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Journal of Physiology (2001), 534.1, pp. 71-85
© Copyright 2001 The Physiological Society

Effects of membrane cholesterol manipulation on excitation-contraction coupling in skeletal muscle of the toad

Bradley S. Launikonis and D. George Stephenson

	ABSTRACT

Top Abstract Introduction Methods Results Discussion References

1. Single mechanically skinned fibres and intact bundles of fibres from the twitch region of the iliofibularis muscle of cane toads were used to investigate the effects of membrane cholesterol manipulation on excitation-contraction (E-C) coupling. The cholesterol content of membranes was manipulated with methyl--cyclodextrin (MCD).
2. In mechanically skinned fibres, depletion of membrane cholesterol with MCD caused a dose- and time-dependent decrease in transverse tubular (t)-system depolarization-induced force responses (TSDIFRs). TSDIFRs were completely abolished within 2 min in the presence of 10 mM MCD but were not affected after 2 min in the presence of a 10 mM MCD-1 mM cholesterol complex. There was a very steep dependence between the change in TSDIFRs and the MCD : cholesterol ratio at 10 mM MCD, indicating that the inhibitory effect of MCD was due to membrane cholesterol depletion and not to a pharmacological effect of the agent. Tetanic responses in bundles of intact fibres were abolished after 3-4 h in the presence of 10 mM MCD.
3. The duration of TSDIFRs increased markedly soon (< 2 min) after application of 10 mM MCD and 10 mM MCD-cholesterol complexes, but the Ca²⁺ activation properties of the contractile apparatus were minimally affected by 10 mM MCD. The Ca²⁺ handling abilities of the sarcoplasmic reticulum appeared to be modified after 10 min exposure to 10 mM MCD.
4. Confocal laser scanning microscopy revealed that the integrity of the t-system was not compromised by either intra- or extracellular application of 10 mM MCD and that a large [Ca²⁺] gradient was maintained across the t-system.
5. Membrane cholesterol depletion caused rapid depolarization of the polarized t-system as shown independently by spontaneous TSDIFRs induced by MCD and by changes in the fluorescence intensity of an anionic potentiometric dye (DiBAC₄(3)) in the presence of MCD. This rapid depolarization of the t-system by cholesterol depletion was not prevented by blocking the Na⁺ channels with TTX (10 µM) or the L-type Ca²⁺ channels with Co²⁺ (5 mM).
6. The results demonstrate that cholesterol is important for maintaining the functional integrity of the t-system and sarcoplasmic reticulum, probably by having specific effects on different membrane proteins that may be directly or indirectly involved in E-C coupling.

	INTRODUCTION

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Cholesterol is an integral component of the membranes of all eukaryotic cells including muscle. In the surface membrane, cholesterol is as high as 35-45 mol % of all lipid molecules in the membrane and represents about 80-90 % of total membrane cholesterol in cells (Yeagle, 1993). Importantly, the cholesterol content of membranes has been shown to affect a variety of membrane proteins, including ion channels, receptors and pumps and it is now clear that some membrane proteins require a certain level of membrane cholesterol for normal functioning (for reviews, see Yeagle, 1985; Bastiaanse et al. 1997). Changing the cholesterol content of membranes alters the bulk characteristics of the bilayer. Thus, increasing the membrane cholesterol content would decrease the 'free volume' proteins have in which to change their conformation for functional purposes and vice versa. Therefore, one could expect that manipulation of cholesterol in a membrane would alter the function of membrane proteins if freedom of movement between conformational forms were restricted or enhanced. The function of the membrane proteins can also be affected by a more specific cholesterol-protein interaction rather than by cholesterol-induced changes of the bulk membrane properties. Such a mode of action has been suggested for the sarcolemmal Ca²⁺ channel (Renaud et al. 1982; Locher et al. 1984; Aepfelbacher et al. 1991; Bastiaanse et al. 1994), the Na⁺-K⁺ exchanger (Claret et al. 1978; Giraud et al. 1981; Yeagle, 1983; Kutryk & Pierce, 1988; Yeagle et al. 1988) and the human oxytocin receptor (Klein et al. 1995; Gimpl et al. 1997).

The process of excitation-contraction coupling (E-C coupling) in skeletal muscle involves signalling between two membrane systems: the transverse tubular (t-) system and the sarcoplasmic reticulum (SR) (Melzer et al. 1995). The two membrane systems differ greatly in their cholesterol content (Jamieson & Robinson, 1977), as is shown by experiments with saponin and -escin, which bind to cholesterol in the membrane and form large non-selective pores (Launikonis & Stephenson, 1997, 1999), but otherwise little is known about whether membrane cholesterol plays any role in E-C coupling.

Unlike saponin and -escin, which bind to membrane cholesterol, the cyclic oligosaccharide methyl--cyclodextrin (MCD) has been shown to act as an efficient agent for manipulating the cholesterol content in membranes (Gimpl et al. 1997). The MCD molecule has hydrophilic groups on the outside, which permit it to dissolve freely in an aqueous environment, while forming a hydrophobic ring structure. Thus MCD acts as a vehicle for the transportation of cholesterol in an aqueous environment, allowing cholesterol to move between the cyclodextrin and the membrane (Bender & Komiyama, 1978; Szejtli, 1982). Therefore MCD allows membrane cholesterol content to be efficiently manipulated in functional cell membranes (Gimpl et al. 1997).

The aim of this study was to examine the role of membrane cholesterol in the E-C coupling process by manipulating cholesterol content with MCD in the t-system and the SR membranes of skeletal muscle fibres. Since cholesterol molecules readily and rapidly 'flip-flop' between the two lipid layers of biological membranes with half-times shorter than several seconds (Lange et al. 1981), MCD can be applied to either side of a biological membrane to manipulate the cholesterol content in that membrane as long as it can come into close proximity to the cholesterol molecules in the lipid bilayer. The presence of an extensive extracellular matrix covering the surface membrane of skeletal muscle fibres can act as a significant barrier for cyclodextrin molecules reaching the outer lipid layer of the plasma membrane. Considering further the complex geometry of the t-system and the ability of intracellular systems to 'buffer' the cholesterol content of the surface membrane, extracellular application of MCD appears considerably less efficient and less reliable than intracellular application of MCD for specifically manipulating the cholesterol content of the t-system and SR in muscle. In this study we aimed therefore to investigate the effect of membrane cholesterol manipulation on E-C coupling when MCD was predominantly applied to the lipid layers of the t-system and SR membrane facing the myoplasmic compartment. For this purpose we used a mechanically skinned fibre preparation where normal voltage control of Ca²⁺ release (Lamb & Stephenson, 1990; Posterino et al. 2000) and SR function are retained (Endo, 1977) while allowing direct access to the myoplasmic environment. Preliminary results have been published elsewhere (Launikonis & Stephenson, 2000).

