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. 2002 Mar 1;539(Pt 2):391–407. doi: 10.1113/jphysiol.2001.013043

Changes in mechanosensitive channel gating following mechanical stimulation in skeletal muscle myotubes from the mdx mouse

Alfredo Franco-Obregón 1, Jeffry B Lansman 1
PMCID: PMC2290167  PMID: 11882673

Abstract

We studied the effects of membrane stretch and voltage on the gating of single mechanosensitive (MS) channels in myotubes from dystrophin-deficient mdx mice. In earlier studies of MS channels in mdx myotubes, we found a novel class of stretch-inactivated channels. In the present experiments, we used a gentle suction protocol to determine whether seal formation damaged the membrane and altered MS channel gating, since dystrophin-deficiency is known to be associated with an increased susceptibility to mechanically induced damage. In some recordings from mdx myotubes, MS channel open probability gradually increased to levels approaching unity following seal formation. In these recordings, channels remained open for the duration of the recording. In other recordings, MS channel open probability remained low after seal formation and applying weak suction evoked conventional stretch-activated gating. Applying strong suction or very positive voltages, however, caused some channels to enter a high open probability gating mode. The shift to a high open probability gating mode coincided with the appearance of stretch-inactivated gating. These findings suggested that mechanical stimulation altered the mechanical properties of the patch causing some MS channels to enter a novel gating mode. In support of this idea, stretch-activated and stretch-inactivated channels were not detected in the same membrane patch and channel inactivation occurred at lower pressures than activation (P1/2, = −13 and −26.5 mmHg, respectively). Other experiments showed that stretch-inactivated gating was not due to a simple loss of MS channel activity from a non-random process such as vesiculation or bleb formation: channel inactivation by suction was readily reversible, stable over tens of minutes, and followed the predictions of the binomial theorem for independent, randomly gating channels. In addition, the voltage-dependent gating of stretch-inactivated channels was similar to that of stretch-activated channels. The results show that MS channels in dystrophin-deficient muscle exist in two distinct gating modes and that mechanical stimuli cause an irreversible conversion between modes. We discuss possible mechanisms for the changes in MS channel gating in relation to the known cytoskeletal abnormalities of mdx muscle and its possible implications for the pathogenesis of Duchenne dystrophy.

Mechanosensitive (MS) channels are found in a wide variety of cells in many different organisms ranging from bacteria to mammals (reviewed by Sackin, 1995; Sachs & Morris, 1998). The identification of MS channels in recordings from cell membranes with the patch clamp technique is based on the observation of changes in the single-channel activity in response to pressure stimuli. Most studies of MS channels have focused on stretch-activated channels, which are the most frequently encountered type of MS channel in recordings from membrane patches. MS channels that are inhibited by the application of a pressure stimulus (stretch-inactivated) have been detected much less frequently, although they are found in a variety of cell types (snail neurones, Morris & Sigurdson, 1989; mouse skeletal muscle, Franco & Lansman, 1990a; toad stomach smooth muscle, Hisada et al. 1993; mammalian hypothalamic neurones, Oliet & Bourque, 1993; rat aortic endothelium, Marchenko & Sage, 1997).

It is generally thought that pressure stimuli open MS channels by stretching the patch membrane, which increases the tension in the plane of the membrane (Gustin et al. 1988; Martinac et al. 1990; Sokabe et al. 1991). Increased membrane tension may be coupled to channel gating through forces exerted in the lipid bilayer (Martinac et al. 1990; Opsahl & Webb, 1994a; Sukharev et al. 1999; Zhang et al. 2000) or through cytoskeletal structures that are directly coupled to the channel protein (Guharay & Sachs, 1984). Much less is known about the gating of stretch-inactivated channels. For example it is not clear whether membrane stretch causes channel closure by acting on structures that are different from those involved in channel opening or whether the submembrane cytoskeleton is coupled to the channel in a manner that favours channel closure. Stretch-inactivated gating might also reflect changes in the mechanical properties or geometry of the patch membrane that affect force transmission or channel expression and/or accessibility. To date, there have been no studies of stretch-inactivated channels that would help address these issues.

One approach to this problem is to use genetic mutants in which specific cytoskeletal proteins are altered or missing. Mouse skeletal muscle possesses stretch-activated channels that dominate recordings from cell-attached patches (Franco & Lansman, 1990b; Haws & Lansman, 1991; Franco-Obregón & Lansman, 1994). The mdx mouse is a mutant that lacks the cytoskeletal protein dystrophin and is a model for Duchenne muscular dystrophy in humans (reviewed by McArdle et al. 1995; Gillis, 1999). Single-channel recordings from mdx myotubes also show activity of stretch-inactivated channels (Franco & Lansman, 1990a; Franco-Obregón & Lansman, 1994). The conductance of stretch-inactivated channels is very similar to stretch-activated channels. Stretch-inactivated channels, however, are persistently open, rather than closed, at rest in the absence of mechanical stimulation.

In previous studies of stretch-inactivated channels in mdx myotubes, relatively large pressures were used to form gigaohm seals. It has become clear that formation of a gigaohm seal can damage the plasma membrane and/or disrupt the cortical cytoskeleton causing changes in MS channel activity (reviewed in Hamill & McBride, 1997). MS channel activity in mdx myotubes may be particularly susceptible to modification by seal-induced damage, since dystrophin is generally thought to provide mechanical reinforcement to the sarcolemma and its absence is associated with increased susceptibility to damage by contraction-induced stresses (reviewed by Petrof, 1998; Gillis, 1999). Thus, it was not clear in these studies whether the increased opening of MS channels in mdx myotubes was due to mechanical damage produced by the electrode during seal formation.

The present experiments were made under conditions designed to minimize membrane damage by using a ‘gentle’ suction protocol to form seals (Hamill & McBride, 1992). We show that when seals are formed using gentle suction, irreversible changes in the gating of MS channels can be detected during the course an experiment. The results indicate that dystrophin does not act directly as a force transmitting element in mechanotransduction but, rather, appears to stabilize interactions between the cortical cytoskeleton and plasma membrane. We discuss these findings in relation to hypotheses concerning the mechanism of muscle damage in Duchenne muscular dystrophy.

METHODS

Muscle cell preparation

Wild-type (C57BL/6J) and mdx (C57BL/10ScSn-mdx) mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA). Mice were killed by cervical dislocation after halothane anaesthesia according to a protocol approved by the UCSF Committee for Animal Research. Myotubes were grown in tissue culture from enriched populations of myoblasts as previously described (Franco & Lansman, 1990b). The hind limbs of 3- to 7-week-old mice were removed and placed in cold (4 °C) Ca2+- and Mg2+-free Hank's buffered saline. After cleaning away bone and connective tissue, pieces of muscle were incubated for 30 min at 37 °C in saline containing 1 % collagenase B (Boehringer-Mannheim) and 0.125 % trypsin (UCSF Tissue Culture Facility) and constantly agitated by stirring. Satellite cells were dissociated by repeatedly triturating the muscle digest through the tip of a pipette. Cell debris was removed by filtering the suspension through a fine-mesh nylon cloth (100 μm). Fibroblasts were selectively removed by preplating the suspension on glass for 1 h and plating the non-adhering cells at a density of ∼3000–5000 cells cm−2 on plastic tissue culture dishes (Falcon). In some experiments, isolated muscle cells were plated on tissue culture dishes that had been coated with 1 μg cm−2 laminin (Sigma). Recordings were made from myotubes ∼2–5 days after the first multinucleated myotubes appeared in culture (∼7–8 days after plating myoblasts). All cultures were maintained in a tissue culture incubator at 37 °C and exposed to 95.0 % air-5.0 % CO2.

