Opioid Pharmacology under the Microscope

Abstract

The powerful analgesic effects of opioid drugs have captivated the interest of physicians and scientists for millennia, and the ability of opioid drugs to produce serious undesired effects has been recognized for a similar period of time (Kieffer and Evans, 2009). Many of these develop progressively with prolonged or repeated drug use and then persist, motivating particular interest in understanding how opioid drugs initiate adaptive or maladaptive modifications in neural function or regulation. Exciting advances have been made over the past several years in elucidating drug-induced changes at molecular, cellular, and physiologic scales of analysis. The present review will highlight some recent cellular studies that we believe bridge across scales and will focus on optical imaging approaches that put opioid drug action "under the microscope." SIGNIFICANCE STATEMENT: Opioid receptors are major pharmacological targets, but their signaling at the cellular level results from a complex interplay between pharmacology, regulation, subcellular localization, and membrane trafficking. This minireview discusses recent advances in understanding the cellular biology of opioid receptors, emphasizing particular topics discussed at the 50th anniversary of the International Narcotics Research Conference. Our goal is to highlight distinct signaling and regulatory properties emerging from the cellular biology of opioid receptors and discuss potential relevance to therapeutics.

Fig. 1.

Agonist-induced signaling and trafficking of opioid receptors. Inactive opioid receptors (gray) become activated (green) after binding to an agonist (A, steps 1 to 2). This enables signaling via G proteins (green ripples, step 2) and triggers phosphorylation of the receptor tail (P) by GRKs, followed by receptor engagement of β-arrestins (step 3) and endocytosis via clathrin-coated pits (step 4). These events inactivate G protein signaling (red receptor) and assure signal termination from the plasma membrane by endocytic removal of receptors. After receptors arrive in early endosomes, they have the capacity to signal again by engaging G proteins in the endosome membrane (green ripples, step 5). Receptors also engage molecular sorting mechanisms in the endosome limiting membrane (not shown), which determine whether internalized receptors are delivered to lysosomes for proteolytic downregulation or are nondestructively recycled to restore surface receptor responsiveness. Many nonpeptide agonist drugs (drug B) are sufficiently membrane-permeant to activate a discrete pool of opioid receptors at the Golgi apparatus and activate receptors from this location (green ripples). Some agonists (drug C) induce receptor reorganization in the plasma membrane to change surface signaling (blue ripples).

Fig. 2.

Cellular basis for sensitive presynaptic neuromodulation by opioid receptors. Opioid receptors inhibit synaptic vesicle (blue circles) exocytosis by locally regulating effectors that are restricted in terminals and positioned in, or adjacent to, individual presynaptic active zones. Opioid receptors are not immobilized at terminals, however, and instead are laterally mobile throughout the axon surface and collisionally couple to effectors at the presynapse. After ligand-induced activation (A), presynaptic opioid receptors undergo phosphorylation (P) and endocytosis directly at terminals. receptors are then are locally recycled and reinserted to the axon surface both within and outside of synapses to replenish the diffusible surface pool. Lateral diffusion of receptors is sufficiently fast for terminals to “sample” agonist-receptor complexes formed outside of synapses. The net effect of these events is to maintain a mobile surface receptor pool that is capable of mediating sensitive signaling at the presynapse by leveraging lateral diffusion and the allosteric nature of opioid receptor signaling by heterotrimeric G proteins.

Fig. 3.

A nonperturbing labeling strategy for opioid receptors using ligand-guided chemistry. Naltrexamine-acylimidazole-Alexa594 binds to the orthosteric binding site of opioid receptors, with the pharmacophore “guiding” covalent coupling to residues located outside of the ligand binding pocket. The coupling reaction releases the pharmacophore (upon washout), leaving the native receptor fluorescently labeled and functional to undergo subsequent activation by an orthosteric agonist after labeling.