Exploring Pharmacological Functions of Alternatively Spliced Variants of the Mu Opioid Receptor Gene, Oprm1, via Gene-Targeted Animal Models

Abstract

The mu opioid receptor has a distinct place in the opioid receptor family, since it mediates the actions of most opioids used clinically (e.g., morphine and fentanyl), as well as drugs of abuse (e.g., heroin). The single-copy mu opioid receptor gene, OPRM1, goes through extensive alternative pre-mRNA splicing to generate numerous splice variants that are conserved from rodents to humans. These OPRM1 splice variants can be classified into three structurally distinct types: (1) full-length 7 transmembrane (TM) carboxyl (C)-terminal variants; (2) truncated 6TM variants; and (3) single TM variants. Distinct pharmacological functions of these splice variants have been demonstrated by both in vitro and in vivo studies, particularly by using several unique gene-targeted mouse models. These studies provide new insights into our understanding of the complex actions of mu opioids with regard to OPRM1 alternative splicing. This review provides an overview of the studies that used these gene-targeted mouse models for exploring the functional importance of Oprm1 splice variants.

Keywords: OPRM1; alternative splicing; animal model; gene targeting; mu opioid receptor; opioid.

Figure 1

Schematic of the OPRM1 gene structures, splice variants, and related knockout (KO) mouse models. (A) The schematic of the human, mouse, and rat OPRM1 gene structures adapted from [9]. Exons and introns are shown by colored boxes and horizontal lines, respectively. Promoters are indicated by arrows. Intron sizes are indicated as kilobases (kb). Exons were numbered based on the published data, not by their genomic location, to keep consistent with the literature. The sequence IDs of the human, mouse, and rat OPRM1 genes in Ensembl are ENSG00000112038, ENSMUSG00000000766, and ENSRNOG00000018191, respectively. Note: the exons in Ensembl or other genome browsers were numbered based on their genomic locations. The exon and intron distances are not drawn to scale. The exon composition of OPRM1 splice variants and the role of these splice variants were described in previous reviews [8,9,11,12]. (B) Schematic of the OPRM1 splice variants and KO mouse models adapted from [48]. Top panel: Three types of receptor structures, full-length 7TM C-terminal and truncated 1TM and 6TM, that were predicted from their transmembrane domains (TMs). Lower panel of the tables: The upper table indicates the number of each type of the splice variants in the mouse, rat, and human OPRM1 genes. The bottom table shows four KO mouse models targeting these three types of variants.

Figure 2

IBNtxA pharmacology (adapted from [101]). (A) IBNtxA analgesia. ED₅₀ values (and 95% confidence limits) obtained from dose–response curves were 0.22 mg/kg (0.13, 0.32) in WT mice and 0.39 mg/kg (0.15, 0.58) in triple-KO mice by using the radiant heat tail-flick assay. (B) Reversal of IBNtxA analgesia by levallorphan. IBNtxA (0.75 mg/kg, s.c.). (C) IBNtxA analgesia in KO mice (IBNtxA, 0.5 mg/kg, s.c.). (D) Respiratory rate. It was assessed in awake, freely moving CD1 mice that had been administered morphine (20 mg/kg, s.c.), IBNtxA (2.5 mg/kg, s.c.), or saline using the MouseOx pulse oximeter system (Starr Life Sciences). (E) Gastrointestinal transit. Morphine (5 mg/kg, s.c.). (F) Physical dependence. After being treated with morphine (10 mg/kg, s.c.) or IBNtxA (1 mg/kg, s.c.) for 10 days, mice were challenged with indicated naloxone. The number of jumps during 15 min was counted. (G) Conditioned place preference in a two-compartment apparatus with IBNtxA (1 mg/kg) or morphine (10 mg/kg).

Figure 3

Gain-of-function study using lentivirus expressing the 6TM variants in a double-exon 1/11 KO mouse model. (A) Distribution of lentiviral-expressed mMOR-1G in the dorsal horn of the spinal cord with an exon 4 antibody. (B) Time course of the spinal EGFP mRNA expression determined by RT-PCR after the lentivirus injection. EGFP was co-expressed in lentivirus as a marker. (C) IBNtxA analgesia. (D) Opioid analgesia with a single dose of IBNtxA (2.5 mg/kg), morphine (10 mg/kg), fentanyl (0.08 mg/kg), buprenorphine (1 mg/kg), or levorphanol (0.8 mg/kg). (E) IBNtxA cumulative dose–response curves. (A–D) are adapted from [86] and (E) is adapted from [48].

Figure 4

Exploring the roles of alternatively spliced mu opioid receptor C-termini in morphine actions using C-terminal truncation mouse models (adapted from [63]). (A) Schematic of the stop codon strategy to generate C-terminal truncation mouse models. Inserted and original stop codons are indicated by yellow and pink bars, respectively. In mE3M, a stop codon was inserted at the end of exon 3. In mE4M and mE7M, a stop codon was inserted at the beginning of exon 4 or exon 7, respectively. (B) Impact of C-terminal truncation on morphine tolerance. *: p < 0.05; ***: p < 0.001; ****: p < 0.0001. (C) Impact of C-terminal truncation on morphine dependence and CPP. *: p < 0.05; **: p < 0.01; #: p < 0.0001.