Single nucleotide polymorphisms in the human mu opioid receptor gene alterbasal G protein coupling and calmodulin binding

The mu opioid receptor (MOR) plays a central role in mediating acute and ch ronic effects of narcotic drugs. Three rare single nucleotide polymorphisms in the hMOR gene have been identified that cause amino acid substitutions in the third intracellular (i3) loop of MOR (R260H, R265H, and S268P). Geno typing 252 individuals of the Coriell collection identified one allele enco ding the R265H-MOR variant and a new variant encoding D274N-MOR. Variants R 260H-, R265H-, and S268P-MOR were constructed and transfected into HEK293 c ells. Morphine stimulated G protein coupling of the three receptor variants to a maximal level approaching that of wild type MOR. In contrast, spontan eous, agonist-independent (basal) MOR signaling, proposed to play a role in opioid tolerance and dependence, was significantly reduced for R260H- and R265H-MOR. Moreover, domains within the i3 loop of MOR have been shown to i nteract with both G proteins and calmodulin (CaM). CaM binding was deficien t for variants R265H- and S268P-MOR, suggesting that domains for G protein coupling and CaM binding overlap partially. Morphine pretreatment significa ntly enhanced basal G protein coupling of wild type MOR, which is thought t o result from release of CaM. In contrast basal G protein coupling activity of the three variants was unaffected by morphine pretreatment consistent w ith diminished CaM regulation, low basal activity, or both. In conclusion, each of the three single nucleotide polymorphisms mapping to the i3 loop of MOR caused substantial changes in basal G protein coupling, CaM binding, o r both. Carriers of the mutant alleles might display altered responses to n arcotic analgesics.