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Updated: Jun 28, 2026

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay
Published on: July 28, 2014
Functional coupling of mu-receptor-Galphai-tethered proteins in AtT20 cells
Billy C Chieng1, David J Lee, Yan P Du
1Pain Management Research Institute, Kolling Institute, University of Sydney, Royal North Shore Hospital, New South Wales, Australia. bchieng@med.usyd.edu.au
Abstract:
Opioid efficacy on mu-receptor may be influenced by various Gi/o-G-protein subunits interacting with intracellular face of receptor. Pertussis toxin-insensitive Galphai1 and Galphai2 subunits tethered with mu-receptor were stably transfected into AtT20 cells to (i) determine coupling of different alpha-subunits on opioid efficacy, and (ii) determine coupling to downstream effectors, for example, calcium and potassium channels. After pertussis toxin, stimulation of [35S]GTP-gamma-S incorporation persisted. Both constructs were able to couple to native calcium and potassium channels, with endomorphins 1 and 2 equally effective. However, pertussis toxin abolished opioid actions on calcium and potassium channels suggesting strong coupling to endogenous G-proteins, and that differences in coupling efficacy to Galphai1 and Galphai2 previously observed are restricted to initial step of signaling cascade.
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