	METHODS

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Isolation and mounting of preparations

Cane toads (Bufo marinus) were stunned with a heavy blow to the head and killed by double pithing under permits granted by the Animal Ethics and Experimentation Committee at La Trobe University. The iliofibularis muscles were removed and prepared as described previously (Lamb & Stephenson, 1990). Isolated fibres were mechanically skinned and attached to a sensitive force transducer (AME875, Horten, Norway) whilst under paraffin oil and the length (L) and diameter (D) measured as described previously (Lamb & Stephenson, 1990). The fibre preparation was then stretched by 20 % above resting level to increase the sensitivity for detection of Ca²⁺ release in any part of the preparation. Fibres were then bathed for 2 min in a standard K⁺-repriming solution containing 50 mM potassium hexamethylenediamine tetraacetate (HDTA; Fluka, Buchs, Switzerland; see below), which mimicked the myoplasmic environment. From this solution the skinned fibre preparation was transferred to different types of solutions as specified in the text.

In other experiments, intact fibre bundles (diameter range: 0.84-1.70 mm) were dissected from the iliofibularis muscle in a physiological solution (PS) containing (mM): NaCl, 115; KCl, 2.5; CaCl₂, 1.8; MgCl₂, 1, Na₂HPO₄, 2.2; NaH₂PO₄, 1 with pH adjusted to 7.15. The preparations were then attached to a Grass force transducer and stimulated at 100 Hz with 1 ms supramaximal pulses.

Solutions

The standard K⁺-repriming solution contained (mM): K⁺, 117; Na⁺, 36; HDTA, 50; free Mg²⁺, 1; MgATP, 7 (total ATP, 8); phosphocreatine, 10; NaN₃, 1; EGTA, 0.025; Hepes, 60 with pH 7.10 and pCa (-log₁₀[Ca²⁺]) 7.1. The Na⁺-depolarizing solution was similar except that all K⁺ was replaced with Na⁺. The low-Mg²⁺ solution was also similar to the K⁺-repriming solution except that MgATP and ionised Mg²⁺ were reduced from 7 to 2 and from 1 to 0.05 mM, respectively, and K⁺ was increased by about 9 mM. For experiments involving caffeine-induced force responses, a standard K⁺-HDTA solution similar in composition to K⁺-repriming solution was used except that it contained caffeine (30 mM) and [EGTA] was increased from 0.025 to 0.5 mM (pCa > 8) (Ca²⁺ release solution). For loading Ca²⁺ into skinned fibres a K⁺-HDTA solution similar to the K⁺-repriming solution at pCa 6.7 containing 0.5 mM CaEGTA/EGTA was used (Ca²⁺ loading solution). The SR Ca²⁺ leak solution was similar to the standard repriming solution but contained 0.5 mM EGTA (pCa > 8) to prevent the SR Ca²⁺ pump from operating. Maximum Ca²⁺ activation was achieved in maximum Ca²⁺ activation solution, which was similar to K⁺-repriming solution but 50 mM HDTA was replaced with 50 mM CaEGTA/EGTA (pCa 4.5). Fibres were finally relaxed in high-EGTA relaxing solution, which contained 50 mM EGTA (pCa > 9) instead of HDTA. All solutions were prepared according to Stephenson & Williams (1981). The final osmolality of all solutions was 250 ± 10 mosmol kg^-1.

MCD (Aldrich, Milwaukee, WI, USA) was dissolved in K⁺-HDTA, K⁺-repriming, Na⁺-depolarizing solution or PS as required. Stock solutions of saponin (50 mg ml^-1; UNILAB, Auburn, NSW, Australia) were freshly prepared in distilled water and added to K⁺-HDTA solutions as required. MCD-cholesterol complexes were prepared following the procedure described by Christian et al. (1997).

Depolarization-induced force responses

The protocol for depolarization-induced force response experiments is described in detail elsewhere (Lamb & Stephenson, 1990). Briefly, depolarization-induced responses were elicited after preparations were first equilibrated in K⁺-repriming solution for 60 s to allow the membrane potential across the sealed t-system to be established close to resting physiological levels, and then the preparation was rapidly transferred to the Na⁺-depolarizing solution, which caused depolarization of the t-system (see Figs 1, 3 and 8).

In some experiments, the Na⁺ and Ca²⁺ channels were blocked with tetrodotoxin (TTX) and Co²⁺, respectively, prior to skinning. For this purpose, iliofibularis muscles were bathed for at least 45 min in PS containing, in addition, one of either 10 µM TTX or 5 mM CoCl₂. Only superficial fibres from treated muscles were used in experiments.

SR Ca²⁺ loading ability and leak experiments

We have previously presented a full description and examples of the protocols for determining SR Ca²⁺ loading ability and Ca²⁺ leak rate (Launikonis & Stephenson, 1997). Briefly, the relative SR Ca²⁺ content was assessed from the relative areas under the 30 mM caffeine-induced force responses which, under our conditions, are directly proportional to the SR Ca²⁺ content and do not require any correction (see Launikonis & Stephenson, 1997). The result is expressed as 'relative response', which is the average area under two to three caffeine-induced force responses after MCD exposure divided by the average of the same number of responses before exposure. The SR Ca²⁺ leak rate was assessed from the areas under the subsequent caffeine-induced force responses following the leak of Ca²⁺ from the SR for two defined time periods (t₁ and t₂ where t₂ < t₁) from the same initial load of Ca²⁺ ([Ca²⁺]_SR). The SR Ca²⁺ leak rate is directly related to the SR Ca²⁺ retention index ([Ca²⁺]_SR,t1/[Ca²⁺]_SR,t2). A SR Ca²⁺ retention index value close to 1 indicates a relatively small SR Ca²⁺ leak rate and a value close to 0 indicates a large SR Ca²⁺ leak rate. In these experiments, we have chosen t values of t₁ = 90 s and t₂ = 30 s and the Ca²⁺ retention index was compared before and after MCD exposure.

Confocal imaging

The fluorescent dye fluo-3 (Molecular Probes, Eugene, OR, USA) was dissolved (1 mM) in PS with raised CaCl₂ (3.5 mM) to compensate for the calcium bound to the dye and administered via a 1 µl microcap pipette (Drummond Scientific, Broomall, PA, USA) to single isolated fibres with an intact sarcolemma under paraffin oil. The PS containing 1 mM fluo-3 was flushed onto and off an intact segment of the fibre for approximately 1 min. Fibres were then either mechanically skinned or left intact and then were placed on a thin coverslip under a droplet of paraffin oil.