Solutions

Physiological saline contained 150 mmNaCl, 5 mm KCl, 1 mm MgCl2, 2.5 mm CaCl2 and 10 mm Hepes. The bathing solution was an isotonic potassium aspartate solution containing 150 mm KOH, 150 mm aspartic acid, 5 mm MgCl2, 10 mm K-EGTA and 10 mm Hepes. The potassium aspartate bathing solution was used to zero the cell's resting potential so that the patch potential would be equal to the applied voltage command. Measuring the single-channel current-voltage relationship before and after patch excision indicated a maximum voltage error of ∼5 mV. The potassium aspartate bathing solution produced no detectable signs of cell deterioration or obvious changes in cell morphology, such as swelling or shrinkage over the duration of the recording. Control recordings made with cells bathed in physiological saline showed no obvious differences in MS channel behaviour. The pH of all solutions was adjusted to 7.5 by adding NaOH. The osmolarity of all solutions was adjusted to 320–330 mosmol l−1 by adding glucose.

Electrophysiological methods

Single-channel activity was recorded from cell-attached patches with the patch recording method (Hamill et al. 1981). Patch electrodes were pulled in two steps from borosilicate haematocrit pipettes (Boralex, Rochester Scientific), coated with Sylgard (Dow Corning), and the tips heat polished with a microforge. Patch electrodes had resistances of 2–4 MΩ when filled with a physiological saline solution and immersed in the potassium aspartate bathing solution. Membrane currents were recorded with a List EPC-7 patch clamp amplifier. Current records were stored on video tape and replayed onto the hard disk of a computer for analysis. Current records were filtered with an eight-pole Bessel filter (-3 dB at 1 kHz) and digitized at 5–10 kHz. All recordings were made from cell-attached patches at a constant holding potential of −60 mV unless otherwise indicated. All experiments were done at room temperature (∼21–25 °C).

Pressure was applied to the patch electrode through the side port of a Teflon electrode holder. Pressures were applied manually using a syringe and were measured with a mercury manometer that was connected in series. A small positive pressure was applied to the electrode when approaching the cell at the start of the experiment. When the electrode tip was within ∼5–10 μm of the cell surface, the pressure was released and allowed to equilibrate with atmospheric pressure. The pressure in the electrode was confirmed to be zero by the absence of visible fluid flow or the movement of small particles at the electrode tip. The electrode was advanced towards the surface of the cell until there was an ∼25 % reduction in the electrode resistance. Seals were formed by applying a negative pressure of ∼2–3 mmHg for 10 s or less. Seals generally formed rapidly on myotubes that formed shortly after fusion of aligned myoblasts. Recordings were made during approximately the first week after differentiated myotubes formed in culture.

Data analysis

Channel open probability was measured by integrating idealized records of channel opening and closing transitions and dividing it by the time integral of the single-channel current. The idealized records were obtained by setting a threshold at half the amplitude of the open channel current and considering an opening event to occur when at least two consecutive sample points crossed this threshold. Since the number of channels varied from patch to patch, the measured open probability is the open probability of each individual channel (po) multiplied by the number of channels (N).

To quantify the pressure-sensitive gating MS channels, channel open probability was measured in the absence of a pressure stimulus and this value was subtracted from the channel open probability that was measured during the application of a constant pressure stimulus. The measurements were normalized to the number of channels in the patch estimated as the maximum number of superimposed openings during maximal activation. Measurements of channel open probability are shown as a function of pressure applied to the electrode. Plots of pressure versus open probability were fitted with a Boltzmann relation of the form:

graphic file with name tjp0539-0391-mu1.jpg

where po is the mean channel open probability, pmax is the maximum channel open probability, P is the pressure applied to the electrode (in mmHg), P1/2 the amount of pressure required to give po = 0.5, and π is the steepness of the relation (in mmHg). The data points were fitted using a non-linear, least-squares algorithm.

RESULTS

Previous recordings from patches on mdx myotubes showed that, following seal formation, the open probability of single MS channels in the absence of a pressure stimulus was either low (Npo < 0.20) or very high (Npo > 0.75) (Franco & Lansman, 1990a; Franco-Obregón & Lansman, 1994). Since these recordings were made by applying prolonged suction to form seals, it was not clear whether mechanical perturbation of the membrane produced abnormally high levels of MS channel activity in some patches. When seals were formed using gentle suction, however, recordings of single MS channel activity from mdx myotubes revealed changes in channel activity that developed slowly following seal formation.

Figure 1A shows an example of the changes in MS channel activity that occurred during the first ∼1.5 min following seal formation on an mdx myotube. Single-channel activity was recorded at a constant holding potential of −60 mV in the absence of a pressure stimulus. Shortly after formation of a gigaohm seal (indicated by the arrowhead), one channel opened for ∼450 ms and subsequently reopened several times over the next ∼10 s. Thereafter, channel activity slowly increased and, within about 1 min, three to four simultaneously open channels could be detected as superimposed current steps. The increase in channel open probability was large, since at least three channels were continuously open at the same time. Moreover, channel open probability remained high for the duration of the recording (see Fig. 5, below). The increase in channel opening following seal formation was not due to a shift in the resting membrane potential because the amplitude of the single-channel current remained constant (data not shown).

Figure 1. Changes in the resting activity of mechanosensitive ion channels following seal formation on mdx myotubes.

Figure 1

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A, recordings from a cell-attached patch on an mdx myotube showing an increase in channel activity following seal formation. Arrowhead indicates the beginning of the recording. Similar increases in activity after seal formation were observed in ∼10 % of recordings from cell-attached patches on mdx, but in fewer than 0.5 % of recordings from wild-type myotubes. B, recordings from two different wild-type myotubes showing that channel activity decreased and remained low following seal formation. Single-channel currents were filtered at 0.5 kHz and sampled at 1.25 kHz.

Figure 5. Reversible stretch-inactivated gating of MS channels in mdx myotubes.

Figure 5

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A, channel open probability (Npo) during consecutive 1 s intervals of a recording lasting ∼13 min. This patch contained only a single channel. Suction was applied to the patch electrode at the indicated times. Mean channel open probability during the first 143 s interval was 0.54 and the first application of −30 mmHg of suction to the electrode for 153 s reduced channel open probability to 0.044. After releasing the pressure applied to the electrode channel open probability returned to 0.85 and a subsequent application of −30 mmHg of suction for 137 s reduced channel open probability to 0.01; open probability returned to 0.63 after suction was released. B, representative current records during the indicated periods. Single-channel records were filtered at 0.5 kHz and sampled at 1.25 kHz.

