Recently, we demonstrated a novel interaction between large-conductance (maxi-K or KCa1.1) and intermediate-conductance (IK1 or KCa3.1) Ca2+-activated K channels: activation of IK1 channels causes the inhibition of maxi-K activity (Thompson J and Begenisich T. J Gen Physiol 127: 159–169, 2006). Here we show that the interaction between these two channels can be regulated by the membrane cholesterol level in parotid acinar cells. Depletion of cholesterol using methyl-β-cyclodextrin weakened, while cholesterol enrichment increased, the ability of IK1 activation to inhibit maxi-K channels. Cholesterol's stereoisomer, epicholesterol, was unable to substitute for cholesterol in the interaction between the two K channels, suggesting a specific cholesterol-protein interaction. This suggestion was strengthened by the results of experiments in which cholesterol was replaced by coprostanol and epicoprostanol. These two sterols have nearly identical effects on membrane physical properties and cholesterol-rich microdomain stability, but had very different effects on the IK1/maxi-K interaction. In addition, the IK1/maxi-K interaction was unaltered in cells lacking caveolin, the protein essential for formation and stability of caveolae. Finally, disruption of the actin cytoskeleton restored the IK1-induced maxi-K inhibition that was lost with cell cholesterol depletion, demonstrating the importance of an intact cytoskeleton for the cholesterol-dependent regulation of the IK1/maxi-K interaction
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