title: Membrane cholesterol modulates dihydropyridine receptor function in mice fetal skeletal muscle cells.
abstract: Caveolae and transverse (T-) tubules are membrane structures enriched in cholesterol and glycosphingolipids. They play an important role in receptor signalling and myogenesis. The T-system is also highly enriched in dihydropyridine receptors (DHPRs), which control excitation-contraction (E-C) coupling. Recent results have shown that a depletion of membrane cholesterol alters caveolae and T-tubules, yet detailed functional studies of DHPR expression are lacking. Here we studied electrophysiological and morphological effects of methyl-beta-cyclodextrin (MbetaCD), a cholesterol-sequestering drug, on freshly isolated fetal skeletal muscle cells. Exposure of fetal myofibres to 1-3 mM MbetaCD for 1 h at 37 degrees C led to a significant reduction in caveolae and T-tubule areas and to a decrease in cell membrane electrical capacitance. In whole-cell voltage-clamp experiments, the L-type Ca(2+) current amplitude was significantly reduced, and its voltage dependence was shifted approximately 15 mV towards more positive potentials. Activation and inactivation kinetics were slower in treated cells than in control cells and stimulation by a saturating concentration of Bay K 8644 was enhanced. In addition, intramembrane charge movement and Ca(2+) transients evoked by a depolarization were reduced without a shift of the midpoint, indicating a weakening of E-C coupling. In contrast, T-type Ca(2+) current was not affected by MbetaCD treatment. Most of the L-type Ca(2+) conductance reduction and E-C coupling weakening could be explained by a decrease of the number of DHPRs due to the disruption of caveolae and T-tubules. However, the effects on L-type channel gating kinetics suggest that membrane cholesterol content modulates DHPR function. Moreover, the significant shift of the voltage dependence of L-type current without any change in the voltage dependence of charge movement and Ca(2+) transients suggests that cholesterol differentially regulates the two functions of the DHPR.
authors.affiliation: LNPC, CNRS UMR 6150, Faculté Médecine Nord, Bd Pierre Dramard, 13916 Marseille Cedex 20, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Pouvreau
authors.forename: Christine
authors.initials: C
authors.lastname: Berthier
authors.forename: Sylvie
authors.initials: S
authors.lastname: Blaineau
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Amsellem
authors.forename: Roberto
authors.initials: R
authors.lastname: Coronado
authors.forename: Caroline
authors.initials: C
authors.lastname: Strube
mesh_terms: D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000818:Animals; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000502:physiology*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002462:Cell Membrane / Q000502:physiology; D002469:Cell Separation; D002784:Cholesterol / Q000502:physiology*; D003505:Cyclodextrins / Q000494:pharmacology; D004558:Electric Stimulation; D005260:Female; D007091:Image Processing, Computer-Assisted; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000502:physiology / Q000648:ultrastructure; D018482:Muscle, Skeletal / Q000166:cytology / Q000502:physiology*; D018408:Patch-Clamp Techniques; D011247:Pregnancy; D047392:beta-Cyclodextrins
chemical_list: D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D003505:Cyclodextrins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D002784:Cholesterol