Cholesterol Depletion Inhibits Store-Operated Calcium Currents and Exocytotic Membrane Fusion in RBL-2H3 Cells

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Abstract
The effects of cholesterol depletion from the plasma membrane with methyl-β-cyclodextrin (MβCD) on exocytotic processes were investigated in rat basophil leukemia cells (RBL-2H3 cells). Pretreatment of the cells with MβCD inhibited antigen-evoked exocytotic release dose-dependently. To elucidate the mechanism of this inhibition, we performed experiments on the effects of MβCD on exocytotic membrane fusion and mobilization of Ca2+ and on the localization of the tyrosine kinase Lyn. Inhibition of degranulation by MβCD was observed even under stimulation with the phorbol ester and calcium ionophore. Therefore, MβCD affected a process downstream of Ca2+ influx, or membrane fusion between the granule and the plasma membrane. Intracellular calcium measurements revealed that MβCD inhibited the Ca2+ increase induced by antigen. Furthermore, we found that MβCD significantly inhibited Ca2+ influx from the extracellular medium through the store-operated calcium channel (SOC) but did not affect Ca2+ release from the intracellular Ca2+ store. Fluorescent image analysis of cells expressing Lyn-YFP showed that treatment with MβCD scarcely affected the localization and lateral mobility of Lyn in the plasma membrane. These results suggest that cholesterol depletion by MβCD decreases degranulation mainly by inhibiting the SOC and membrane fusion between the secretory granules and the plasma membrane in mast cells.