Regulation of the mu opioid receptor, as in all G protein coupled receptors, is accomplished in a variety of ways that are still being discovered or appreciated. Thus the response of the mu receptor to a drug depends on the structure of the drug itself which determines both its potency (concentration of half maximum response) and its intrinsic activity (level of maximum response). The structure of the receptor further affects its responses, as we have previously shown that a single mutation can enhance specific activities of drugs. In addition there are other dynamic factors, only recently appreciated, that include the density of receptors on the cell membrane and the cycling (trafficking) of these receptors as they become internalized and recycled to the plasma membrane.Our chief efforts over the past year have been in characterizing by laser scanning confocal microscopy the cellular localization of mu opioid receptors stably expressed on CHO cells. Our radioligand binding experiments have shown that these cells express 100,000 receptors per cell, and we have demonstrated that the fluorescent probe fluorescein naloxone (FNAL) is capable of labeling the mu receptors on these living cells. We have shown that confocal microscopy not localizes the receptors but is capable of providing quantitative kinetic estimates of probe association and dissociation rates that we corroborated by radioligand binding. While the fluorescent antagonist FNAL is mostly confined to the plasma membrane in a fairly uniform way as expected for an antagonist, another antagonist, 6-BNX, is unexpectedly internalized and shows punctate clustering only in transfected cells. To our knowledge this is the first instance of an antagonist to any G protein coupled receptors causing this internalization and clustering. Using fluorescent probes of cell membrane and lysosomes, we hope to identify, by colocalization, the compartments to which these 6-BNX labeled receptors are migrating. Temperature studies are also imminent that should distinguish cell surface from internalization kinetic components. - mu opioid receptor partial agonist intrinsic activity radioligand binding confocal microscopy Xenopus oocyte voltage clamp
