Functional Characteristics of the Naked Mole Rat μ-Opioid Receptor

Abstract

While humans and most animals respond to µ-opioid receptor (MOR) agonists with analgesia and decreased aggression, in the naked mole rat (NMR) opioids induce hyperalgesia and severe aggression. Single nucleotide polymorphisms in the human mu-opioid receptor gene (OPRM1) can underlie altered behavioral responses to opioids. Therefore, we hypothesized that the primary structure of the NMR MOR may differ from other species. Sequencing of the NMR oprm1 revealed strong homology to other mammals, but exposed three unique amino acids that might affect receptor-ligand interactions. The NMR and rat oprm1 sequences were cloned into mammalian expression vectors and transfected into HEK293 cells. Radioligand binding and 3'-5'-cyclic adenosine monophosphate (cAMP) enzyme immunoassays were used to compare opioid binding and opioid-mediated cAMP inhibition. At normalized opioid receptor protein levels we detected significantly lower [3H]DAMGO binding to NMR compared to rat MOR, but no significant difference in DAMGO-induced cAMP inhibition. Strong DAMGO-induced MOR internalization was detectable using radioligand binding and confocal imaging in HEK293 cells expressing rat or NMR receptor, while morphine showed weak or no effects. In summary, we found minor functional differences between rat and NMR MOR suggesting that other differences e.g. in anatomical distribution of MOR underlie the NMR's extreme reaction to opioids.

pKa of opioid ligands as a discriminating factor for side effects

“…MOR-expressing HEK 293 cells were cultured in flasks with a growth area of 175 cm². Cells were washed twice with ice-cold assay buffer (Trizma® Preset Crystals, 50 mM, pH 7.4) (Sigma-Aldrich), then harvested from the culture flask in 10 ml ice-cold assay buffer, homogenized and centrifuged twice at 42,000 g for 20 min at 4 °C as described previously 24,27,28 . Protein concentration was determined according to the Bradford method 29 .…”

Opioid receptor signaling, analgesic and side effects induced by a computationally designed pH-dependent agonist

“…HEK293 cells stably expressing rat MOR were cultured in flasks with a growth area of 175 cm². Cells were washed twice with ice-cold assay buffer (Trizma® Preset Crystals, 50 mM, pH 7.4) (Sigma-Aldrich), then scratched from the culture flask in 10 ml ice cold assay buffer, homogenized and centrifuged twice at 42,000 g for 20 min at 4 °C as described previously 7 , 19 , 20 . Protein concentration was determined according to the Bradford method 21 .…”

Production of G protein‐coupled receptors in an insect‐based cell‐free system

“…For binding experiments MOR expressing cells were cultured in flasks with a growth area of 175 cm 2 . Cells were washed with ice‐cold Trizma (50 mM, pH 7.4) (Sigma–Aldrich), scraped off with a cell scraper, homogenized and centrifuged twice at 42.000 g for 20 min at 4°C as described previously (Busch‐Dienstfertig et al, ; Spahn et al, , ). Protein concentration was determined using the Bradford method (Bradford, ).…”

“…Protein concentration was determined using the Bradford method (Bradford, ). Binding experiments with labeled MOR ligands [ 3 H]‐D‐Ala2, N‐MePhe4, Gly‐ol]‐enkephalin (DAMGO) and—[ 3 H]‐naloxone (NLX), respectively, were carried out according to a modified protocol (Busch‐Dienstfertig et al, ). Briefly, 100 μg of cell membranes were prepared and incubated for 90 min in assay buffer (50 mM Trizma, pH 7.4) with increasing doses of [ 3 H]‐DAMGO (0.5–16 nM) (47.1 Ci/mmol) and [ 3 H]‐NLX (0.9–15 nM) (58.2 Ci/mmol) (Perkin Elmer, Waltham, MA), respectively, for saturation binding studies or with 4 nM [ 3 H]‐DAMGO for single point binding studies in the absence or presence of 10 μM unlabeled NLX to determine nonspecific binding.…”