Membrane cholesterol content influences binding properties of muscarinic M2 receptors and differentially impacts activation of second messenger pathways

We investigated the influence of membrane cholesterol content on preferential and non-preferential signaling through the M2 muscarinic acetylcholine receptor expressed in CHO cells. Cholesterol depletion by 39% significantly decreased the affinity of M2 receptors for [3H]-N-methylscopolamine ([3H]-NMS) binding and increased Bmax in intact cells and membranes. Membranes displayed two-affinity agonist binding sites for carbachol and cholesterol depletion doubled the fraction of high-affinity binding sites. In intact cells it also reduced the rate of agonist-induced receptor internalization and changed the profile of agonist binding from a single site to two affinity states. Cholesterol enrichment by 137% had no effects on carbachol Emax of cAMP synthesis inhibition and on cAMP synthesis stimulation and inositolphosphates (IP) accumulation at higher agonist concentrations (non-preferred pathways). On the other hand, cholesterol depletion significantly increased Emax of cAMP synthesis inhibition or stimulation without change in potency, and decreased Emax of IP accumulation. Noteworthy, modifications of membrane cholesterol had no effect on membrane permeability, oxidative activity, protein content, or relative expression of Gs, Gi/o, and Gq/11 alpha subunits. These results demonstrate distinct changes of M2 receptor signaling through both preferential and non-preferential G-proteins consequent to membrane cholesterol depletion that occur at the level of receptor/G-protein/effector protein interactions in the cell membrane. The significant decrease of IP accumulation by cholesterol depletion was also observed in cells expressing M3 receptors and by both cholesterol depletion and enrichment in cells expressing M1 receptors indicating relevance of reduced Gq/11 signaling for the pathogenesis of Alzheimer's disease.