Single-nucleotide polymorphism in the human mu opioid receptor gene alters beta-endorphin binding and activity: possible implications for opiate addiction

Abstract

Opioid drugs play important roles in the clinical management of pain, as well as in the development and treatment of drug abuse. The mu opioid receptor is the primary site of action for the most commonly used opioids, including morphine, heroin, fentanyl, and methadone. By sequencing DNA from 113 former heroin addicts in methadone maintenance and 39 individuals with no history of drug or alcohol abuse or dependence, we have identified five different single-nucleotide polymorphisms (SNPs) in the coding region of the mu opioid receptor gene. The most prevalent SNP is a nucleotide substitution at position 118 (A118G), predicting an amino acid change at a putative N-glycosylation site. This SNP displays an allelic frequency of approximately 10% in our study population. Significant differences in allele distribution were observed among ethnic groups studied. The variant receptor resulting from the A118G SNP did not show altered binding affinities for most opioid peptides and alkaloids tested. However, the A118G variant receptor binds beta-endorphin, an endogenous opioid that activates the mu opioid receptor, approximately three times more tightly than the most common allelic form of the receptor. Furthermore, beta-endorphin is approximately three times more potent at the A118G variant receptor than at the most common allelic form in agonist-induced activation of G protein-coupled potassium channels. These results show that SNPs in the mu opioid receptor gene can alter binding and signal transduction in the resulting receptor and may have implications for normal physiology, therapeutics, and vulnerability to develop or protection from diverse diseases including the addictive diseases.

Figures

Figure 1

DNA sequences showing the A118G polymorphism. Examples of DNA sequence are shown from individuals homozygous for the most common allele (Left), heterozygous (Center), and homozygous for the A118G variant allele (Right). The arrows indicate the position of nucleotide 118, with the adjacent sequences shown.

Figure 2

Binding of endogenous opioid peptides to the most common and A118G mu opioid receptors. Membrane preparations from cells expressing either the most common (■) or the A118G (▵) receptors were used in binding experiments to displace the [3H]DAMGO binding. Shown are examples of displacement binding for four endogenous peptides: Met-enkephalin, dynorphin A, β-endorphin, and endomorphin-1.

Figure 3

Comparison of the most common and the A118G variant mu opioid receptors in coupling to G protein-activated, inwardly rectifying K+ (GIRK) channels. (A) Example of current trace showing the experimental protocol and calculation method for the agonist-induced response. Oocytes were clamped at a holding potential of −80 mV and superfused with different solutions as indicated. Imax, maximum K+ currents evoked by DAMGO at a saturating concentration (100 nM). ITest, K+ currents evoked by the test dose of agonists. The trace example shows the response of the A118G variant receptor to 20 nM β-endorphin. (B) Dose-response curves of receptor activation. The tested concentrations of agonists ranged from 0.1 nM to 1 μM. The response to a test dose is expressed as the fraction of the maximum activation by 100 nM DAMGO (ITest/Imax). Data are presented as mean ± SEM (n = 4–5). All oocytes were used only once to avoid desensitization.