Figure 1B shows examples of MS channel activity recorded from two different patches on wild-type myotubes. In contrast to mdx myotubes, seal formation on wild-type myotubes caused an immediate increase in channel activity that persisted only a short time (< 10 s), whereupon channels closed abruptly and remained closed, opening only briefly (∼40–50 ms) during the remainder of the recording. Evidently, formation of a gigaohm seal can have very different effects on subsequent channel activity in patch recordings from wild-type and mdx myotubes. Notably, seal formation on mdx myotubes often leads to a large increase in channel open probability that develops slowly and is irreversible over the time scale of the recording. The changes in MS channel gating occur even though care was taken to minimize membrane damage by forming seals using minimal suction. Slow, irreversible changes in MS channel gating were not detected in recordings from wild-type myotubes.

In some recordings from patches on mdx myotubes, large inward currents were recorded at a constant holding potential. In earlier studies of MS channels in mdx myotubes, we mistakenly attributed these currents to seal breakdown. Closer examination, however, revealed that the large currents were due to the presence in a patch of many MS channels each having a very high open probability. Figure 2 shows an example of this type of recording. In the absence of suction, there was initially a large inward current at the holding potential (Fig. 2A, the asterisk marks the zero current level). Discrete fluctuations in the single-channel current were not apparent. Subsequently, applying −40 mmHg of suction reduced the inward current sufficiently so that the discrete opening of two to four individual channels could be resolved (Fig. 2B). Channel activity was reduced even further with −60 mmHg suction (Fig. 2C). The effects of suction were fully reversible (see Fig. 5, below). The effects of suction on high open probability MS channels are similar to those described in earlier studies and attributed to stretch-inactivated channels (Franco & Lansman, 1990a). Evidently, MS channels with a high resting open probability can be detected immediately after seal formation or may appear slowly during an experiment, even when seals are formed using gentle suction to minimize membrane damage.

Figure 2. Presence of hot spots of channel activity in mdx myotubes.

Figure 2

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In this experiment, the patch was held at −40 mV. The zero current level is indicated with an asterisk (*). A, patch current recorded before and immediately after the application of −40 mmHg of suction to the electrode. Horizontal scale bar = 10 s. B, single channel currents recorded during maintained application of −40 mmHg. C, single-channel currents recorded during maintained application of −60 mmHg. Records of single-channel activity were filtered at 0.5 kHz and sampled at 1.25 kHz. Horizontal scale = 20 s.

Initially, we were interested in comparing the mechanosensitivity of stretch-activated and stretch-inactivated channels in mdx myotubes. It became clear, however, that suction had additional effects on the behaviour of MS channels. Figure 3 shows a recording from an mdx myotube in which application of suction after seal formation produced an irreversible change in MS channel gating. In this experiment, MS channel activity was low immediately after forming a seal with gentle suction (B, first pair, 0 mmHg). Application of −5 mmHg of suction (B, second pair, −5 mmHg) produced a large increase in open probability. After releasing the suction (B, third pair, 0 mmHg), however, channel open probability remained high. When suction was applied a second time (B, fourth pair, −15 mmHg), channel activity was reduced, rather than increased, by the pressure stimulus. Upon terminating the suction stimulus, channel activity promptly increased to a high level (B, fifth pair, 0 mmHg).

Figure 3. Irreversible changes in MS channel gating in mdx myotubes by suction pulses.

Figure 3

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A, channel open probability (Npo) measured in consecutive 300 ms sweeps. The bars indicate the time during which the indicated pressure stimulus was applied to the patch electrode. B, representative current records obtained during the experiment. Npo = 0.04 at the beginning of the experiment, 0.20 after applying −5 mmHg, 0.15 after subsequently releasing the pressure stimulus, 0.01 after application of a second pressure stimulus of −15 mmHg, and 0.10 after releasing the pressure stimulus.

The graph in Fig. 3A shows a plot of channel open probability during consecutive 1.3 s sweeps for the entire recording which lasted ∼10 min. The sweep-by-sweep plot of channel open probability shows how the effects of suction depended on the patch's history of mechanical stimulation. The first application of suction (-5 mmHg, indicated by the first bar) increased channel activity while the suction stimulus was maintained. On the other hand, the second application of suction (-15 mmHg, indicated by the second bar) greatly suppressed channel activity. The graph also shows that resting activity in the absence of suction was increased after the first application of 5 mmHg of suction and it returned to this high level after the application of 15 mmHg of suction, despite the change in the effects of suction during the pulse. This type of behaviour can be explained if suction has two effects on MS channel activity. The first involves a stretch-induced increase in channel open probability. The second involves an irreversible effect of stretch in which, once channels are in the high open probability state, the gating mechanism becomes modified so that channels close only with the application of additional suction.

In these experiments, 7/44 patches showed a suction-induced change from stretch-activated to stretch-inactivated gating (Table 1). The conversion occurred with suction in the range of 5–20 mmHg, although this was not systematically studied because the process was irreversible. The conversion process also occurred infrequently enough to make it impractical to study. Conversion was detected in only one patch of 57 on wild-type myotubes. In addition, we found that seals on wild-type myotubes were less stable.

Table 1.

Responses of single MS channels in mdx myotubes to pressure

Response to pressure No. of patches
Wild-type mdx
(n = 57) (n = 49)
Stretch-insensitive 15 17
Stretch-activated 40 11
Stretch-inactivated 1 14
Converted 1 7
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The results suggest low levels of suction are sufficient to cause a change in MS channel gating in mdx myotubes in which channels enter a high open probability state that is energetically favoured at resting membrane tensions. In normal myotubes, depolarizing the membrane to positive potentials causes a large increase in channel open probability (Franco & Lansman, 1990b; Franco-Obregón & Lansman, 1994). If entry into a high open probability state is required for the gating conversion, then membrane depolarization might be expected to produce a similar change in gating. This possibility was tested by applying voltage steps to positive potentials (+50 to +80 mV) where the open probability of MS channels is maximal (Franco & Lansman, 1990b).

Figure 4A shows a recording from a wild-type myotube in which a voltage step to +80 mV was applied to the patch. Channels opened rapidly during the voltage step. After returning the patch potential to the holding potential, channels closed rapidly as expected for a voltage-dependent gating process. In contrast to the reversible voltage-dependent gating of MS channels in wild-type myotubes, Fig. 4B shows that a strong positive voltage step produced an irreversible increase in channel open probability in mdx myotubes. As shown in Fig. 4B, a voltage step to +80 mV caused channels to open without an appreciable delay. Channels, however, failed to close after repolarization and remained open for the duration of the recording. In this recording, the high open probability state was associated with the appearance of a subconductance level that was particularly prominent at a holding potential of −60 mV (asterisk) and is shown at higher time resolution in the inset. Thus, either strong depolarization or negative pressures larger than those used routinely to form gigaohm seals cause changes in MS channel gating in mdx myotubes.

