Membrane cholesterol content modulates activation of volume-regulated anion current in bovine endothelial cells

Activation of volume-regulated anion current (VRAC) plays a key role in the maintenance of cellular volume homeostasis. The mechanisms, however, that regulate VRAC activity are not fully understood. We have examined whether V RAC activation is modulated by the cholesterol content of the membrane bila yer. The cholesterol content of bovine aortic endothelial cells was increas ed by two independent methods: (a) exposure to a methyl-beta-cyclodextrin s aturated with cholesterol, or (b) exposure to cholesterol-enriched lipid di spersions. Enrichment of bovine aortic endothelial cells with cholesterol r esulted in a suppression of VRAC activation in response to a mild osmotic g radient, but not to a strong osmotic gradient. Depletion of membrane choles terol by exposing the cells to methyl-beta-cyclodextrin not complexed with cholesterol resulted in an enhancement of VRAC activation when the cells we re challenged with a mild osmotic gradient. VRAC activity in cells challeng ed with a strong osmotic gradient were unaffected by depletion of membrane cholesterol. These observations show that changes in membrane cholesterol c ontent shift VRAC sensitivity to osmotic gradients. Changes in VRAC activat ion were not accompanied by changes in anion permeability ratios, indicatin g that channel selectivity was not affected by the changes in membrane chol esterol. This suggests that membrane cholesterol content affects the equili brium between the closed and open states of VRAC channel rather than the ba sic pore properties of the channel. We hypothesize that changes in membrane cholesterol modulate VRAC activity by affecting the membrane deformation e nergy associated with channel opening.