Membrane cholesterol removal promotes a low agonist affinity state of mu opioid receptors

Abstract

Previously, we have shown that removal of plasma membrane cholesterol by the cholesterol‐sequestering agent methyl‐β‐cyclodextrin (MβCD) results in a loss of potency of the mu opioid agonist DAMGO to stimulate GTPγ35S binding or to inhibit adenylyl cyclase. Here, we test the hypothesis that this decrease in potency is due to an uncoupling of the mu opioid receptor from G protein. Approximately half of the mu opioid receptors stably expressed in HEK293 cells were present on the plasma membrane, as determined by whole cell binding. Removal of plasma membrane cholesterol with MβCD significantly decreased the maximum binding of the mu opioid agonist 3H‐DAMGO in a cell membrane preparation. This decrease in agonist binding was partially reversed when cholesterol was added back to the membrane, and could not be explained by changes in total receptor number as measured by binding of the opioid antagonist 3H‐diprenorphine. Rather, MβCD treatment induced the appearance of a low affinity state of the mu opioid receptor as shown by competition of DAMGO with 3H‐diprenorphine. This induced low affinity state was mimicked by the addition of sodium and GTPγS, which uncouples G proteins from receptors. Thus, removal of plasma membrane cholesterol by MβCD decreased the high affinity state of mu opioid receptors which could be explained by an uncoupling of receptor from G protein. Supported by DA07267, DA23339, GM07767, DA04087.