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

M2 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-[3H]methylscopolamine (NMS) is only 50% of that for [3H]quinuclidinylbenzilate (QNB), yet unlabeled NMS exhibits high affinity for all of the sites labeled by [3H]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;41:5588-604]. In contrast, M2 receptor extracted from Sf9 membranes exhibited no shortfall in the capacity for [3H]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 [3H]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 M2 muscarinic receptor.