For confocal imaging of the t-system of fibres that had been mechanically skinned, the fibres were viewed under the confocal laser scanning microscope (CLSM; Leitz) and then exposed to K⁺-HDTA solution containing 0.1 mM EGTA in the absence (control) or presence of 10 mM MCD for 10 min. Other skinned fibres were treated with 100 µg ml^-1 saponin in the presence of relaxing solution containing 0.5 mM EGTA for 10 min. After the 10 min treatments the fibres were viewed again under the CLSM to investigate the integrity of the sealed t-system. For examination of the t-system of intact fibres, the whole muscle was pretreated for 1 h in PS in the absence (control) or presence of 30 mM MCD prior to isolation of single fibres. After such treatment, single fibres were placed under paraffin oil and loaded with fluo-3 as described above for viewing under the confocal microscope (see Fig. 6).

The confocal microscope was also used to determine the state of the t-system membrane potential using the potentiometric dye, bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC₄(3); Molecular Probes). This dye has an overall negative charge and will accumulate in membrane-bound compartments that are positively charged. Therefore, when the t-system is normally polarized, the dye will be preferentially distributed within the sealed t-system. The dye (4 µM) was included in all solutions to which the preparations were exposed. All intracellular solutions used in the DiBAC₄(3) experiments contained 4 mM EGTA_total (pCa 7.1) to provide an adequate buffer for Ca²⁺ in the resting physiological range.

The ability of the preparations to establish a membrane potential and subsequently be depolarized was determined by exposure of DiBAC₄(3)-loaded preparations to K⁺-repriming and Na⁺-depolarizing solutions. The effect of MCD on the membrane potential was investigated by bathing preparations in K⁺-repriming solution in the presence of 10 mM MCD (see Fig. 7) and qualitatively analysing the fluorescence pattern. All confocal images were stored on an optical disk for later analysis. All experiments were conducted at 22 ± 2 °C.

Analysis of results

In the text, mean values ± standard error of the mean are given; n is the number of fibres. Student's t test was used to determine statistical significance (P) where appropriate. GraphPad Prism software (GraphPad Software, Inc., San Diego, CA, USA) was used to fit data to various functions.

	RESULTS

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Effect of MCD on E-C coupling

Figure 1 shows a representative example of the effect of 10 mM MCD applied in the Na⁺-depolarizing solution on the subsequent depolarization-induced force responses. After exposure to 10 mM MCD in the presence of Na⁺-depolarizing solution a subsequent depolarization of the t-system resulted in a much reduced peak size compared to that prior to treatment. Exposure to 10 mM MCD in Na⁺-depolarizing solution for 15, 30, 60 and 120 s reduced the amplitude of the subsequent depolarization-induced responses to 51.0 ± 14.0 % (n = 5), 33.1 ± 6.8 % (n = 5), 7.2 ± 2.8 % (n = 3) and 2.1 ± 1.1 % (n = 5) of that prior to treatment, respectively (Fig. 2A). It should also be noted that a large release of Ca²⁺ from the SR could be induced by lowering [Mg²⁺] from 1 to 0.05 mM after exposure to 10 mM MCD for up to 2 min, indicating that the SR could not have been considerably depleted of Ca²⁺ (Figs 1, 3 and 8). In control experiments exposure of the preparation to Na⁺-depolarizing solution for 2 min did not affect the ability of the preparation to respond to t-system depolarization (amplitude was 97.4 ± 3.0 % (n = 3) of that of responses prior to the beginning of the control experiment; Fig. 2).

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Figure 1. Effect of MCD on E-C coupling in mechanically skinned fibres from toad muscle Force responses induced by t-tubule depolarization in Na+-depolarizing solution (Depol) were almost abolished after a 2 min exposure to 10 mM MCD in the presence of Na+-depolarizing solution. Note that MCD was washed off the preparation in Na+-depolarizing solution before transfer to K+-repriming solution. Lowering [Mg2+] from 1 to 0.05 mM after exposure to MCD could induce a large force response, indicating substantial Ca2+ release from the SR. Note that immediately following exposure to low [Mg2+] the preparation was briefly exposed to high-EGTA relaxing solution. The trace at the end of the panel represents the response to maximum Ca2+ activating solution (Max) at pH 7.10 and with 1 mM Mg2+. Artifacts on the force trace between solution changes are due to the transfer of the preparation through the air. Calibration bars: horizontal, 2 s during depolarization-induced and low-Mg2+-induced force responses and 30 s elsewhere; vertical, 50 µN. Fibre dimensions: L = 1.4 mm, D = 28 µm.

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Figure 2. Summary of the effect of MCD on depolarization-induced force responses in toad skinned fibres A, summary of results from 21 toad skinned fibres showing the decrease in the ability of preparations to respond to depolarization of the sealed t-system after exposure to 10 mM MCD for different periods of time. B, summary of results obtained with 18 toad skinned fibres showing the decrease in the ability of preparations to respond to depolarization of the t-system after 2 min exposure to different concentrations of MCD. The data were fitted by linear regression. The results in A and B were determined using the protocol described in Fig. 1 and are expressed as a percentage of the height of depolarization-induced force responses prior to exposure to MCD or to an identical solution without MCD (control). Values of n are given in parentheses. The continuous line in A is an exponential function of type y = Aexpbt, which best fitted the data points, where A = 97.3, b = -0.040 s-1 and r = 0.90.

Figure 2B shows the effect of 2 min treatments of toad preparations with different MCD concentrations (in Na⁺-depolarizing solution) on the response of skinned fibre preparations to t-tubule depolarization after 1 min repriming in the K⁺-repriming solution. The data points were fitted by linear regression. Since the rate of cholesterol efflux induced by cyclodextrins has been shown to be linear over this concentration range for short periods of exposure (Yancey et al. 1996), the results shown in Fig. 2B suggest that a greater efflux of cholesterol from skinned fibres in the presence of higher [MCD] exerted an increased inhibitory action on E-C coupling.

In Fig. 2A, the data points were fitted by a simple exponential function with a rate constant of 0.040 ± 0.010 s^-1 (n = 21). In contrast to the effect of 10 mM MCD alone on t-system depolarization-induced force responses, exposure of skinned fibres to 10 mM MCD complexed with cholesterol in a ratio of 10 :1 for 2 min did not significantly affect the ability of the preparations to respond to t-system depolarization, as illustrated in Fig. 3. The response to Na⁺-induced depolarization was 90.2 ± 9.2 % (n = 4) of the response prior to treatment. Interestingly, a relatively small decrease in the amount of cholesterol complexed with MCD inhibited the ability of skinned fibres to respond to t-tubule depolarization after a 2 min exposure. Thus 10 mM total MCD : cholesterol ratios of 11:1, 12:1 and 20:1 reduced depolarization-induced force responses to 59.7 ± 15.1 % (n = 5), 2.5 ± 1.4 % (n = 3) and 2.1 ± 2.1 % (n = 3), respectively, of the size of responses prior to treatment (Fig. 4). These results show that 10 mM MCD alone and 10 mM MCD-cholesterol complexes with MCD : cholesterol ratios of 12:1 and 20:1 exerted their inhibitory action on the ability of toad skinned fibres to respond to t-system depolarization by removing membrane cholesterol, whereas the amount of cholesterol in the 10 mM MCD-1 mM cholesterol complex was sufficent to prevent such action. This result indicates that MCD per se does not have a pharmacological effect on the preparation but acts via cholesterol depletion.

