Regulation of the cell swelling-activated chloride conductance by cholesterol-rich membrane domains

Aim: The role of high cholesterol‐containing microdomains in the signal transduction cascade leading to the activation of volume‐regulated anion channels (VRACs) was studied. Methods: Osmotic cell swelling‐induced efflux of 125I− was determined in human epithelial Intestine 407 cells and in skin fibroblasts obtained from healthy controls or Niemann–Pick type C (NPC) patients. Cellular cholesterol content was modulated by pre‐incubation with 2‐hydroxypropyl‐β‐cyclodextrin in the presence of acceptor lipid vesicles. Results: Osmotic cell swelling of human Intestine 407 cells leads to the rapid activation of a compensatory anion conductance. Treatment of the cells with cyclodextrin enhanced the response to submaximal hypotonic stimulation by approx. twofold, but did not further increase the efflux elicited by a saturating stimulus. In contrast, the volume‐sensitive anion efflux was markedly inhibited when cholesterol‐loaded cyclodextrin was used. Potentiation of the response by cholesterol depletion was maintained in caveolin‐1 deficient Caco‐2 colonocytes as well as in sphingomyelinase‐treated Intestine 407 cells, indicating that cholesterol‐rich microdomains are not crucially involved. However, treatment of the cells with progesterone, an inhibitor of NPC1‐dependent endosomal cholesterol trafficking, not only markedly reduced the hypotonicity‐provoked anion efflux, but also prevented its potentiation by cyclodextrin. In addition, the volume‐sensitive anion efflux from human NPC skin fibroblasts was significantly smaller when compared with control fibroblasts. Conclusions: The results support a model of regulatory volume decrease involving recruitment of volume‐sensitive anion channels from intracellular compartments to the plasma membrane.