Mediation of opioid analgesia by a truncated 6-transmembrane GPCR

Abstract

The generation of potent opioid analgesics that lack the side effects of traditional opioids may be possible by targeting truncated splice variants of the μ-opioid receptor. μ-Opioids act through GPCRs that are generated from the Oprm1 gene, which undergoes extensive alternative splicing. The most abundant set of Oprm1 variants encode classical full-length 7 transmembrane domain (7TM) μ-opioid receptors that mediate the actions of the traditional μ-opioid drugs morphine and methadone. In contrast, 3-iodobenzoyl-6β-naltrexamide (IBNtxA) is a potent analgesic against thermal, inflammatory, and neuropathic pain that acts independently of 7TM μ-opioid receptors but has no activity in mice lacking a set of 6TM truncated μ-opioid receptor splice variants. Unlike traditional opioids, IBNtxA does not depress respiration or result in physical dependence or reward behavior, suggesting it acts through an alternative μ-opioid receptor target. Here we demonstrated that a truncated 6TM splice variant, mMOR-1G, can rescue IBNtxA analgesia in a μ-opioid receptor-deficient mouse that lacks all Oprm1 splice variants, ablating μ-opioid activity in these animals. Intrathecal administration of lentivirus containing the 6TM variant mMOR-1G restored IBNtxA, but not morphine, analgesia in Oprm1-deficient animals. Together, these results confirm that a truncated 6TM GPCR is both necessary and sufficient for IBNtxA analgesia.

Figure 3. Lentivirus rescue of IBNtxA analgesia.

(A) Opioid analgesia. Analgesia was determined in groups of mice (n = 6–13) at the stated time. (*P < 0.0001 compared with week 0; ANOVA followed by Tukey). (B) IBNtxA cumulative dose–response curves were carried out in exon 1/exon 11 KO mice with lentivirus vector alone (n = 4), lenti–mMOR-1G (ED₅₀ 1.1 mg/kg [95% CI, 0.72–1.53], n = 18) and WT mice (ED₅₀ 0.42 mg/kg [95% CI, 0.29–0.58], n = 16). (C) Single doses of IBNtxA (2.5 mg/kg, s.c. n = 5), morphine (10 mg/kg, s.c. n = 7), fentanyl (0.08 mg/kg, s.c. n = 7), buprenorphine (1 mg/kg, s.c. n = 7), ketocyclazocine (2 mg/kg, s.c. n = 7), or levorphanol (0.8 mg/kg, s.c. n = 7) were administered to groups of either WT or exon 1/exon 11 KO mice infected with lenti–mMOR-1G. The mice were assessed for analgesia. Another group of mice received IBNtxA with levallorphan (2.5 mg/kg, s.c. n = 7). ANOVA shows that the IBNtxA group and the ketocyclazocine group were significantly different (*P < 0.001 and **P < 0.006 by ANOVA, respectively). The IBNtxA WT and lenti–mMOR-1G animals were not significantly different from each other but were different from both the exon 1/exon 11 KO alone and the lenti–mMOR‑1G/levallorphan groups (Tukey). Ketocyclazocine in WT and lenti–mMOR-1G were not significantly different from each other but were different from exon 1/exon 11 alone (Tukey).

Figure 2. Lentivirus expression in the spinal cord following intrathecal administration.

(A) Distribution of lentiviral-expressed mMOR-1G (lenti–mMOR-1G) and EGFP. An exon 4 antibody was used for staining mMOR-1G. Nuclei were stained with DAPI. Scale bars: 100 μm in first three columns and 10 μm in last column. (B and C) Receptor binding in the spinal cord of exon 1/exon 11 KO mice without and following lenti–mMOR-1G (percent of WT; mean ± SEM from 3–4 pooled samples. Each pool contains the spinal cord from 2–3 mice). Significant differences between WT, exon 1/exon 11 KO, and lenti–mMOR-1G groups were seen with ANOVA for ¹²⁵I-IBNtxA and ³H-DAMGO (*P < 0.001) (A) but not ³H-DPDPE and ³H-U69,593 (B). For ¹²⁵I-IBNtxA, the exon 1/exon 11 KO and the lenti–mMOR-1G groups differed from WT (P < 0.001) and from each other (P < 0.05; Tukey). For ³H-DAMGO, the exon 1/exon 11 KO and the lenti–mMOR-1G groups differed from WT (P < 0.001) but not from each other (P > 0.05).

Figure 1. Gene-targeting exons 1 and 11 in the Oprm1 gene.

(A) Schematic of the targeting strategy. The coding and its adjacent intron regions of exons 1 and 11 were replaced with the tdTomato/BGHpolyA (tdT/pA)/PGK-Neo (neo) and ZsGree/SVpolyA (ZsG/pA) cassettes, respectively. The expected EcoRV-digested fragment lengths for WT and targeted alleles are indicated by arrows. The 5′ probe is indicated by a short line. LoxP and Flp sites are shown by triangles and diamonds, respectively. P, PI-SecI; E, EcoRV. (B) Southern blot analysis with the 5′ probe. (C) RT-PCR using RNAs from brain and appropriate primers. Each line represents data from 1 mouse. +/+, WT; +/–, heterozygous; –/–, homozygous.