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Figure 3. Effect of MCD-cholesterol complexes on E-C coupling in mechanically skinned fibres from toad muscle Force responses induced by t-tubule depolarization (Depol) were not significantly affected by 10 mM MCD-1 mM cholesterol in the presence of Na+-depolarizing solution. Note that MCD was washed out of the preparation in Na+-depolarizing solution before transfer to K+-repriming solution. Also, lowering [Mg2+] from 1 to 0.05 mM after exposure to MCD-cholesterol induced a substantial Ca2+ release from the SR. Artifacts on the force trace between solution changes are due to the transfer of the preparation through the air. Calibration bars: horizontal, 2 s during depolarization-induced and low-Mg2+-induced force responses and 30 s elsewhere; vertical, 50 µN. Fibre dimensions: L = 1.5 mm, D = 48 µm.

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Figure 4. Summary of depolarization-induced force responses after 2 min exposure to 10 mM MCD-cholesterol complexes in different ratios The data points were best fitted to a Hill curve with variable slope of type: y = 100/((EC50)nH + [MCD : cholesterol]nH), where nH = 32.5, EC50 = 11.08, r = 0.90. The result is expressed as a percentage of the height of depolarization-induced force responses prior to 2 min exposure to each MCD-cholesterol complex. Values of n are given in parentheses.

To determine the site of action of cholesterol depletion-mediated inhibition of depolarization-induced force responses in toad skinned fibres, the effect of MCD on the various steps in E-C coupling (contractile apparatus, SR and the t-system and components thereof) were examined separately.

Effects of MCD on the contractile apparatus

As shown in Fig. 1 and Fig. 3, exposure to 10 mM MCD did not abolish the ability of the contractile apparatus to develop force in the presence of [Ca²⁺]. However, there was the possibility that MCD did affect to some degree the sensitivity to Ca²⁺ of the contractile apparatus. This was investigated by assessing the effect of 10 mM MCD on submaximal and maximal Ca²⁺ activation of the contractile apparatus of mechanically skinned fibres before, during and after exposure to MCD. Preparations to be examined were initially treated with Triton X-100 (3 % v/v) for 5 min to destroy all membraneous compartments within the preparation but leave the contractile apparatus intact. This was done to stop the SR interfering with any Ca²⁺ movements. Note that the Triton treatment per se does not affect the sensitivity to Ca²⁺ in mechanically skinned skeletal muscle fibres (Fryer et al. 1995). Preparations were then exposed to a series of well-buffered Ca²⁺ solutions of various pCa values (prepared from maximum Ca²⁺ activation and high-EGTA relaxing solutions) in the presence and absence of MCD.

The presence of 10 mM MCD in solutions caused a small reversible shift in the sensitivity of the contractile apparatus to Ca²⁺ to higher pCa values (shift of 0.025 ± 0.005 pCa units; paired data, P < 0.05, n = 3) and reversibly potentiated the maximum Ca²⁺-activated force by 3.1 ± 1.8 % (paired t test, P = 0.03, n = 4). Therefore, it can be concluded that the presence of 10 mM MCD in solution will have a rather small potentiating effect on Ca²⁺-activated force development.

Effect of MCD on SR

In order to determine whether MCD treatment affected SR function, experiments were performed to estimate the passive Ca²⁺ leak rate and the Ca²⁺ loading ability before and after 2 and 10 min exposure to MCD (see Methods). When the SR was loaded with Ca²⁺ close to capacity in the Ca²⁺ loading solution (10 min loading time), MCD treatment (10 min, 10 mM) did not cause any change in the shape or in the area under the 30 mM caffeine-induced force response (relative response: 0.948 ± 0.08; n = 5). This showed that MCD treatment does not alter either the time course of caffeine-induced release from the SR or the SR capacity in the Ca²⁺ loading solution. Treatment of the skinned fibre preparation with 10 mM MCD for 2 min also did not significantly alter the rate of SR Ca²⁺ loading at submaximal levels. However, treatment with 10 mM MCD for 10 min caused a significant decrease in SR Ca²⁺ loading rate, as evidenced by the marked reduction in the area under the caffeine-induced force responses following submaximal SR loading for 2 min in the SR Ca²⁺ loading solution after the MCD treatment. A summary of these results is presented in Fig. 5, which suggests that 10 min treatment with 10 mM MCD decreases the SR Ca²⁺ loading rate. Interestingly, the SR Ca²⁺ retention index, measured from the ratio of the areas under the caffeine-induced force responses obtained when the SR was submaximally loaded with Ca²⁺ to a certain level and then allowed to leak its Ca²⁺ contents for 90 and 30s (see Methods), did not significantly alter after 2 min exposure to MCD, but increased significantly (P < 0.03) after 10 min exposure to 10 mM MCD, from 0.74 ± 0.06 (n = 3) to 0.94 ± 0.01 (n = 4). Since an increase in the SR Ca²⁺ retention index directly implies a decrease in the SR Ca²⁺ leak rate, one can estimate that after the 10 min treatment with MCD, the rate of Ca²⁺ loss from the SR was reduced by a factor of 4.3 from 26 ± 6 to 6 ± 1 % SR Ca²⁺ content min^-1. Therefore, the decrease in the SR Ca²⁺ loading rate induced by 10 min treatment with MCD cannot be explained by increased loss of Ca²⁺ from the SR during loading, implying that the SR Ca²⁺ pump must have become progressively inhibited by exposure to 10 mM MCD. The inhibition of the Ca²⁺ pump by a 10 min treatment with MCD may also explain the decrease in the passive Ca²⁺ leak rate because a large fraction of the Ca²⁺ leak is via the SR Ca²⁺ pump (Duke & Steele, 1998; Macdonald & Stephenson, 2001) and, therefore, inhibition of the Ca²⁺ pump would also be expected to decrease Ca²⁺ leak via the pump. The fact that after 10 min treatment with MCD, the SR Ca²⁺ pump rate appears to have decreased by a factor of 4.1 ± 1.0 (n = 4; paired results), which is similar to the factor by which the SR Ca²⁺ leak rate decreases, explains why the SR capacity in the Ca²⁺ loading solution did not significantly change after the 10 min treatment with MCD, because when the SR is loaded to capacity, the pump rate must equal the leak rate and, if both rates change in proportion, the steady-state level to which the SR fills with Ca²⁺ will not change.

