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 B_max 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 E_max 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 E_max of cAMP synthesis inhibition or stimulation without change in potency, and decreased E_max of IP accumulation. Noteworthy, modifications of membrane cholesterol had no effect on membrane permeability, oxidative activity, protein content, or relative expression of G_s, G_i/o, and G_q/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 G_q/11 signaling for the pathogenesis of Alzheimer's disease.