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Single nucleotide polymorphisms in the human mu opioid receptor gene alter basal G protein coupling and calmodulin
D Wang1, J M Quillan, K Winans
1Department of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446, USA.
Abstract:
The mu opioid receptor (MOR) plays a central role in mediating acute and chronic 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). Genotyping 252 individuals of the Coriell collection identified one allele encoding the R265H-MOR variant and a new variant encoding D274N-MOR. Variants R260H-, R265H-, and S268P-MOR were constructed and transfected into HEK293 cells. Morphine stimulated G protein coupling of the three receptor variants to a maximal level approaching that of wild type MOR. In contrast, spontaneous, 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 interact with both G proteins and calmodulin (CaM). CaM binding was deficient for variants R265H- and S268P-MOR, suggesting that domains for G protein coupling and CaM binding overlap partially. Morphine pretreatment significantly enhanced basal G protein coupling of wild type MOR, which is thought to result from release of CaM. In contrast basal G protein coupling activity of the three variants was unaffected by morphine pretreatment consistent with 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, or both. Carriers of the mutant alleles might display altered responses to narcotic analgesics.
Insights
Rare genetic variations in the mu opioid receptor (MOR) affect its signaling and calmodulin binding. These changes in the i3 loop may alter how individuals respond to narcotic analgesics.
Area of Science:
- Pharmacology
- Genetics
- Molecular Biology
Background:
- The mu opioid receptor (MOR) is crucial for the effects of opioid drugs.
- Single nucleotide polymorphisms (SNPs) in the hMOR gene can alter MOR function.
- The third intracellular (i3) loop of MOR is involved in G protein and calmodulin (CaM) interactions.
Purpose of the Study:
- To investigate the functional consequences of three rare SNPs in the hMOR gene.
- To determine how these SNPs affect MOR signaling, G protein coupling, and CaM binding.
- To explore potential implications for responses to narcotic analgesics.
Main Methods:
- Genotyping of 252 individuals to identify MOR variants.
- Construction and transfection of MOR variants (R260H, R265H, S268P) into HEK293 cells.
- Assessing G protein coupling and CaM binding of wild-type and variant MORs, with and without morphine pretreatment.
Main Results:
- Three known SNPs (R260H, R265H, S268P) and one new variant (D274N-MOR) were identified.
- Morphine-stimulated G protein coupling was similar for variants and wild-type MOR.
- Basal MOR signaling was reduced for R260H- and R265H-MOR variants.
- CaM binding was deficient for R265H- and S268P-MOR variants.
- Morphine pretreatment did not affect basal G protein coupling of the variants.
Conclusions:
- SNPs in the MOR i3 loop significantly alter basal G protein coupling and/or CaM binding.
- These functional changes suggest that individuals carrying these mutant MOR alleles may exhibit altered responses to opioid medications.
- The findings highlight the importance of genetic variations in MOR for personalized pain management.