Format

Send to

Choose Destination
Mol Pharmacol. 2013 Nov;84(5):687-95. doi: 10.1124/mol.113.088096. Epub 2013 Aug 19.

Caged naloxone reveals opioid signaling deactivation kinetics.

Author information

1
Howard Hughes Medical Institute, Department of Neurobiology, Harvard Medical School, Boston, Massachusetts (M.R.B., R.C.S., B.L.S.); Vollum Institute, Oregon Health & Science University, Portland, Oregon (J.T.W.); and Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia (L.D.L.).

Abstract

The spatiotemporal dynamics of opioid signaling in the brain remain poorly defined. Photoactivatable opioid ligands provide a means to quantitatively measure these dynamics and their underlying mechanisms in brain tissue. Although activation kinetics can be assessed using caged agonists, deactivation kinetics are obscured by slow clearance of agonist in tissue. To reveal deactivation kinetics of opioid signaling we developed a caged competitive antagonist that can be quickly photoreleased in sufficient concentrations to render agonist dissociation effectively irreversible. Carboxynitroveratryl-naloxone (CNV-NLX), a caged analog of the competitive opioid antagonist NLX, was readily synthesized from commercially available NLX in good yield and found to be devoid of antagonist activity at heterologously expressed opioid receptors. Photolysis in slices of rat locus coeruleus produced a rapid inhibition of the ionic currents evoked by multiple agonists of the μ-opioid receptor (MOR), but not of α-adrenergic receptors, which activate the same pool of ion channels. Using the high-affinity peptide agonist dermorphin, we established conditions under which light-driven deactivation rates are independent of agonist concentration and thus intrinsic to the agonist-receptor complex. Under these conditions, some MOR agonists yielded deactivation rates that are limited by G protein signaling, whereas others appeared limited by agonist dissociation. Therefore, the choice of agonist determines which feature of receptor signaling is unmasked by CNV-NLX photolysis.

PMID:
23960100
PMCID:
PMC3807075
DOI:
10.1124/mol.113.088096
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center