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

Bradley S. Launikonis and D. George Stephenson

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.

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