High-affinity naloxone binding to filamin a prevents mu opioid receptor-Gs coupling underlying opioid tolerance and dependence

• Published in: PloS one Vol. 3; no. 2; p. e1554
• Main Authors: Wang, Hoau-Yan, Frankfurt, Maya, Burns, Lindsay H
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 06.02.2008; Public Library of Science (PLoS)
• Subjects: Affinity; Alanine; Analgesia; Analogs; Anesthesiology and Pain Management/Basic Science of Pain Management; Animals; Antagonists; Binding sites; Brain; Brain Chemistry; Cell Line, Tumor; Cholera; Contractile Proteins - metabolism; Contractile Proteins - physiology; Corpus Striatum - metabolism; Coupling; Cyclic adenosine monophosphate; Dopamine; Dose-Response Relationship, Drug; Downstream effects; Drug abuse; Drug Tolerance; Filamins; Fragments; G proteins; Humans; Immunoglobulins; Lysine; Melanoma; Microfilament Proteins - metabolism; Microfilament Proteins - physiology; Morphine; Naloxone; Naloxone - pharmacology; Naltrexone; Narcotic Antagonists; Narcotics; Neostriatum; Neuroscience/Neuronal Signaling Mechanisms; Opioid receptors (type mu); Pain perception; Peptides; Physiology; Prevention; Proteins; Rats; Receptors; Receptors, Opioid, mu - physiology; Rodents; Scaffolding; Vibrio cholerae; New York; United States > US
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0001554

Ultra-low-dose opioid antagonists enhance opioid analgesia and reduce analgesic tolerance and dependence by preventing a G protein coupling switch (Gi/o to Gs) by the mu opioid receptor (MOR), although the binding site of such ultra-low-dose opioid antagonists was previously unknown. Here we show that with approximately 200-fold higher affinity than for the mu opioid receptor, naloxone binds a pentapeptide segment of the scaffolding protein filamin A, known to interact with the mu opioid receptor, to disrupt its chronic opioid-induced Gs coupling. Naloxone binding to filamin A is demonstrated by the absence of [(3)H]-and FITC-naloxone binding in the melanoma M2 cell line that does not contain filamin or MOR, contrasting with strong [(3)H]naloxone binding to its filamin A-transfected subclone A7 or to immunopurified filamin A. Naloxone binding to A7 cells was displaced by naltrexone but not by morphine, indicating a target distinct from opioid receptors and perhaps unique to naloxone and its analogs. The intracellular location of this binding site was confirmed by FITC-NLX binding in intact A7 cells. Overlapping peptide fragments from c-terminal filamin A revealed filamin A(2561-2565) as the binding site, and an alanine scan of this pentapeptide revealed an essential mid-point lysine. Finally, in organotypic striatal slice cultures, peptide fragments containing filamin A(2561-2565) abolished the prevention by 10 pM naloxone of both the chronic morphine-induced mu opioid receptor-Gs coupling and the downstream cAMP excitatory signal. These results establish filamin A as the target for ultra-low-dose opioid antagonists previously shown to enhance opioid analgesia and to prevent opioid tolerance and dependence.