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Figure 5. The effect of treatment with 10 mM MCD on SR Ca2+ loading ability (relative response) and SR Ca2+ retention (Ca2+ retention index) Summary of results showing the Ca2+ retention index (; [Ca2+]SR,90s/[Ca2+]SR,30s; see Methods) and the Ca2+ loading ability (; Relative response; see Methods) after 0, 2 and 10 min exposure to 10 mM MCD. The data points for and were fitted to simple exponential and linear functions, respectively. Results are from 5 () and 9 () fibres. The data points at time 0 correspond to control responses either prior to exposure to 10 mM MCD () or when the preparations were exposed to K+-HDTA solution without 10 mM MCD for 10 min ().

Integrity, [Ca²⁺] gradient and membrane potential of the t-system after MCD exposure

The loss of ability to respond to t-system depolarization following 2 min exposure to 10 mM MCD must be due to changes occurring at the level of the t-system because neither the SR function nor the Ca²⁺ activation properties of the contractile apparatus appear to be impaired by such treatment. Such changes may involve disruption of the integrity of the t-system and/or loss of Ca²⁺ from the t-system leading to voltage sensor inactivation. In order to find out whether such situations occur, the membrane-impermeant form of the Ca²⁺-sensitive fluophore fluo-3 has been trapped in the sealed t-system or in the t-system of intact fibres kept under oil. It has previously been observed that toad intact fibres exposed to fluo-3 display a banded fluorescence pattern in confocal optical sections at the level of the Z-line (Lamb et al. 1995). Also, in fibres exposed to fluo-3 and then mechanically skinned, the same banded fluorescence pattern persists for more than 1 h (Lamb et al. 1995). These results indicate that fluo-3 remains trapped in the t-system and therefore can be used to examine the integrity of [Ca²⁺] gradient across the t-system under various conditions. In Fig. 6 we show a mechanically skinned fibre that had been loaded with fluo-3 both before (Fig. 6A) and after 10 min exposure to 10 mM MCD (Fig. 6B). Note that fluo-3 remains trapped in the preparation in both instances. This result indicates that the integrity of the t-system as visualized by confocal microscopy was not disrupted by a depletion of cholesterol molecules from this membrane which prevented the preparation from responding to t-system depolarization (Fig. 1 and Fig. 2).

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Figure 6. The t-system of intact and mechanically skinned fibres maintains a [Ca2+] gradient and remains intact after exposure to MCD but not saponin A and B, confocal images of a toad skinned fibre before (A) and after (B) exposure to 10 mM MCD for 10 min. C and D, confocal images of two different intact toad fibres loaded with fluo-3 that had been exposed to physiological solution for 1 h in the absence (C) or presence (D) of 30 mM MCD prior to isolation from the iliofibularis muscle. Note the banded fluorescence pattern within the fibre, indicating that fluo-3 and Ca2+ remained trapped in the t-system in A-D. E and F, confocal images of a toad skinned fibre that had been loaded with fluo-3 before (E) and after (F) exposure to 100 µg ml-1 saponin for 10 min. Note that the transverse fluorescence-banding pattern is no longer present in F, indicating that fluo-3 had been lost from the preparation. Scale bar: 10 µm.

As MCD only has access to t-system cholesterol from the cytoplasmic side of the membrane in skinned fibres, the integrity of the t-system after MCD exposure on the extracellular surface of intact fibres was also investigated. Whole iliofibularis muscles were immersed in a toad physiological solution in the presence and absence (control) of 30 mM MCD for 1 h. [MCD] was increased from 10 to 30 mM in these experiments in an attempt to facilitate the diffusion of MCD into the t-system. Also, fibres were taken from the surface of the muscle to ensure that MCD had diffused into the t-system of preparations that were used in the subsequent examination under the confocal microscope.

After the 1 h treatment, single intact fibres were loaded with fluo-3 and kept under oil and examined under the confocal microscope. Keeping intact fibres under oil ensured that the fluo-3 remained trapped in the t-system as long as the integrity of the t-system had not been compromised by MCD exposure. It was found that the integrity of the t-system was not affected by 30 mM MCD treatment, since similarly strong fluo-3 fluorescence signals were obtained with control (Fig. 6C) and 30 mM MCD-treated (Fig. 6D) fibres, clearly indicating that the structure of the t-system was not compromised. A similar result was observed in all control and treated fibres (n = 4 for both treatments).

A control experiment was performed to visualize a disrupted t-system by exposing the preparation to saponin, which causes the formation of pores in the t-system membrane and loss of fluo-3 and [Ca²⁺] gradients across the t-system membrane (Bangham & Horne, 1962; Glauert et al. 1962; Endo & Iino, 1980; Launikonis & Stephenson, 1999). In Fig. 6E and F are shown confocal images of a preparation that had been loaded with fluo-3 (Fig. 6E) and subsequently exposed to 100 µg ml^-1 saponin in a relaxing solution for 10 min (Fig. 6F) after mechanical skinning. No distinct fluorescence-banding pattern exists across this preparation after saponin treatment (Fig. 6F). A similar result was obtained in two other preparations.

In order to directly observe if depolarization of the t-system occurs following exposure to 10 mM MCD, toad skinned fibres loaded with the anionic potentiometric dye DiBAC₄(3) were examined under the confocal microscope (Fig. 7). The existence of a membrane potential across the t-system of skinned fibres can be visualized by the reversible change in the fluorescence signal emitted from the preparation when the K⁺-HDTA solution is replaced with the Na⁺-HDTA solution and vice versa. This is shown in Fig. 7 where the fluorescence signal emitted from a depolarized preparation that had been exposed to Na⁺-HDTA solution (4 mM EGTA_total; pCa 7.1) (Fig. 7A) increased markedly within about 10 s after the preparation was transferred to K⁺-HDTA solution (4 mM EGTA_total; pCa 7.1). Note that the structure of the t-system is clearly evident only when the t-system is normally polarized because only then would sufficient dye have accumulated in the t-system compartment since DiBAC₄(3) is an anionic dye that would have distributed predominantly in positively charged compartments compared with the myoplasmic environment. Similar results were obtained in two other preparations. Exposure to 10 mM MCD in the presence of K⁺-HDTA solution (4 mM EGTA_total; pCa 7.1) caused depolarization of the t-system, as indicated by the decrease in fluorescence intensity (Fig. 7C). Note that the confocal image represented in Fig. 7C was taken after about 2 min in the presence of MCD and a very weak fluorescence pattern in the t-system persisted, indicating that the membrane potential difference across the t-system was not completely abolished.

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Figure 7. The t-system membrane is depolarized by exposure to MCD Confocal laser scanning microscopy of a skinned fibre of toad that had been exposed to DiBAC4(3) in the presence of Na+-HDTA solution (A), K+-HDTA solution (B) and 10 mM MCD in the presence of K+-HDTA solution (C). Note that in B the preparation fluoresced intensely and the t-system was clearly evident, indicating that a membrane potential (more positive in the t-system lumen) was established in K+-HDTA solution. The ill-defined fluorescence pattern in C indicates that no significant membrane potential could be maintained in K+-HDTA solution when 10 mM MCD was present. Scale bar: 10 µm.

