title: Design and functional evaluation of an optically active μ-opioid receptor.
abstract: The use of opioids, which achieve therapeutic analgesia through activation of μ-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 μ-opioid receptor that could be photoactivated to trigger intracellular signaling. A prototype optoactive μ-opioid receptor (optoMOR) was designed by replacing the intracellular domains from rhodopsin with those of the native μ-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 μ-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 (Ca(2+)) 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 μ-opioid receptor in HEK293 cells. Photoactivation of optoMOR decreased the Ca(2+) influx and inhibited the forskolin-induced cAMP generation, activation of CREB, and BDNF levels in optoMOR-expressing cells similar to the activation of native μ-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.
authors: Department of Biomedical Engineering, University of Florida, Gainesville, Florida 32607, USA.
authors: Philip A
authors: PA
authors: Barish
authors: Ying
authors: Y
authors: Xu
authors: Jianxin
authors: J
authors: Li
authors: Jiao
authors: J
authors: Sun
authors: Yagna P R
authors: YP
authors: Jarajapu
authors: William O
authors: WO
authors: Ogle
mesh_terms: D000701:Analgesics, Opioid / Q000494:pharmacology; D019208:Brain-Derived Neurotrophic Factor / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D062308:Optogenetics; D010775:Photic Stimulation; D017450:Receptors, Opioid, mu / Q000819:agonists / Q000235:genetics* / Q000378:metabolism; D012243:Rhodopsin / Q000235:genetics
chemical_list: D000701:Analgesics, Opioid; D019208:Brain-Derived Neurotrophic Factor; C115163:CREB1 protein, human; D017362:Cyclic AMP Response Element-Binding Protein; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D012243:Rhodopsin