Vol. 298, Issue 1, 116-121, July 2001

Stimulation of Guanosine-5'-O-(3-[³⁵S]thio)triphosphate Binding in Digitonin-Permeabilized C6 Rat Glioma Cells: Evidence for an Organized Association of µ-Opioid Receptors and G Protein

Andrew Alt, Iain J. McFadyen, Charles D. Fan, James H. Woods and John R. Traynor

Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan

The guanosine-5'-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPS) 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]GTPS binding over basal in a naloxone-sensitive manner. Relative µ-agonist efficacy was DAMGO > fentanyl  morphine > 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 -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]GTPS binding stimulated by DAMGO was observed, but the t_1/2 for [³⁵S]GTPS 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.