Opioid Receptors are Organized into Nanodomains in the Plasma Membrane

Abstract

Opioid receptors (ORs) are essential for mediating some of the normal functions of our respiratory, immune, and neuroendocrine systems. ORs are the subjects of numerous pharmacological studies because they are involved in important physiological processes such as pain regulation; reward and motivation processing; and drug addiction and abuse. While the signaling cascades that are responsible for the receptors’ physiological effects are well understood, little is known about the physical organization of these receptors on cell membranes and the impact of that organization on signaling outcomes. To clarify the lateral organization of ORs at a molecular level, we investigated three ORs variants: kappa opioid receptor (KOP), mu opioid receptor (MOPWT), and a naturally occurring MOP receptor variant (MOPN40D). We transiently transfected COS-7 cells with our functional constructs (OR tagged with photoactivatable GFP) and used quantitative super-resolution microscopy methods to delineate their nanoscale distribution. Spatial auto-correlation curves with two characteristic correlation lengths indicate that ORs exhibit distinct and complex lateral organization: long-distance correlations indicate that opioid receptors are organized into nano-domains; short-distance correlations indicate that receptor organization within these nano-domains is not random. Additionally, we found that domain radius and occupancy increase in the following order: MOPN40D, MOPWT, and KOP. These results were validated by ensemble-averaged Monte Carlo simulations. Finally, using a clustering algorithm and the domain size obtained from analysis, we estimated the number of molecules partitioned in domains versus the number of free molecules in the plasma membrane. KOP had the largest and MOPN40D had the smallest fraction of molecules in domains. These results provide new insight into OR nano-organization and complement our ongoing Fluorescence Correlation Spectroscopy measurements.