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Membrane microviscosity modulates mu-opioid receptor conformational transitions and agonist efficacy
P J Emmerson1, M J Clark, F Medzihradsky
1Department of Pharmacology, University of Michigan Medical School, Ann Arbor, USA.
Abstract:
The influence of membrane microviscosity on mu-opioid agonist and antagonist binding, as well as agonist efficacy, was examined in membranes prepared from SH-SY5Y cells and from a C6 glioma cell line stably expressing the rat mu-opioid receptor (C6mu). Addition of cholesteryl hemisuccinate (CHS) to cell membranes increased membrane microviscosity and reduced the inhibitory effect of sodium and guanine nucleotides on the affinity of the full agonists sufentanil and [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO) for the mu-opioid receptor. Binding of the antagonists [3H]naltrexone and [3H]diprenorphine and the partial agonist nalbuphine was unaffected by CHS. The effect of CHS on agonist binding was reversed by subsequent addition of cis-vaccenic acid, suggesting that the effect of CHS is the result of increased membrane microviscosity and not a specific sterol-receptor interaction. CHS addition increased the potency of DAMGO to stimulate guanosine-5'-O-(3-[35S]thio)triphosphate binding by fourfold, whereas the potency of nalbuphine was unaffected. However, nalbuphine efficacy relative to that of the full agonist DAMGO was strongly increased in CHS-treated membranes compared with that in control membranes. Membrane rigidification also resulted in an increased efficacy for the partial agonists meperidine, profadol, and butorphanol relative to that of DAMGO as measured by agonist-stimulated GTPase activity in control and CHS-modified membranes. These findings support a regulatory role for membrane microviscosity in receptor-mediated G protein activation.
Insights
Altering membrane microviscosity affects mu-opioid receptor binding and efficacy. Increased microviscosity enhances partial agonist efficacy, suggesting a role in G protein activation.
Area of Science:
- Pharmacology
- Biochemistry
- Cell Biology
Background:
- Membrane microviscosity influences receptor function.
- The role of microviscosity in mu-opioid receptor pharmacology is not fully understood.
Purpose of the Study:
- To investigate the impact of membrane microviscosity on mu-opioid receptor binding and agonist efficacy.
- To elucidate the relationship between membrane physical properties and G protein-coupled receptor (GPCR) signaling.
Main Methods:
- Utilized SH-SY5Y cells and C6mu cells expressing rat mu-opioid receptors.
- Manipulated membrane microviscosity using cholesteryl hemisuccinate (CHS) and cis-vaccenic acid.
- Assessed ligand binding (agonists and antagonists) and G protein activation (GTPγS binding and GTPase activity).
Main Results:
- Increased membrane microviscosity reduced sodium/guanine nucleotide inhibition of full agonist binding.
- CHS did not affect antagonist or partial agonist binding.
- CHS increased full agonist potency and significantly enhanced partial agonist efficacy.
- Membrane rigidification increased efficacy of multiple partial agonists relative to a full agonist.
Conclusions:
- Membrane microviscosity plays a regulatory role in mu-opioid receptor-mediated G protein activation.
- Modulating membrane fluidity can alter the functional output of GPCRs, impacting drug response.