Figure 4. Changes in MS channel gating produced by strong depolarizations in mdx, but not wild-type, myotubes.

Figure 4

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A, a voltage step was applied to a cell-attached patch on a wild-type myotube from a holding potential of −80 mV to +80 mV. After repolarization to −80 mV, channel activity rapidly returned to low levels. B, during a recording from an mdx myotube, a voltage step to +80 mV caused channels to open during the voltage pulse. After returning the membrane potential to −80 mV, channel activity remained high throughout the remainder of the recording. The lower records in B are the currents after repolarization shown on an expanded time scale at −60 mV (*). Single-channel records were filtered at 0.5 and 2 kHz and sampled at 1.25 and 5 kHz. The dashed line indicates the amplitude of the dominant subconductance level at −60 mV.

MS channels with high open probability in mdx myotubes are inhibited by suction. Whether the ability of suction to inhibit MS channel activity represents an intrinsic gating mechanism or some change in the configuration or geometry of the patch membrane is not clear. For example suction may have caused formation of a membrane vesicle within the electrode. Vesicle formation may be associated with the development of a large series resistance that reduces the voltage across the channel, thereby attenuating the single-channel current. Alternatively, vesicle formation may cause membrane containing MS channels to be drawn into an electrically inaccessible compartment. These types of changes, however, would be expected to be more or less irreversible. In addition, changes in patch configuration or geometry would be expected to cause changes in gating that are either non-random or co-operative (e.g. Silberberg & Magleby, 1997; Gil et al. 1999). We therefore tested whether the effects of suction could be explained in terms of a reversible and random gating process.

Figure 5 shows that stretch-inactivated gating of MS channels in mdx myotubes is reversible and remarkably robust. Figure 5 (left) shows a plot of channel open probability for consecutive 1 s intervals during a 10 min recording. The patch contained only a single channel, as judged by the absence of superimposed openings in the records. Applying −30 mmHg of suction suppressed channel activity almost completely. After releasing the suction, channel activity returned rapidly to levels approaching unity. Figure 5 (right) shows representative currents recorded during the application of suction and following the termination of the pressure stimulus. Note that, in the absence of suction, channel openings lasted many tens of seconds, in striking contrast to channel openings in normal myotubes (Franco & Lansman, 1990a, b). In the absence of suction, MS channel activity showed only minimal rundown during the 10 min that this recording lasted. The stability of stretch-inactivated gating contrasts sharply with gating of stretch-activated channels in either wild-type or mdx fibres which is often not fully reversible and generally runs down during prolonged mechanical stimulation (e.g. Franco-Obregón & Lansman, 1994, Fig. 8).

Figure 8. Effect of membrane potential on mechanosensitive channels in mdx myotubes.

Figure 8

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A, single-channel currents recorded from mdx myotubes at the indicated patch holding potentials. B, the effect of patch holding potential on channel open probability in recordings of mechanosensitive channel activity from mdx myotubes showing high (•) and low (○) levels of activity. Channel open probability increased ∼e-fold per 36 and ∼37 mV with depolarization for stretch-activated and stretch-inactivated channels, respectively.

We also tested whether channel inhibition by suction was consistent with a model in which pressure affects the opening and closing of independently gating channels. Such a test would provide additional evidence that the apparent channel inactivation by suction was not due to vesiculation or some other non-random change in membrane configuration. The traces in Fig. 6A were obtained from a patch containing three channels as judged by the maximum number of superimposed openings that were observed during a ∼10 min recording. The top, middle and bottom traces show the channel activity during ∼65 s sweeps during application of 0, 20 or 40 mmHg of suction, respectively. The graphs in Fig. 6B show the probability that 0, 1, 2 or 3 channels were open during the recording. The open probability at each conductance level was measured as the open time at each level divided by the total open time (filled bars). The open bars represent the predictions of the binomial theorem for three independent, randomly gating channels (details in figure legend). The experimental measurements are quite close to the predictions of the binomial theorem, suggesting that channel gating in response to pressure is random rather than co-operative.

Figure 6. The channel inactivation mechanism acts on independently gating channels in multi-channel patches.

Figure 6

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A, single-channel records from a cell-attached patch recording from a mdx myotube in a patch containing three channels with either 0, −20 or −40 mmHg suction applied to the electrode. Single-channel records were filtered at 0.5 kHz and sampled at 1.25 kHz. B, comparison of the effect of suction on channel open probability with the predictions of the binomial theorem (□) for the gating of N independent channels. If there are N independent channels in the patch, then the probability that r of those channels are simultaneously open should follow a binomial distribution such that:
graphic file with name tjp0539-0391-mu2.jpg
where r is the number of channels open at one time, and p is the probability that any one channel is open.

In recordings from mdx myotubes, we found that ∼57 % (28/49) of the patches had MS channels with a low probability of opening in the absence of suction (Table 1). These patches consisted of those in which channel activity was insensitive to suction (stretch-insensitive, ∼35 %, 17/49) and those that showed conventional stretch-activated gating (∼22 %, 11/49). The existence of a relatively stable population of stretch-activated channels in mdx myotubes is interesting, since it implies that the absence of dystrophin alone is not sufficient to produce changes in MS channel gating in mdx myotubes. We never observed stretch-activated and stretch-inactivated channels within the same membrane patch. The existence of distinct populations of stretch-activated and stretch-inactivated channels suggests that MS channel gating behaviour may reflect the local mechanical properties of the patch membrane, which may vary at different sites on the muscle surface. Stretch-activated and stretch-inactivated channels were not found to be associated with the position of the electrode on the membrane surface nor with any obvious morphological landmark, such as myonuclei.

Two general hypotheses might account for the existence of distinct modes of MS channel gating. Perturbation of the membrane by the recording electrode may have changed the orientation of the channel within the membrane and its coupling to membrane lipids or cytoskeletal structures with little or no change in the mechanical properties of the patch membrane. In this type of model, the energy required to gate either stretch-activated or stretch-inactivated channels would be similar because it reflects the energy required to deform the membrane. Alternatively, seal formation or other stimuli may cause a change in the organization, composition or geometry of the patch membrane. Such a change might alter the mechanical properties of the patch and, hence, the energy required to deform the membrane. As a first step towards distinguishing between these two mechanisms, we compared the sensitivity to pressure of stretch-activated and stretch-inactivated channels in mdx myotubes.

Figure 7 shows the effects of the magnitude of the pressure stimulus on the open probability of stretch-activated and stretch-inactivated channels. The records on the top show the two types of reversible MS channel gating recorded from membrane patches on mdx myotubes. Stretch-activated channels have a very low open probability at rest. In response to suction, they open rapidly and then close quickly when the pressure stimulus is terminated (Fig. 7A, left). By contrast, stretch-inactivated channels have a very high open probability at rest, close rapidly when suction is applied, and reopen after releasing the suction (Fig. 7A, right). We quantified the relationship between pressure and channel open probability to determine whether there are differences in the energetics of gating. Although it would be preferable to determine the effects of membrane tension, rather than pressure, on channel gating (e.g. Sokabe et al. 1991), the optics used in these experiments did not have sufficient resolution to observe the patch membrane. The results, nonetheless, showed clear differences in the pressure dependence of stretch-activated and stretch-inactivated gating.

