Cholesterol as a determinant of cooperativity in the M2 muscarinic cholinergic receptor

M-2 muscarinic receptor extracted from Sf9 cells in cholate-NaCl differs from that extracted from porcine sarcolemma. The latter has been shown to exhibit an anomalous pattern in which the capacity for N-[H]methylscopolamine (NMS) is only 50% of that for [H-3]quinuclidinylbenzilate (QNB), yet unlabeled NMS exhibits high affinity for all of the sites labeled by [H-3]QNB. The effects can be explained in terms of cooperativity within a receptor that is at least tetravalent [Park PS, Sum CS, Pawagi AB, Wells JW. Cooperativity and oligomeric status of cardiac muscarinic cholinergic receptors. Biochemistry 2002;41:5588-6041. In contrast, M-2 receptor extracted from Sf9 membranes exhibited no shortfall in the capacity for [H-3]NMS at either 30 or 4 degrees C, although there was a time-dependent inactivation during incubation with [3H]NMS at 30 degrees C; also, any discrepancies in the affinity of NMS were comparatively small. The level of cholesterol in Sf9 membranes was only 4% of that in sarcolemmal membranes, and it was increased to about 100% by means of cholesterol- methyl- beta -cyclodextrin. M2 receptors extracted from treated Sf9 membranes were stable at 30 and 4 degrees C and resembled those from heart. Cholesterol induced a marked heterogeneity detected in the binding of both radioligands, including a shortfall in the apparent capacity for [3H]NMS, and there were significant discrepancies in the apparent affinity of NMS as estimated directly and via the inhibition of [3 H]QNB. The data can be described quantitatively in terms of cooperative effects among six or more interacting sites. Cholesterol therefore appears to promote cooperativity in the binding of antagonists to the M-2 muscarinic receptor. (C) 2007 Elsevier Inc. All rights reserved.