Depolarizing effect of MCD on the t-system

The experiments described above indicate that MCD-mediated cholesterol efflux causes depolarization of the t-system without disrupting the integrity of or the [Ca²⁺] gradient across this membrane (Fig. 6 and Fig. 7). It appears, therefore, that depletion of membrane cholesterol is affecting t-system excitability via an effect on membrane protein(s) essential to the normal maintenance of the membrane potential. To test this hypothesis further, the effect of membrane cholesterol depletion on t-system function was observed whilst applying MCD directly to the fully polarized t-system and results were contrasted with those from preparations exposed to MCD with briefly depolarised, chronically depolarized and repolarizing t-systems.

In these experiments it was observed that exposing polarized fibres to 10 mM MCD (n = 4) in the presence of K⁺-repriming solution resulted in a transient force response after only a few seconds exposure to this solution (Fig. 8B), whereas no response was seen in briefly depolarized fibres (n = 18) exposed to 10 mM MCD in the presence of Na⁺-depolarizing solution (see Fig. 1). Furthermore, after the sealed t-system was chronically depolarized by exposure to 50 µg ml^-1 saponin, no force response could be induced by 10 mM MCD in K⁺-repriming solution for up to 5 min in four fibres examined (eg. Fig. 8C). These results independently and unequivocally show that cholesterol depletion causes t-system depolarization (Fig. 7 and Fig. 8).

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Figure 8. Effect of MCD-induced cholesterol depletion of the t-system on mechanically skinned fibres from toad muscle A transient force response was elicited in K+-repriming solution in the presence of 10 mM MCD about 12 s after transfer from Na+-depolarizing solution (A) or almost immediately following transfer from K+-repriming solution (B). C, after disruption of the sealed t-system with 50 µg ml-1 saponin, no response could be elicited in the presence of MCD. Lowering [Mg2+] from 1 to 0.05 mM after exposure to MCD could still induce a substantial Ca2+ release from the SR. Note that immediately following exposure to low [Mg2+] the preparation was briefly exposed to high-EGTA relaxing solution. The trace at the end of each panel represents the response to maximum Ca2+ activating solution (Max). Artifacts on the force trace between solution changes are due to the transfer of the preparation through the air. Calibration bars: horizontal, 2 s during depolarization-induced, low-Mg2+-induced and saponin-induced force responses and 30 s elsewhere; vertical, 50 µN. Fibre dimensions: A, L = 1.5 mm, D = 43 µm; B, L = 2.5 mm, D = 43 µm; C, L = 1.9 mm, D = 38 µm.

It was also found that the time taken for initiation of the transient force response in the presence of 10 mM MCD in K⁺-repriming solution was dependent on the state of polarization of the t-system immediately prior to MCD exposure. That is, fibres transferred from K⁺-repriming solution to MCD in the K⁺-repriming solution (i.e. with a polarized t-system) required a significantly shorter period of time to initiate a transient force response (5.0 ± 1.8 s; n = 4; Fig. 8B) than fibres transferred from Na⁺-depolarizing solution (i.e. with a depolarized t-system) to MCD in the K⁺-repriming solution (15.8 ± 0.8 s; n = 4; Fig. 8A) (t test: P < 0.002). The 10 s difference between the times to initiation of the force response is consistent with the time required for the sealed t-system of skinned fibres to polarize to sufficiently negative potentials for normal E-C coupling (Lamb & Stephenson, 1990) and shows that MCD treatment causes rapid depolarization of the t-system if the t-system is well polarized, but the effect is markedly delayed if the t-system is depolarized, because it allows full repolarization of the t-system to occur before causing it to depolarize.

Also, importantly, the response of the preparations to depolarization of the sealed t-system was significantly altered after a short period (< 2 min) of exposure to MCD (e.g. < 1 min in Fig. 8A). Thus, the duration of the depolarization-induced force responses was considerably lengthened immediately following exposure to 10 mM MCD in the preparation represented in Fig. 8A. There was a marked increase in the duration of depolarization-induced force responses in other preparations. On average, the response duration measured at half-height increased by a factor of 2.9 ± 0.8 compared to responses prior to 10 mM MCD exposure (P < 0.005; n = 18). For this evaluation, after treatment with cyclodextrin, only responses with a peak size which was at least 20 % of that of initial responses were considered. There was also an increase in the duration of depolarization-induced force responses in preparations exposed to 10 mM MCD- cholesterol complexes with MCD : cholesterol ratios of 10 : 1 (see Fig. 3) and 11 : 1, by factors of 1.74 ± 0.44 (P > 0.1; n = 3) and 1.51 ± 0.14 (P < 0.05; n = 5), respectively. As argued in the Discussion, these results suggest that depletion of cholesterol caused by MCD does decrease the rate of inactivation of the voltage sensors.

Effect of MCD after blocking Na⁺ and Ca²⁺ channels in the t-system

The possibility that Na⁺ channels and the entry of Ca²⁺ through the Ca²⁺ channel/dihydropyridine (DHP) receptor may have been playing a role in the depolarization effect induced by MCD was examined by blocking Na⁺ channels with TTX and Ca²⁺ channels with Co²⁺. This was achieved by soaking iliofibularis muscles in physiological solution containing 10 µM TTX for approximately 4 h or in PS containing 5 mM Co²⁺ for 45 min (in separate experiments; see Methods). Fibres exposed to TTX and Co²⁺ were capable of depolarization-induced force responses similar to untreated fibres, as observed previously (Lamb & Stephenson, 1990). In TTX-treated fibres, two preparations exposed to 10 mM MCD for 90 s lost the ability to respond to t-system depolarization, which is similar to results shown in Fig. 2A. Two Co²⁺-treated fibres also completely lost the ability to respond to t-system depolarization after 75 s exposure to 10 mM MCD, and one fibre treated with 10 mM MCD for 18 s could only produce depolarization-induced force responses which were 25 % of the response prior to treatment. This shows that Na⁺ channels and Ca²⁺ entry through Ca²⁺ channel/DHP receptors are not likely to be involved in the MCD-mediated effect on t-system depolarization observed in this study.

Effect of MCD on intact fibres

To determine whether MCD has an effect on the excitability of intact iliofibularis muscles, the ability of bundles of these fibres to contract upon electrical stimulation was tested after exposure to 10 mM MCD. Tetanic responses (stimulated at 100 Hz) in three bundles of intact fibres were initially slightly potentiated upon exposure to 10 mM MCD and then gradually decreased until they were almost completely abolished after 270 min. In contrast, control experiments with three preparations in the absence of MCD showed that these preparations produced more than 75 % of initial tetanic force after a similar period of time. A summary of these results is shown in Fig. 9. It is important to note that during exposure to MCD, the baseline level of force of intact bundles did not change, indicating that the sarcolemma remained a selective barrier to the Ca²⁺ present in the physiological solution. These results complement the observations made with the skinned fibre preparations shown in Figs 1, 2, 6 and 7.

