Membrane cholesterol content modulates ClC-2 gating and sensitivity to oxidative stress.
ClC-2 is a broadly expressed member of the voltage-gated ClC chloride channel family. In this study, we aimed to evaluate the role of the membrane lipid environment in ClC-2 function, and in particular the effect of cholesterol and ClC-2 distribution in membrane microdomains. Detergent-resistant and detergent-soluble microdomains (DSM) were isolated from stably transfected HEK293 cells by a discontinuous OptiPrep gradient. ClC-2 was found concentrated in detergent-insoluble membranes in basal conditions and relocalized to DSM upon cholesterol depletion by methyl-beta-cyclodextrin. As assessed by patch clamp recordings, relocalization was accompanied by acceleration of the activation kinetics of the channel. A similar distribution and activation pattern were obtained when cells were treated with the oxidant tert-butyl hydroperoxide and after ATP depletion. In both cases activation was prevented by cholesterol enrichment of cells. We conclude that the cholesterol environment regulates ClC-2 activity, and we provide evidence that the increase in ClC-2 activity in response to acute oxidative or metabolic stress involves relocalization of this channel to DSM.
Hinzpeter
Alexandre
A
INSERM, U806, Université Paris-Descartes, Faculté de Médecine René Descartes, F-75015 Paris, France.
Fritsch
Janine
J
Borot
Florence
F
Trudel
Stéphanie
S
Vieu
Diane-Lore
DL
Brouillard
Franck
F
Baudouin-Legros
Maryvonne
M
Clain
Jérôme
J
Edelman
Aleksander
A
Ollero
Mario
M
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D018118:Chloride Channels / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating; D008563:Membrane Lipids / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism; D008564:Membrane Potentials; D018384:Oxidative Stress; D018408:Patch-Clamp Techniques; D051381:Rats
D018118:Chloride Channels; D008563:Membrane Lipids; D002784:Cholesterol