Membrane Microviscosity Modulates μ‐Opioid Receptor Conformational Transitions and Agonist Efficacy

: The influence of membrane microviscosity on μ‐opioid agonist and antagonist binding, as well as agonist efficacy, was examined in membranes prepared from SH‐SY5Y cells and from a C6 glioma cell line stably expressing the rat μ‐opioid receptor (C6μ). Addition of cholesteryl hemisuccinate (CHS) to cell membranes increased membrane microviscosity and reduced the inhibitory effect of sodium and guanine nucleotides on the affinity of the full agonists sufentanil and [D‐Ala2,N‐MePhe4,Gly‐ol5]enkephalin (DAMGO) for the μ‐opioid receptor. Binding of the antagonists [3H]naltrexone and [3H]diprenorphine and the partial agonist nalbuphine was unaffected by CHS. The effect of CHS on agonist binding was reversed by subsequent addition of cis‐vaccenic acid, suggesting that the effect of CHS is the result of increased membrane microviscosity and not a specific sterol—receptor interaction. CHS addition increased the potency of DAMGO to stimulate guanosine‐5′‐O‐(3‐[35S]thio)triphosphate binding by fourfold, whereas the potency of nalbuphine was unaffected. However, nalbuphine efficacy relative to that of the full agonist DAMGO was strongly increased in CHS‐treated membranes compared with that in control membranes. Membrane rigidification also resulted in an increased efficacy for the partial agonists meperidine, profadol, and butorphanol relative to that of DAMGO as measured by agonist‐stimulated GTPase activity in control and CHS‐modified membranes. These findings support a regulatory role for membrane microviscosity in receptor‐mediated G protein activation.