A. Cromer Cytoplasmic ATP-sensing Domains Regulate Gating of Skeletal Muscle ClC-1 Chloride Channels J. Biol. Chem., September 16, 2005; 280(37): 32452 - 32458. [Abstract] [Full Text] [PDF]
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G. N. Filatov, M. J. Pinter, and M. M. Rich Resting Potential-dependent Regulation of the Voltage Sensitivity of Sodium Channel Gating in Rat Skeletal Muscle In Vivo J. Gen. Physiol., July 25, 2005; 126(2): 161 - 172. [Abstract] [Full Text] [PDF]
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 A. K. Hansen, T. Clausen, and O. B. Nielsen Effects of lactic acid and catecholamines on contractility in fast-twitch muscles exposed to hyperkalemia Am J Physiol Cell Physiol, July 1, 2005; 289(1): C104 - C112. [Abstract] [Full Text] [PDF]
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) and tetanic force (•). Data show means ± SEM from three muscles with 100% force corresponding to 0.32 ± 0.01 N. (B) Representative M-wave traces from a single muscle obtained at the time points indicated by a, b, and c in A.
), pH 6.8 + Cl– (
), pH 7.4 –Cl– (•), pH 6.8 – Cl– (
), respectively. For group average, see 
