Cholesterol-dependent regulation of adenosine A2A receptor-mediated anion secretion in colon epithelial cells

Cholesterol affects diverse biological processes, in many cases by modulating the function of integral membrane proteins. In this study we have investigated the role of cholesterol in the adenosine-dependent regulation of ion transport in colonic epithelial cells. We observed that methyl-β-cyclodextrin (MβCD), a cholesterol-sequestering molecule, enhanced adenosine A2A receptor-activated transepithelial short circuit current (Isc), but only from the basolateral side. Cholesterol is a major constituent of membrane microdomains, called lipid rafts that also contain sphingolipids. However, studies with the sphingomyelin-degrading enzyme, sphingomyelinase, and the cholesterol-binding agent, filipin, indicated that the change in the level of cholesterol alone was sufficient to control the adenosine-modulated Isc. Cholesterol depletion had a major effect on the functional selectivity of A2A receptors. Under control conditions, adenosine activated Isc more potently than the specific A2A agonist, CGS-21680, and the current was inhibited by XE991, an inhibitor of cAMP-dependent K+ channels. Following cholesterol depletion, CGS-21680 activated Isc more potently than adenosine, and the current was inhibited by clotrimazole, an inhibitor of Ca2+-activated K+ (IK1) channels. Co-immunoprecipitation experiments revealed that A2A receptors associate with IK1 channels following cholesterol depletion. These results suggest that cholesterol content in colonic epithelia affects adenosine-mediated anion secretion by controlling agonist-selective signaling.