Figure 7.

Figure 7

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A, examples of reversible stretch-activated (left) and stretch-inactivated (right) gating of MS channel in mdx myotubes. B, relationship between pressure and channel open probability for stretch-activated (left) and stretch-inactivated (right) channels. Data points are the mean ± s.e.m. open probabilities measured for stretch-activated (n = 6) and stretch-inactivated channels (n = 14). The relationship between the amount of pressure applied to the patch electrode and channel open probability was fitted with a Boltzmann relation of the form po = pmax/[1 + exp(P−- P1/2)/π], where po is the mean channel open probability, pmax is the maximum channel open probability, P is the pressure applied to the electrode (mmHg), P1/2 the amount of pressure to give po = 0.5, and π is the steepness of the relation. The data points were fitted using a non-linear, least-squares algorithm. For stretch-activated channels in mdx myotubes P1/2 = −36.5 mmHg and the steepness of the relationship, π = −6.0. For stretch-activated channels in wild-type myotubes P1/2 = −48 mmHg and the steepness of the relationship, π = −8.5. For stretch-inactivated channels P1/2 = −13 mmHg and π = span

Channel open probability was measured during a continuous recording of single-channel activity at each of the pressures indicated. Open probability was plotted as a function of pressure. The relationship between pressure and channel open probability for stretch-activated channels in mdx myotubes is shown in the left panel of Fig. 7B. The data was well fitted with a Boltzmann relation (dotted line) with a P1/2 of 26.5 mmHg and steepness (π) of −6 (details in figure legend). This is similar to the pressure-sensitivity of stretch-activated channels in intact flexor digitorum fibres acutely isolated from mdx mice (P1/2 = −20 mmHg, π = 5 mmHg, Franco-Obregón & Lansman, 1994). The pressure dependence of the gating of stretch-inactivated channels (Fig. 7B, right) was also well fitted by a Boltzmann relation (dotted line, P1/2 = ∼-13). The filled circles in the right panel of Fig. 7B represent the open probability of stretch-inactivated channels that were present in the patch initially, while the open circles represent open probability measured after conversion to the stretch-inactivated gating mode. The pressure dependence of gating of pre-existing and converted stretch-inactivated channels was similar.

Evidently, stretch-inactivated gating occurs at lower pressures than stretch-inactivated gating (P1/2 = ∼-13 and −26.5 mmHg, respectively). The shift of the pressure versus channel open probability curve for stretch-inactivated channels to lower pressures suggests that the mechanical properties of patch membrane changed so that mechanical energy is transferred more efficiently to the channel. This could reflect, for example, a change in which more of the total membrane tension falls across structures directly involved in conveying mechanical forces to the channel. Since the steepness of the relationship between pressure and channel open probability does not change, the actual energy required to open or close MS channels is the same. This would argue against the existence of large changes in the orientation and/or coupling of the channel to force-conveying membrane structures.

In this and previous studies, the analysis of stretch-activated gating in dystrophin-deficient muscle was restricted to pressures ≤-20 mmHg, since seal breakdown occurred at higher pressures. Interestingly, we found that patches containing stretch-inactivated channels could routinely be subjected to pressures exceeding −40 mmHg without evidence of seal breakdown (e.g. Fig. 2C). Even with repeated application of high pressures, patches containing stretch-inactivated channels were remarkably robust (e.g. Fig. 5). This observation, and the finding that stretch-inactivated gating is shifted to lower pressures relative to stretch-activated gating (above), supports the idea that there are important differences in the mechanical properties of patches containing stretch-activated and stretch-inactivated channels.

The open probability of MS channels in wild-type skeletal muscle increases with depolarization (Guharay & Sachs, 1985; Franco & Lansman, 1990b; Franco-Obregón & Lansman, 1994). In myotubes from the C2 mouse cell line, MS channel open probability reaches values close to 100 % at ∼+80 mV (Franco & Lansman, 1990b). One possible explanation for the large increase in MS channel open probability in mdx myotubes is that there is a shift in the voltage dependence of channel opening to more negative potentials. This might occur, for example, if changes in the configuration of the patch membrane altered the orientation of the voltage sensor in the membrane field. To evaluate this possibility, we investigated the effects of voltage on the gating of stretch-activated and stretch-inactivated channels in mdx myotubes.

Figure 8 shows records of the activity of stretch-activated (Fig. 8A, left) and stretch-inactivated channels (Fig. 8A, right) in mdx myotube at different holding potentials. Because stretch-inactivated channels have a very high open probability at negative holding potentials, membrane depolarization would be expected to produce only negligible increases in channel opening. To resolve the voltage-dependent gating mechanism of stretch-inactivated channels, a small amount of suction (∼2 mmHg) was applied to the electrode to reduce channel open probability. Under these conditions, channel open probability was low at a holding potential of −80 mV (Fig. 8A, right, fourth trace), but became progressively larger as the membrane potential was made more positive.

The graphs in Fig. 8B are plots of channel open probability as a function of the holding membrane potential for the stretch-activated (left) and stretch-inactivated (right) channels. The slopes of the relationship between membrane potential and open probability were similar for the two MS channel gating modes (∼e-fold per ∼36 and ∼38 mV for stretch-activated and stretch-inactivated gating, respectively). In addition, we found that the increase in channel opening with depolarization for both types of MS channels resulted from an increase in burst duration (data not shown) which were obtained as the mean open time obtained after excluding rapid channel closures (< 5 ms, see Methods, Franco & Lansman, 1990b). The results suggest that voltage-dependent gating of stretch-inactivated channels is similar to stretch-activated channels when the resting open probability of stretch-inactivated channels is reduced by suction.

Applying suction to reduce channel open probability, however, may have altered the voltage dependence of channel opening. This might occur, for example, if suction caused a change in patch configuration that modified either the orientation of the voltage sensor or the electric field across the membrane. We, therefore, measured the voltage dependence of channel opening of stretch-activated and stretch-inactivated channels in mdx myotubes in the absence of suction. Figure 9A shows the effects of the holding membrane potential on MS channel open probability in a number of recordings from mdx myotubes. The open symbols represent recordings of stretch-activated channels that showed a low open probability in the absence of suction, while the filled symbols are from recordings from stretch-inactivated channels that showed a high open probability in the absence of suction. The voltage dependence of channel opening was again similar for both types of channel (∼e-fold per ∼36 ± 7 and ∼38 ± 5 mV, n = 6 and 5 for stretch-activated and stretch-inactivated gating, respectively).

Figure 9.

