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Truncated μ-Opioid Receptors With 6 Transmembrane Domains Are Essential for Opioid Analgesia

Lu, Zhigang PhD*†; Xu, Jin MD; Xu, Mingming MS; Rossi, Grace C. PhD§; Majumdar, Susruta PhD; Pasternak, Gavril W. MD, PhD; Pan, Ying-Xian MD, PhD

doi: 10.1213/ANE.0000000000002538
Pain and Analgesic Mechanisms: Original Laboratory Research Report

BACKGROUND: Most clinical opioids act through μ-opioid receptors. They effectively relieve pain but are limited by side effects, such as constipation, respiratory depression, dependence, and addiction. Many efforts have been made toward developing potent analgesics that lack side effects. Three-iodobenzoyl-6β-naltrexamide (IBNtxA) is a novel class of opioid active against thermal, inflammatory, and neuropathic pain, without respiratory depression, physical dependence, and reward behavior. The μ-opioid receptor (OPRM1) gene undergoes extensive alternative precursor messenger ribonucleic acid splicing, generating multiple splice variants that are conserved from rodents to humans. One type of variant is the exon 11 (E11)–associated truncated variant containing 6 transmembrane domains (6TM variant). There are 5 6TM variants in the mouse OPRM1 gene, including mMOR-1G, mMOR-1M, mMOR-1N, mMOR-1K, and mMOR-1L. Gene-targeting mouse models selectively removing 6TM variants in E11 knockout (KO) mice eliminated IBNtxA analgesia without affecting morphine analgesia. Conversely, morphine analgesia is lost in an exon 1 (E1) KO mouse that lacks all 7 transmembrane (7TM) variants but retains 6TM variant expression, while IBNtxA analgesia remains intact. Elimination of both E1 and E11 in an E1/E11 double KO mice abolishes both morphine and IBNtxA analgesia. Reconstituting expression of the 6TM variant mMOR-1G in E1/E11 KO mice through lentiviral expression rescued IBNtxA but not morphine analgesia. The aim of this study was to investigate the effect of lentiviral expression of the other 6TM variants in E1/E11 KO mice on IBNtxA analgesia.

METHODS: Lentiviruses expressing 6TM variants were packaged in HEK293T cells, concentrated by ultracentrifugation, and intrathecally administered 3 times. Opioid analgesia was determined using a radiant-heat tail-flick assay. Expression of lentiviral 6TM variant messenger ribonucleic acids was examined by polymerase chain reaction (PCR) or quantitative PCR.

RESULTS: All the 6TM variants restored IBNtxA analgesia in the E1/E11 KO mouse, while morphine remained inactive. Expression of lentiviral 6TM variants was confirmed by PCR or quantitative PCR. IBNtxA median effective dose values determined from cumulative dose–response studies in the rescued mice were indistinguishable from wild-type animals. IBNtxA analgesia was maintained for up to 33 weeks in the rescue mice and was readily antagonized by the opioid antagonist levallorphan.

CONCLUSIONS: Our study demonstrated the pharmacological relevance of mouse 6TM variants in IBNtxA analgesia and established that a common functional core of the receptors corresponding to the transmembrane domains encoded by exons 2 and 3 is sufficient for activity. Thus, 6TM variants offer potential therapeutic targets for a distinct class of analgesics that are effective against broad-spectrum pain models without many side effects associated with traditional opioids.

From the *The Affiliated Hospital of Nanjing University of Chinese Medicine, First College of Clinical Medicine, and Key Laboratory of Acupuncture and Medicine Research of Ministry of Education Nanjing University of Chinese Medicine, Nanjing, China; Department of Neurology and the Molecular Pharmacology Program, Memorial Sloan-Kettering Cancer Center, New York, New York; and §Department of Psychology, Long Island University, Post Campus, Brookville, New York.

Published ahead of print October 4, 2017.

Accepted for publication August 24, 2017.

Funding: This work was supported, in part, by grants from a National Natural Science Foundation of China grant (81673412) to Z.L., the Peter F. McManus Charitable Trust, Mr William H. Goodwin and Mrs Alice Goodwin and the Commonwealth Foundation for Cancer Research, the Experimental Therapeutics Center of Memorial Sloan-Kettering Cancer Center, and the National Institute on Drug Abuse of the National Institutes of Health (DA06241 and DA07242 to G.W.P., and DA029244, DA013997, and DA04585 to Y.-X.P.), a core grant from the National Cancer Institute of the National Institutes of Health (CA08748) to the Memorial Sloan-Kettering Cancer Center.

The authors declare no conflicts of interest.

Reprints will not be available from the authors.

Address correspondence to Ying-Xian Pan, MD, PhD, Department of Neurology and the Molecular Pharmacology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10065. Address e-mail to

© 2018 International Anesthesia Research Society
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