Design and functional evaluation of an optically active μ-opioid receptor

The use of opioids, which achieve therapeutic analgesia through activation of mu-opioid receptors, are limited in the management of chronic pain by adverse effects including tolerance and addiction. Optogenetics is an emerging approach of designing molecular targets that can produce cell-specific receptor-mediated analgesia with minimal side effects. Here we report the design and functional characterization of a chimeric mu-opioid receptor that could be photoactivated to trigger intracellular signaling. A prototype optoactive mu-opioid receptor (optoMOR) was designed by replacing the intracellular domains from rhodopsin with those of the native mu-opioid receptor and was transiently expressed in human embryonic kidney (HEK293) cells. Expression and distribution of the protein were confirmed by immunocytochemistry. The signal-transduction mechanisms induced by photoactivation of the optoMOR were evaluated and compared with the native mu-opioid receptor stimulation by an agonist, D-Ala(2), N-MePhe(4), Gly-ol-enkephalin (DAMGO). Cells were depolarized by extracellular potassium and the depolarization-induced calcium (Ca2+) influx was quantified by using Fura-2 imaging. The forskolin-stimulated adenylate cyclase/cAMP cascade was evaluated by ELISA or western blotting of brain-derived neurotrophic factor (BDNF) and the phosphorylation of cAMP response element binding protein (CREB). The optoMOR protein distribution was observed intracellularly and on the plasma membrane similar to the native mu-opioid receptor in HEK293 cells. Photoactivation of optoMOR decreased the Ca2+ influx and inhibited the forskolin-induced cAMP generation, activation of CREB, and BDNF levels in optoMOR-expressing cells similar to the activation of native mu-opioid receptor by DAMGO. Thus the current study has accomplished the design of a prototype optoMOR and characterized the cellular signaling mechanisms activated by light stimulation of this receptor. Published by Elsevier B.V.