Figure 9

Open in a new tab

A, voltage dependence of stretch-activated (○) and stretch-inactivated (•) channels in mdx myotubes in the absence of suction. Channel open probability increased ∼e-fold per 36 ± 7 mV (s.d., n = 6) and ∼38 ± 5 mV (s.d., n = 5) with depolarization for stretch-activated and stretch-inactivated channels, respectively. The voltage dependence of stretch-inactivated channels having a high open probability at rest could not be studied at very positive potentials, since open probability approached unity. B, membrane potential and stretch act independently on the gating of MS channels in mdx myotubes. The filled symbols represent a recording from an mdx myotubes which contained several channels. Channel activity increased ∼e-fold per 50, 38 and 40 mV of depolarization with 0 (•), −20 (▪) and −40 (▴) mmHg suction applied to the patch electrode, respectively. The experiment shown in open symbols (□) represents the voltage dependence of stretch-activated channel activity recorded from an mdx myotube with −10 mmHg applied to the electrode. Channel activity increased ∼e-fold per 37 mV depolarizatspan

Studies of MS channels in Xenopus oocytes suggest that voltage-dependent activation reflects a voltage-dependent displacement of the patch membrane (Gil et al. 1999). If voltage-dependent activation of MS channels in mdx myotubes were due to voltage-dependent patch displacement, then changes in voltage would have progressively less effect when the membrane is deformed by applying suction. To test this possibility, we measured the voltage dependence of opening of stretch-inactivated channels in the presence of different amounts of suction. We expected that voltage-dependent gating would be minimal when applying large amounts of suction.

Figure 9B show the results of a recording from a single patch on an mdx myotube that contained several channels. Channel activity was high in the absence of suction (filled circles). Applying −20 mmHg (filled squares) or −40 mmHg (filled triangles) of suction greatly reduced channel activity. In the presence of maintained suction, however, membrane depolarization still increased channel open probability. Channel open probability increased ∼e-fold per 50, 38 and 40 mV of depolarization with 0, −20, and −40 mmHg applied suction. For comparison, the open symbols in Fig. 9B show the changes in channel open probability of a stretch-activated channel in a wild-type myotube in the presence of a constant −10 mmHg pressure stimulus. The voltage dependence of channel opening was similar in the presence of −20 and −40 mmHg of suction. In the absence of suction, however, the slope was somewhat less steep. The reduced slope at 0 mmHg may reflect experimental error rather than a real change in voltage dependence. In the absence of suction, open probability is close to unity at rest and so depolarization would be expected to produce relatively small changes in open probability that would be difficult to resolve by our methods. In the light of possible measurement errors, it seems reasonable to conclude that suction did not produce large changes in the voltage dependence of activation, particularly at the higher pressures. Thus, the effects of voltage and pressure on channel gating are independent rather than additive, in contrast to the results of Gil et al. (1999).

DISCUSSION

In this study, we show that there is a remarkable diversity of MS channel behaviour in mdx myotubes. In some patches, channels rarely open in the absence of suction, while in others, channels are open almost continuously (Franco & Lansman, 1990b). We now show that in some patches, MS channel opening increases dramatically following seal formation and this process is essentially irreversible over the time course of the recording. Strong suction or positive voltage steps also caused a large and irreversible increase in MS channel open probability. Irreversible increases in MS channel open probability following seal formation were missed in earlier studies. The use of large pressures to form gigaohm seals may have caused such changes to go undetected by causing a more rapid and complete shift of channels into a high open probability gating mode. However, when care is taken to minimize mechanical perturbation of the plasma membrane by the recording electrode, slow changes in MS channel opening can be observed following seal formation in the absence of an applied pressure stimulus.

Previous studies of MS channels in other preparations have shown that large pressures applied during seal formation or during repetitive stimulation influence the level of MS channel activity (reviewed by Hamill & McBride, 1997). In Xenopus oocytes, for example, the use of large pressures to form seals (>-20 mmHg) reduces MS channel activity during subsequent episodes of mechanical stimulation (Hamill & McBride, 1992). In snail neurones, however, repeated application of large suction steps (-60 to −80 mmHg) shortened the delay before channel opening and increased the current amplitude (Small & Morris, 1994; Wan et al. 1999). In both studies, however, changes in MS channel activity were detected only in response to relatively high pressures. Moreover, mechanical stimulation did not change the basic MS channel activation gating mechanism. In mdx myotubes, by contrast, mechanical stimulation caused both an irreversible increase in channel open probability and a shift to a stretch-inactivated gating mechanism. In the subsequent sections, we consider possible mechanisms that might account for these parallel changes in MS channel gating behaviour.

McBride and Hamill (1992) have also found changes in MS channel gating in mdx myotubes using a rapid pressure clamp method to activate MS currents. They found that during pressure steps >∼20 mmHg, MS channels failed to close normally and remained open throughout the maintained pressure step. This contrasts with the behaviour of MS currents in normal muscle which shows pronounced adaptation during a pressure step (McBride & Hamill, 1992). Loss of adaptation of MS currents in mdx muscle, however, was reversible and channels frequently exhibited normal adaptation during subsequent pressure steps. The maintained MS channel opening described here differs in that it occurs with only minimal mechanical perturbation following seal formation and is essentially irreversible. An irreversible loss of MS channel adaptation has been described in patch recordings from Xenopus oocytes following repeated mechanical stimulation that can be seen to decouple the membrane from the underlying cytoskeleton (Hamill & McBride, 1992). This suggested that failure of channel closure was due to the loss of some cytoskeletal element whose viscoelastic properties normally relaxes membrane tension during maintained pressure steps.

The increase in MS channel opening described here may reflect a mechanically induced decoupling of the cytoskeleton from the plasma membrane of mdx myotubes. Although we have no direct evidence for this, such a mechanism is consistent with the known cytoskeletal abnormalities of mdx skeletal muscle and its increased sensitivity to mechanical damage (reviewed by Gillis, 1999). To produce persistent channel opening, however, a decoupling mechanism would require the disruption or loss of a viscoelastic element associated with the membrane that normally causes channel closure by relaxing membrane tension. We believe that the spectrin skeleton may act as such an element in mechanotransduction (below). Nonetheless, the conditions under which the cytoskeleton decouples form the membrane are not well defined, particularly in dystrophin-deficient muscle. A number of experimental variables, such as electrode composition or shape and state of cytoskeletal assembly in myotubes grown in tissue culture are likely to influence the pressure sensitivity, extent and reversibility of cytoskeletal decoupling.

