Key result
Cholesterol depletion, oxidative stress, and ATP depletion accelerated ClC-2 activation kinetics and relocalized the channel to detergent-soluble microdomains in HEK293 cells.
Why the study?
Does membrane cholesterol content modulate ClC-2 gating and sensitivity to oxidative stress in HEK293 cells?
Does membrane cholesterol content modulate ClC-2 gating and sensitivity to oxidative stress in HEK293 cells?
Membrane cholesterol environment regulates ClC-2 chloride channel activity, and its response to acute oxidative or metabolic stress involves relocalization to detergent-soluble microdomains.
May modulate ClC-2 under oxidative stress in vitro; leaves open relevance to cardiac ion channel regulation.
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.
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Hinzpeter et al. (2006) studied this question. Cholesterol depletion, oxidative stress, and ATP depletion vs. Basal conditions and cholesterol enrichment was evaluated on ClC-2 relocalization and activation kinetics. Cholesterol depletion, oxidative stress, and ATP depletion accelerated ClC-2 activation kinetics and relocalized the channel to detergent-soluble microdomains in HEK293 cells.
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