PT - JOURNAL ARTICLE AU - Andrew Alt AU - Iain J. McFadyen AU - Charles D. Fan AU - James H. Woods AU - John R. Traynor TI - Stimulation of Guanosine-5′-<em>O</em>-(3-[<sup>35</sup>S]thio)triphosphate Binding in Digitonin-Permeabilized C6 Rat Glioma Cells: Evidence for an Organized Association of μ-Opioid Receptors and G Protein DP - 2001 Jul 01 TA - Journal of Pharmacology and Experimental Therapeutics PG - 116--121 VI - 298 IP - 1 4099 - http://jpet.aspetjournals.org/content/298/1/116.short 4100 - http://jpet.aspetjournals.org/content/298/1/116.full SO - J Pharmacol Exp Ther2001 Jul 01; 298 AB - The guanosine-5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding assay for the determination of relative opioid efficacy has been adapted to measure G protein activation in digitonin-permeabilized C6 rat glioma cells expressing a cloned μ-opioid receptor. The μ-agonist [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) caused a 3-fold increase in [35S]GTPγS binding over basal in a naloxone-sensitive manner. Relative μ-agonist efficacy was DAMGO &gt; fentanyl ≥ morphine &gt; buprenorphine. Nalbuphine showed no efficacy. G protein activation by receptors has been predicted to occur by random encounter. In this model a reduction in the number of receptors will decrease the rate of G protein activation but not the maximum number of G proteins activated. To test this model C6 μ cells were treated with the irreversible μ-antagonist β-funaltrexamine (10 nM) prior to permeabilization. This reduced the number of μ-opioid receptors determined with [3H]diprenorphine to 23 ± 3% of control with no change in affinity. A commensurate reduction (to 29 ± 10% of control) in the level of [35S]GTPγS binding stimulated by DAMGO was observed, but thet1/2 for [35S]GTPγS binding remained unchanged. Thus, random encounters of receptor and G protein failed to occur in this permeabilized cell preparation. A model that assumes an organized association of G proteins with receptors better describes the activation of G proteins by opioid μ-receptors. The American Society for Pharmacology and Experimental Therapeutics