It is not known exactly how dystrophin-deficiency might render the interactions between the submembrane cytoskeleton and sarcolemma more sensitive to mechanical stimuli. Dystrophin deficiency is known to be associated with changes in the mechanical properties of the muscle membrane (e.g. Pasternak et al. 1995; Rybakova et al. 2000). Dystrophin is concentrated at the sarcolemma in strands running along the fibre axis and at costameres, regions of the sarcolemma that overlie the Z and M lines (Masuda et al. 1992; Minetti et al. 1992; Porter et al. 1992). Costameres are thought to participate in the lateral transmission of contraction-induced stresses to the extracellular matrix and adjacent fibres via filaments that connect the contractile apparatus to the sarcolemma (Pierobon-Bormioli, 1981; Street, 1983). These filaments are thought to regulate membrane folding and, therefore, contribute to the mechanical stability of the membrane. Costameres are enriched in β-spectrin, which colocalizes with dystrophin in a two-dimensional lattice (Craig & Pardo, 1983; Pardo et al. 1983; Porter et al. 1992; Dmytrenko et al. 1993). Morphological studies show that the absence of dystrophin is associated with the loss of β-spectrin from the sarcolemma over M lines and longitudinal strands (Williams & Bloch, 1999). The loss of β-spectrin leaves regions of the sarcolemma without structural support from the cytoskeleton. It seems reasonable to assume that the membrane in these regions may be more susceptible to disruption by mechanical stimulation during patch recordings. Thus, local abnormalities in the spectrin skeleton may give rise to mechanically sensitive spatial domains in which MS channel gating behaviour is altered. Hot spots of MS channel activity encountered during recordings from mdx myotubes (Fig. 2) may reflect the clustering of MS channels in such spatially restricted domains.

It is interesting to consider how the cytoskeletal abnormalities in mdx muscle might lead to persistent MS channel opening. The submembrane cytoskeleton normally organizes the plasma membrane into spatially distinct microdomains by binding to integral membrane proteins (Gruen & Wolfe, 1982). The cytoskeleton thus serves to constrain the bilayer and increase its local curvature (Zeman et al. 1990; reviewed by Petrov & Usherwood, 1994). When constrained to a high local curvature, the tension across the membrane as determined by LaPlace's Law is small and most of the global tension is supported by the more rigid cytoskeleton. In dystrophin-deficient muscle, the spectrin-based cytoskeleton is likely to perform this function. We speculate that abnormalities in the spectrin cytoskeleton in dystrophin-deficient muscle renders the microdomain structure of the sarcolemma sensitive to disruption by mechanical stimuli. If seal formation in regions of sarcolemma with an altered cytoskeletal network disrupts its microdomain structure, the reduction in local bilayer curvature would result in most of the global membrane tension falling across the bilayer. The increase in membrane tension across the bilayer would be expected to be associated with a large and, presumably, irreversible increase in MS channel open probability. Since increases in MS channel open probability also occur following gentle seal formation, the resting tension produced by adhesive interactions between the electrode glass and membrane lipid in the absence of pressure (Opsahl & Webb, 1994b) may be sufficient to disrupt local membrane structure. On the other hand, large pressures or positive voltage steps may cause much more rapid changes in local membrane geometry.

Several other mechanisms might explain the persistent MS channel opening. For example mechanical stimulation might release a diffusible second messenger that increases MS channel activity, but it is not obvious how suction would subsequently reduce channel activity. Moreover, the high open probability gating mode persists in excised patches (data not shown) making the participation of a diffusible channel modulator unlikely. Alternatively, mechanical stimulation might produce slow changes in the composition of the lipid bilayer such that membrane lipids adjacent to the channel selectively stabilize the open conformation of the channel protein. Changes of this sort could be produced by adhesive interactions between the membrane and electrode that selectively retain certain components to the electrode glass, producing changes in the composition of non-adhered membrane in the electrode tip. Mechanical stimulation might also directly modify the folding state of the channel protein in dystrophin-deficient muscle. Both types of mechanisms would imply that dystrophin plays a role in restraining lipids within the membrane and in protecting MS channels from unfolding forces generated within the bilayer.

Mechanism of stretch-inactivated gating

Our results indicate that the cytoskeletal protein dystrophin is not directly involved in the transmission of the mechanical forces that gate MS channels in skeletal muscle. Recordings from mdx myotubes show a relatively stable population of stretch-activated channels that open rapidly in response to suction and close after termination of the stimulus. Moreover, the pressure dependence of channel opening shows that the energies required for stretch-activated and stretch-inactivated gating are similar (Fig. 7B). This can be interpreted as indicating that the same structure conveys membrane tension to the channel for both forms of mechanosensitive gating. This would be equivalent to a gating spring whose stiffness remains the same whether channels are in the stretch-activated or stretch-inactivated gating mode. The discussion above leads to the conclusion that the gating spring resides in the membrane bilayer.

The conclusion that dystrophin does not participate directly in mechanotransduction, however, must be stated with some reservation, since the expression utrophin, an autosomal homologue of dystrophin (Tinsley et al. 1992), may compensate for the loss of dystrophin (Tinsley et al. 1996). Utrophin is expressed at high levels in fetal and neonatal muscle where it is localized beneath the sarcolemma (Khurana et al. 1991; Schofield et al. 1993). Utrophin is also expressed at earlier stages of development than dystrophin (Lin & Burgunder, 2000) and so may be present in myotubes used in these experiments. Further experiments are necessary to evaluate the contribution of utrophin to the functional diversity of MS channels in mdx myotubes.

We believe that disruption of the spectrin skeleton that normally supports membrane tension causes an increase in the tension supported by the lipid bilayer and a corresponding increase in MS channel open probability. It is not, however, obvious how changes in local membrane structure also cause a parallel change in the mechanism of MS gating. There are two general models that could explain reversible stretch-inactivated gating in dystrophin-deficient muscle. Both models assume that membrane tension is coupled to MS channel gating through forces exerted in the lipid bilayer and that the membrane is in a high tension state. In one model, which we term the fluid monolayer model, applying suction would draw excess lipid from the inner monolayer which would cause a relaxation of the stress in the bilayer. This type of mechanism, while physically plausible, seems inconsistent with a stretch-inactivated gating process that is rapid, readily reversible, and stable over many minutes. This model is also difficult to reconcile with observations that MS channels retain their basic stretch-activated gating mechanism in pure lipid bilayers (Sukharev et al. 1999) or in cytoskeleton-deficient membrane vesicles (Zhang et al. 2000).

A second type of model for stretch-inactivated gating is one in which mechanical stimuli recruits new membrane from a previously inaccessible membrane compartment, thereby reducing the high bilayer tension and causing channel closure. Seal formation has been shown to cause vesicles to form from the plasma membrane (Sokabe & Sachs, 1990), but shear-induced vesiculation is not likely to produce a readily reversible gating process. Alternatively, recruitment of membrane might occur by the fusion of intracellular secretory vesicles with the patch membrane (Zorec & Tester, 1993; Homann, 1998). A secretion mechanism, however, would require the existence of a stable population of release-ready vesicles capable of undergoing repeated cycles of fusion and endocytosis without appreciable delay. This would be necessary to account for the rapid and reversible changes in channel opening in response to suction (Fig. 5). A simple fusion model, therefore, seems unlikely, particularly since we have observed stretch-inactivated gating of MS channels in membrane patches in the absence of divalent cations (data not shown).

