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Volume 617 of the series Methods in Molecular Biology pp 421-435

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Molecular Assays for Characterization of Alternatively Spliced Isoforms of the Mu Opioid Receptor (MOR)

  • Pavel GrisAffiliated withCenter for Neurosensory Disorders, University of North Carolina
  • , Philip ChengAffiliated withCenter for Neurosensory Disorders, University of North Carolina
  • , John PiersonAffiliated withCenter for Neurosensory Disorders, University of North Carolina
  • , William MaixnerAffiliated withCenter for Neurosensory Disorders, Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill
  • , Luda DiatchenkoAffiliated withCenter for Neurosensory Disorders, Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill Email author 

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Abstract

Mu-opioid receptor (MOR) belongs to a family of heptahelical G-protein-coupled receptors (GPCRs). Studies in humans and rodents demonstrated that the OPRM1 gene coding for MOR undergoes extensive alternative splicing afforded by the genetic complexity of OPRM1. Evidence from rodent studies also demonstrates an important role of these alternatively spliced forms in mediating opiate analgesia via their differential signaling properties. MOR signaling is predominantly Gia coupled. Release of the α subunit from G-protein complex results in the inhibition of adenylyl cyclase/cAMP pathway, whereas release of the βγ subunits activates G-protein-activated inwardly rectifying potassium channels and inhibits voltage-dependent calcium channels. These molecular events result in the suppression of cellular activities that diminish pain sensations. Recently, a new isoform of OPRM1, MOR3, has been identified that shows an increase in the production of nitric oxide (NO) upon stimulation with morphine. Hence, there is a need to describe molecular techniques that enable the functional characterization of MOR isoforms. In this review, we describe the methodologies used to assay key mediators of MOR activation including cellular assays for cAMP, free Ca2+, and NO, all of which have been implicated in the pharmacological effects of MOR agonists.

Key words

Alternative splicing OPRM1 Opioid Calcium cAMP Nitric oxide Fluo-4 Fluo-3 GPCR FSK Capsaicin