Abstract
Background and PurposeAdverse side effects of conventional opioids can be avoided if ligands selectively activate peripheral opioid receptors in injured tissue. Injury and inflammation are typically accompanied by acidification. In this study, we examined influences of low pH and mutation of the ionizable amino acid residue H2976.52 on μ‐opioid receptor binding and signalling induced by the μ‐opioid receptor ligands fentanyl, DAMGO, and naloxone.Experimental ApproachHEK 293 cells stably transfected with μ‐opioid receptors were used to study opioid ligand binding, [35S]‐GTPγS binding, and cAMP reduction at physiological and acidic pH. We used μ‐opioid receptors mutated at H2976.52 to A (MOR‐H2976.52A) to delineate ligand‐specific interactions with H2976.52.Key ResultsLow pH and the mutant receptor MOR‐H2976.52A impaired naloxone binding and antagonism of cAMP reduction. In addition, DAMGO binding and G‐protein activation were decreased under these conditions. Fentanyl‐induced signalling was not influenced by pH and largely independent of H2976.52.Conclusions and ImplicationsOur investigations indicate that low pH selectively impairs μ‐opioid receptor signalling modulated by ligands capable of forming hydrogen bonds with H2976.52. We propose that protonation of H2976.52 at acidic pH reduces binding and subsequent signalling of such ligands. Novel agonists targeting opioid receptors in injured tissue might benefit from lack of hydrogen bond formation with H2976.52.
Highlights
The μ-opioid receptor is the clinically most important member of the opioid receptor family of class A GPCRs
We have investigated the role of pH and H2976.52 on ligand binding, G-protein activation, and downstream signalling by Effect Fentanyl-induced cAMP reduction
Our data suggest that low pH impairs binding and signalling selectively for ligands that can form hydrogen bond networks with the μ-opioid receptor histidine residue, H2976.52
Summary
We focused on the amino acid histidine 2976.52 (H2976.52, superscript indicates the Ballesteros–Weinstein numbering; Ballesteros & Weinstein, 1995), which is critically involved in μ-opioid receptor ligand binding (Huang et al, 2015; Koehl et al, 2018; Manglik et al, 2012) This residue is topologically conserved across species (including humans) and class A GPCRs, and homologous histidines convey proton sensing to several proton-activated receptors (Liu et al, 2010; Ludwig et al, 2003). To assess ligand binding potential, we used naloxone, a μ-opioid receptor antagonist that is likely to form a hydrogen bond network with H2976.52 based on its structural similarity to β-funaltrexamine (Manglik et al, 2012), DAMGO, an μ-opioid receptor agonist that was shown to form hydrogen bonds with H2976.52 (Koehl et al, 2018), and fentanyl, a highly selective μ-opioid receptor agonist The latter does not include a hydroxyl group (Figure 1), an important interaction partner of H2976.52 via hydrogen bonds (Dosen-Micovic, Ivanovic, & Micovic, 2006; Manglik et al, 2016). Novel injury-specific opioid agonists might benefit from lack of hydrogen bond formation with H2976.52
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