Alternatively, suction may recruit membrane from surface invaginations of the plasma membrane. In skeletal muscle, these compartments include vacuoles (Gonzalez-Serratos et al. 1978; Fraser et al. 1998; Lännergren et al. 1999) and caveolae (Rayns et al. 1968; Dulhunty & Franzini-Armstrong, 1975). Dulhunty and Franzini-Armstrong (1975) showed that stretching frog skeletal muscle fibres cause caveolae to open up, a reversible process that contributes to the increase in membrane surface area at large sarcomere lengths (Dulhunty & Franzini-Armstrong, 1975). The density off caveolae in amphibian muscle is ∼37 per mm2 (Dulhunty & Franzini-Armstrong, 1975). If the area of the patch membrane within the electrode is 10–12 mm2, a patch may contain many hundreds of caveolae. The surface membrane of dystrophic muscle is highly invaginated, possessing many more vacuoles and caveolae than normal muscle (Libelius et al. 1979; Bonilla et al. 1981; Malouf & Wilson, 1986).

The mechanisms that control the formation and stability of vacuoles and caveolae are not well understood. In rat myotubes, spectrin appears to participate in the formation and stabilization of vacuoles (Herring et al. 2000). Abnormalities in the spectrin skeleton in dystrophin-deficient muscle may cause surface invaginations to be less stable. The resting tension across the membrane is determined, in part, by forces that stabilize surface invaginations. When this force is exceeded by applying suction, vacuoles and/or caveolae in dystrophin-deficient muscle may provide a source of membrane which reduces membrane tension. The existence of vacuoles and/or caveolae alone cannot explain stretch-inactivated gating, since normal muscle possesses these features yet rarely exhibit this gating mode. Rather, cytoskeletal defects in dystrophin-deficient muscle may render surface invaginations sufficiently labile to reconfigure in response to moderate changes in tension.

Relationship to the pathogenesis of Duchenne muscular dystrophy

Although an absence of dystrophin in skeletal muscle has been known for more than a decade to be the primary genetic defect in Duchenne dystrophy, it is still unclear how its absence leads to progressive muscle degeneration. Two general hypotheses have been proposed to account for muscle degeneration in Duchenne dystrophy. Early reports showed that muscle fibres from humans with Duchenne dystrophy have discrete lesions in the sarcolemma and underlying sarcoplasm (Mokri & Engel, 1975; Schmalbruch, 1975; Carpenter & Karpati, 1979). Later it was shown that dystrophin-deficient muscle is more susceptible to membrane damage produced by contraction-induced stresses (Weller et al. 1990; Petrof et al. 1993; Moens et al. 1993). These observations and the finding that dystrophin links the actin-based cortical cytoskeleton with the extracellular matrix (reviewed in Straub & Campbell, 1997) suggested the idea that dystrophin plays a structural role by supporting and distributing the stresses that develop within the sarcolemma during muscle contraction.

While conceptually appealing, the ‘structural hypothesis’ in its simplest form is not consistent with a number of important findings. First, there is little evidence that the membrane of dystrophic muscle is actually weaker than normal muscle as judged by measurements of lytic tension or by pipette aspiration (e.g. Franco & Lansman, 1990b; Hutter et al. 1991). Second, while muscle contraction causes lesions in the membrane of dystrophin-deficient muscle, these lesions are rapidly repaired (Carpenter & Karpati, 1979). In the mdx mouse, these repaired fibres remain relatively resistant to degeneration, despite the fact that they are subject to injury throughout the life of the animal (Dimario et al. 1991). Moreover, plasma membrane lesions that disrupt the membrane permeability barrier and presumably cause Ca2+ leakage do not cause fibres to become hypercontracted, a sign of Ca2+ overload, and do not necessarily lead to fibre necrosis (Straub et al. 1997). Finally, mutations that affect the actin binding domain of dystrophin and would be expected to reduce significantly the ability of dystrophin to redistribute contractile forces cause virtually no muscle pathology (Rafael et al. 1996).

An alternative hypothesis is that dystrophin deficiency leads to an increase in the activity of specific membrane Ca2+ channels and, consequently, to an elevation of cytoplasmic Ca2+ levels. There have been numerous studies of Ca2+ homeostasis in dystrophic muscle and these have been reviewed in detail (Gillis, 1996, 1999). There have been two general problems with the Ca2+ hypothesis. First, several investigators have been unable to demonstrate elevated levels of cytosolic Ca2+ in dystrophin-deficient muscle (e.g. Head, 1993; Pressmar et al. 1994; Collet et al. 1999). Second, the mechanism by which an absence of dystrophin might modify membrane Ca2+ channels is not clear, since dystrophin is not known to be associated with any ion channel proteins. Several studies, however, have shown that elevated cytosolic Ca2+ levels can be detected in dystrophin-deficient muscle that is allowed to undergo repeated cycles of contraction (Imbert et al. 1995) or exposed to hyposmotic stress (Imbert et al. 1996; Leijendekker et al. 1996). Moreover, hyposmotic-induced increases in cytosolic Ca2+ that occurs in mdx myotubes (Leijendekker et al. 1996) and myotubes from humans with Duchenne muscular dystrophy (Imbert et al. 1996) are blocked by the lanthanide cation, Gd3+, an inhibitor of both stretch-activated (Yang & Sachs, 1989; Franco & Lansman, 1990b) and stretch-inactivated channels (Franco et al. 1991) in skeletal muscle. Increased Ca2+ entry through Gd3+-sensitive channels in dystrophin-deficient fibres has also been reported by Tutdibi et al. (1999) using the Mn2+ quench technique to monitor divalent cation entry. Identifying the channels that mediate Ca2+ entry is made difficult by the fact that Gd3+ is not a selective inhibitor of MS channels, but blocks other types of Ca2+ channels (e.g. Lansman, 1990). Nonetheless, the available data are consistent with the idea that Ca2+ entry into dystrophin-deficient muscle involves mechanosensitive ion channels whose activity is increased by membrane stress.

Our results provide new information that may help in understanding the pathophysiology of dystrophin deficiency, particularly in how contraction-induced membrane stresses might be coupled to increased Ca2+ entry. The available data suggest that contraction-induced stresses may not cause muscle degeneration by directly damaging the cell membrane. Rather, contraction-induced stresses may disrupt membrane structure in spectrin-deficient regions in muscle lacking dystrophin and, hence, alter MS channel gating in these regions. MS channels might also selectively cluster at these sites producing hot spots of activity. High rates of Ca2+ entry through MS channels with altered gating could cause a persistent weakening of the plasma membrane that is not easily repaired. Alternatively, high rates of Ca2+ entry could modify Ca2+-sensitive signalling pathways that are localized at the membrane near sites of entry. Recently, domains of elevated submembrane Ca2+ have been detected using the activity of Ca2+-activated K+ channels as probes of local Ca2+ concentrations (Mallouk et al. 2000). Whether contraction-induced stresses are sufficient to produce changes like those that occur during patch recordings remains an open question. Answers to this question will require non-invasive measurements of mechanosensitive ion fluxes in dystrophic muscle subjected to stress.

Acknowledgments

We thank Dr Ellen Lumpkin for helpful comments on the manuscript. This work was supported by grants from the NIH, Muscular Dystrophy Association, and Office of Army Research.

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