Bs. Launikonis et Dg. Stephenson, Effects of membrane cholesterol manipulation on excitation-contraction coupling in skeletal muscle of the toad, J PHYSL LON, 534(1), 2001, pp. 71-85
1. Single mechanically skinned fibres and intact bundles of fibres from the
twitch region of the iliofibularis muscle of cane toads were used to inves
tigate the effects of membrane cholesterol manipulation on excitation-contr
action (E-C) coupling. The cholesterol content of membranes was manipulated
with methyl-beta -cyclodextrin (M beta CD).
2. In mechanically skinned fibres, depletion of membrane cholesterol with M
beta CD caused a dose-and time-dependent decrease in transverse tubular (t
)-system depolarization-induced force responses (TSDIFRs). TSDIFRs were com
pletely abolished within 2 min in the presence of 10 mar M beta CD but were
not affected after 2 min in the presence of a 10 mM M beta CD-1, mar chole
sterol complex. There was a very steep dependence between the change in TSD
IFRs and the M beta CD : cholesterol ratio at 10 mM M beta CD, indicating t
hat the inhibitory effect of M beta CD was due to membrane cholesterol depl
etion and not to a pharmacological effect of the agent. Tetanic responses i
n bundles of intact fibres were abolished after 3-4 h in the presence of 10
mM M beta CD.
3. The duration of TSDIFRs increased markedly soon (< 2 min) after applicat
ion of 10 mM M beta CD and 10 mM M beta CD-cholesterol complexes, but the C
a2+ activation properties of the contractile apparatus were minimally affec
ted by 10 mM M beta CD. The Ca2+ handling abilities of the sarcoplasmic ret
iculum appeared to be modified after 10 min exposure to 10 mM M beta CD.
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 M beta CD and that a large [Ca2+] gradient was maintained across the
t-system.
5. Membrane cholesterol depletion caused rapid depolarization of the polari
zed t-system as shown independently by spontaneous TSDIFRs induced by M bet
a CD and by changes in the fluorescence intensity of an anionic potentiomet
ric dye (DiBAC(4)(3)) in the presence of M beta CD. This rapid depolarizati
on of the t-system by cholesterol depletion was not prevented by blocking t
he Na+ channels with TTX (10 muM) or the L-type Ca2+ channels with Co2+ (5
mar).
6. The results demonstrate that cholesterol is important for maintaining th
e functional integrity of the t-system and sarcoplasmic reticulum, probably
by having specific effects on different membrane proteins that may be dire
ctly or indirectly involved in E-C coupling.