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Figure 9. Summary of the effect of MCD on tetanic force responses in bundles of intact muscle fibres Tetanic force responses were abolished in the presence () but not in the absence () of 10 mM MCD. Results are from 3 preparations in both cases. The result is expressed as a percentage of the height of the tetanic force response immediately prior to exposure to MCD or just prior to the beginning of the control experiment. The continuous lines are exponential functions of types which best fitted the data points: y = Aexp(-bt) + c () and y = (A + 100)exp(-b1t) - Aexp(-b2t) (), where A = 28.9 () and 341.8 (), b = 0.012 min-1, b1 = 0.016 min-1, b2 = 0.022 min-1; c = 72.6 and r = 0.71 () and 0.95 ().

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

Importance of membrane cholesterol to E-C coupling

This study has used the cholesterol-depleting agent MCD to investigate the role of membrane cholesterol in functional muscle fibres. MCD is a relatively large molecule of about 1.3 kDa and its efficiency at extracting cholesterol from membranes depends on its ability to come into close proximity to the cholesterol molecules in the lipid bilayer and to the cholesterol molecules directly associated with membrane proteins. The removal of cholesterol with MCD is thus dependent on the accessibility of the cyclodextrin molecules to the various cellular membranes and membrane proteins. It has been argued in the Introduction that the accessibility of MCD to the surface membranes of skeletal muscle fibres should be greater if the cyclodextrin is applied intracellularly than extracellularly. This view is supported by observations made in this study, where exposure of the cytoplasmic side of the t-system in skinned fibres to 10 mM MCD for < 2 min resulted in an almost complete loss of the ability of the preparation to respond to t-system depolarization (Fig. 1 and Fig. 2), while in intact bundles of fibres application of 10 mM MCD for 3-4 h was required before almost complete loss of excitability was achieved (Fig. 9). The increase in the time needed for MCD to cause depolarization in the intact bundles of fibres can be explained by impaired accessibility of the cyclodextrin to the t-system when applied extracellularly (longer diffusion times of MCD from the extracellular solution into the intact t-system and across the basal lamina). Furthermore, extracellular application of MCD can lead to slower cholesterol depletion from the surface membranes, considering that the intracellular environment can act as a cholesterol reservoir (buffer) for cellular membranes and that there is a rapid 'flip-flop' movement of cholesterol molecules between the lipid leaflets in membranes (Lange et al. 1981). As far as we are aware, this is the first time that cholesterol has been depleted from cells by the intracellular application of MCD (i.e. applied to skinned muscle fibres).

The effects of cholesterol removal with MCD were time and concentration dependent (Fig. 2), showing that in these experiments, the removal of cholesterol was not a steady-state process. Nevertheless, the results obtained in this study clearly demonstrate that cholesterol is necessary for maintaining the functional integrity of the skeletal muscle. In particular, this study shows that depletion of t-system cholesterol with MCD causes depolarization and loss of fibre excitability, as well as attenuation of the rate of inactivation of the voltage sensors, and that depletion of SR cholesterol impairs the function of the SR Ca²⁺ pump.

The ability of the skinned fibre preparation to respond to t-system depolarization was preserved when the preparation was exposed to an MCD-cholesterol complex with an MCD : cholesterol ratio of 10 :1 and a total cyclodextrin concentration of 10 mM (Fig. 4). This result demonstrates not only that 10 mM MCD per se has no direct effect on the measured functional properties of the muscle preparation, but also that cholesterol exchange is reversible and rather rapid at the site responsible for this loss of E-C coupling. If the process were not reversible, then, in accordance with results shown in Fig. 2, one would have expected the 9 mM excess MCD to have depleted the cholesterol from the preparation and thus caused a marked loss in the t-system depolarization-induced response. However, this was not the case. Furthermore, the very sharp transition in the effect of the MCD- cholesterol complex on the ability of the skinned fibre preparation to respond to t-system depolarization when the MCD : cholesterol ratio was increased from 10 :1 to 12 :1 at a constant cyclodextrin concentration (10 mM) suggests that cholesterol can be finely buffered with MCD at the site responsible for the loss of E-C coupling.

Next, the Discussion will be focused on the possible mechanisms responsible for the observed effects of cholesterol removal from the t-system and SR membranes on the functional properties of the skeletal muscle fibre.

Cholesterol depletion affects t-system membrane potential

Evidence presented here shows that depletion of cholesterol by MCD affects the ability of the t-system to develop and maintain a normal membrane potential. This could be caused by either a change in the properties of membrane proteins essential to normal maintenance of the electrical potential difference or to the disruption of the lipid bilayer or integrity of the t-system. A rapid disruption of the normally polarized t-system is adequate to activate the voltage sensor and initiate Ca²⁺ release, as shown by exposure to 50 µg ml^-1 saponin (e.g. Fig. 8C; see also Endo & Iino, 1980; Lamb & Stephenson, 1990). Confocal images of toad fibres presented in Fig. 6B and D show that the integrity of the t-system and its ability to maintain a large [Ca²⁺] gradient are not impaired following cholesterol extraction in the presence of 10 mM MCD for 10 min (skinned fibres treated) and 30 mM MCD for 1 h (whole muscle treated), even though the loss of excitability of the skinned fibre preparation occurred within 2 min. This indicates that cholesterol is not required in the plasma membrane to maintain the integrity of the t-system and that the [Ca²⁺] gradient across the t-tubules remains when the membrane is depleted of cholesterol. This result is also consistent with the fact that intact fibre bundles did not develop a contracture during prolonged exposure to MCD (up to 270 min), indicating that the high [Ca²⁺] in the extracellular solution was not accessible to the contractile apparatus. The depolarization of the t-system following extraction of cholesterol in the presence of MCD was also shown by confocal microscopy in conjunction with the potentiometric dye DiBAC₄(3) (Fig. 7) and by the transient force response induced by direct application of MCD to the polarized preparation (Fig. 8B). Taken together, the results indicate that MCD-induced cholesterol depletion must be affecting the normal functioning of membrane proteins essential for the maintenance of the membrane potential.

