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 M-2 muscarinic acetylcholine receptor expressed in CHO cells. Cholesterol depletion by 39% significantly decreased the affinity of M-2 receptors for [H-3]-N-methylscopolamine ([H-3]-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 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 M-2. 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 M-3 receptors and by both cholesterol depletion and enrichment in cells expressing M, receptors indicating relevance of reduced G(q/11) signaling for the pathogenesis of Alzheimer's disease. (C) 2009 Elsevier B.V. All rights reserved.