Experiments performed in this study suggest that the depolarization of the t-system is not due to increased Ca²⁺ permeability and/or loss of Ca²⁺ from the t-system. Specifically Co²⁺, which is known to block the Ca²⁺ flow through the voltage sensor/DHP receptor/L-type Ca²⁺ channel, does not prevent the depolarizing effect of MCD-mediated cholesterol depletion. Also, experiments with fluo-3 indicate that the [Ca²⁺] gradient is maintained after cholesterol depletion (Fig. 6) and a contracture did not develop in intact fibres exposed to high extracellular MCD and Ca²⁺ (Fig. 9). Furthermore, blocking the Na⁺ channels with TTX did not prevent the depolarizing effect of MCD-mediated cholesterol depletion. Thus, it is unlikely that either Na⁺ or Ca²⁺ channels per se in the surface membrane play a major role in the cholesterol-induced depolarization of the t-system. A cholesterol depletion-dependent reduction in Na⁺-K⁺ pump activity (Claret et al. 1978; Giraud et al. 1981; Yeagle, 1983; Kutryk & Pierce, 1988; Yeagle et al. 1988) may be important but cannot be the sole factor involved because the t-system can be repolarized and stay polarized in the presence of 10 mM MCD (Fig. 8A), long after 10 mM MCD induces depolarization of an already polarized t-system (Fig. 8B).

There is evidence to suggest that specific types of K⁺ channels are dependent on membrane cholesterol content (Bolotina et al. 1989; Martens et al. 2000). Martens and colleagues (2000) found that the voltage-gated delayed rectifier Kv2.1 channel, which targets itself to cholesterol-enriched microdomains in membranes, known as lipid rafts, but not the Kv4.1 channel, was affected by cyclodextrin-mediated membrane cholesterol depletion. This suggests that specific interactions of cholesterol with channel proteins are important to normal channel function in some cases. The loss of t-system excitability observed in this study following membrane cholesterol depletion could be explained fully by inhibitory effects of cholesterol depletion on K⁺ channels. One should also bear in mind that caveolae (associated with lipid rafts) have a high cholesterol content and contain a number of channels in their inactive configuration (Anderson, 1998). Removal of cholesterol may render these channels within caveolae active, and this could contribute to changes in membrane permeability for various ions and thus to t-system depolarization.

Cholesterol depletion affects the voltage sensor/DHP receptor

Evidence from this study suggests that the function of the voltage sensor/DHP receptor/L-type Ca²⁺ channel may also have been affected by membrane cholesterol depletion. Thus, the time course of depolarization-induced force responses following cholesterol depletion by MCD was increased significantly (e.g. Fig. 8A). This effect could be due to a slowing of the inactivation of the voltage sensor or to a marked inhibition of the SR Ca²⁺ pump (Bakker et al. 1996). Since the reduction in the SR Ca²⁺ pump activity would have been much too small (Fig. 5) when this effect was obvious (after less than 1 min in 10 mM MCD; Fig. 8A), it follows that treatment with MCD slowed down the inactivation of the voltage sensor, resulting in a more prolonged Ca²⁺ release from the SR and hence a more prolonged force response. The MCD-induced slowing down of inactivation of the voltage sensors cannot be due to a slower depolarization of the t-system when K⁺ is substituted with Na⁺ in the depolarizing solution because, as shown earlier, the t-system becomes less selective for K⁺ than for Na⁺ after MCD treatment and therefore, when Na⁺ is substituted for K⁺, it would be expected to depolarize faster and increase the rate of inactivation of the voltage sensors rather than decrease it. Taken together, these results show that the voltage sensor/DHP receptor/L-type Ca²⁺ channel is more sensitive to membrane cholesterol manipulation than the proteins responsible for membrane excitability, suggesting that a direct cholesterol-protein interaction may be required for the normal functioning of the voltage sensor. Alternatively, the loss of membrane cholesterol may change the bilayer thickness and consequently this could result in an alteration of protein function due to a change in hydrophobic coupling between the bilayer and the transmembrane segments of the voltage sensor (Lundbæk et al. 1996). Other studies have conclusively shown that membrane cholesterol manipulation alters the function of the Ca²⁺ channels in surface membranes (Renaud et al. 1982; Locher et al. 1984; Aepfelbacher et al. 1991; Bastiaanse et al. 1994).

In a previous study, we showed that the depolarization-induced force responses of mechanically skinned fibres from iliofibularis muscle exposed to low concentrations of saponin and -escin did not increase in duration (Launikonis & Stephenson, 1999), suggesting that these cholesterol-interacting agents did not affect the voltage sensors. Since saponin and -escin complex with cholesterol without removing it from membranes (Bangham & Horne, 1962; Glauert et al. 1962), while MCD actually removes cholesterol from membranes, it is conceivable that the presence of cholesterol, more so that the orientation of cholesterol in the membrane, is important to voltage sensor function.

Cholesterol depletion affects the SR function

The SR has a significantly lower cholesterol content than the t-system (Jamieson & Robinson, 1977; Launikonis & Stephenson, 1997 and 1999), totalling less than 2 % of the SR lipid (Meissner & Fleischer, 1971). Consistent with this, the functional properties of the SR were only marginally altered following exposure of the skinned fibre preparation to 10 mM MCD for 2 min, while the t-system became chronically depolarized. Nevertheless, further depletion of SR membrane cholesterol after 10 min exposure to 10 mM MCD resulted in a decreased SR Ca²⁺ loading rate and Ca²⁺ leak rate (Fig. 5). The decreased SR Ca²⁺ loading rate without an increase in the Ca²⁺ leak rate points to the fact that the SR Ca²⁺ pump was inhibited following cholesterol depletion of the SR membrane. Importantly, the inhibition of the SR Ca²⁺ pump can also explain the decrease in the SR Ca²⁺ leak rate, as the Ca²⁺ leak through the SR Ca²⁺ pump represents a major pathway for Ca²⁺ loss from the SR (Duke & Steele, 1998; Macdonald & Stephenson, 2001). Thus, inhibition of the SR Ca²⁺ pump could reduce the Ca²⁺ leak rate in addition to lowering the rate of Ca²⁺ accumulation into the SR. In previous studies, an increase in membrane cholesterol content did not affect the SR Ca²⁺ pump (Warren et al. 1975; Johansson et al. 1981; Simmonds et al. 1982), but another study (Madden et al. 1981) suggests that the SR Ca²⁺ pump may also be inhibited following an increase in SR membrane cholesterol.

The results obtained in this study do not allow a critical evaluation of whether cholesterol depletion from the SR membranes also affects the functional properties of the ryanodine receptor/Ca²⁺ release channel, although a previous study, where saponin was used to complex cholesterol in the SR membrane, suggested that the function of the SR Ca²⁺ release channels may be modulated by cholesterol (Launikonis & Stephenson, 1997).

Concluding remarks

The results of this study have shown that MCD, a specific cholesterol-depleting agent, caused loss of fibre excitability in toad muscle fibres by depolarizing the t-system. The maintenance of the integrity of and [Ca²⁺] gradient across the t-system after membrane cholesterol depletion indicated that the loss of fibre excitability must be due to the effects of cholesterol depletion on the function of membrane proteins, including K⁺ channels and the voltage sensor. The SR function was also affected by the depletion of membrane cholesterol via a decrease in the function of the SR Ca²⁺ pump.

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Acknowledgements

We thank Dr G. D. Lamb fo