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Endogenous, plant-derived, synthetic cannabinoids bind to CB1R, initiating the inhibitory G-protein (G\n                    <jats:sub>i<\/jats:sub>\n                    ) and the \u03b2-arrestin signaling pathways. Within the G\n                    <jats:sub>i<\/jats:sub>\n                    signaling pathway, CB1R activates G protein-gated, inwardly-rectifying potassium (GIRK) channels. The \u03b2-arrestin pathway reduces CB1R expression on the cell surface through receptor internalization. Because of their association with analgesia and drug tolerance, GIRK channels and receptor internalization are of interest to the development of pharmaceuticals. This research used immortalized mouse pituitary gland cells transduced with a pH-sensitive, fluorescently-tagged human CB1R (AtT20-SEPCB1) to measure GIRK channel activity and CB1R internalization. Cannabinoid-induced GIRK channel activity is measured by using a fluorescent membrane-potential sensitive dye. We developed a kinetic imaging assay that visualizes and measures CB1R internalization. All cannabinoids stimulated a GIRK channel response with a rank order potency of WIN55,212-2\u2009&gt;\u2009(\u00b1)CP55,940\u2009&gt;\u2009\u0394\n                    <jats:sup>9<\/jats:sup>\n                    -THC\u2009&gt;\u2009AEA. Efficacy was expressed relative to (\u00b1)CP55,940 with a rank order efficacy of (\u00b1)CP55,940\u2009&gt;\u2009WIN55, 212-2\u2009&gt;\u2009AEA\u2009&gt;\u2009\u0394\n                    <jats:sup>9<\/jats:sup>\n                    -THC. All cannabinoids stimulated CB1R internalization with a rank order potency of (\u00b1)CP55,940\u2009&gt;\u2009WIN55, 212-2\u2009&gt;\u2009AEA\u2009&gt;\u2009\u0394\n                    <jats:sup>9<\/jats:sup>\n                    -THC. Internalization efficacy was normalized to (\u00b1)CP55,940 with a rank order efficacy of WIN55,212-2\u2009&gt;\u2009AEA\u2009&gt;\u2009(\u00b1)CP55,940\u2009&gt;\u2009\u0394\n                    <jats:sup>9<\/jats:sup>\n                    -THC. (\u00b1)CP55,940 was significantly more potent and efficacious than AEA and \u0394\n                    <jats:sup>9<\/jats:sup>\n                    -THC at stimulating a GIRK channel response; no significant differences between potency and efficacy were observed with CB1R internalization. No significant differences were found when comparing a cannabinoid\u2019s GIRK channel and CB1R internalization response. In conclusion, AtT20-SEPCB1 cells can be used to assess cannabinoid-induced CB1R internalization. While cannabinoids display differential G\n                    <jats:sub>i<\/jats:sub>\n                    signaling when compared to each other, this did not extend to CB1R internalization.\n                  <\/jats:p>","DOI":"10.1038\/s41598-024-68451-2","type":"journal-article","created":{"date-parts":[[2024,8,7]],"date-time":"2024-08-07T08:05:46Z","timestamp":1723017946000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Comparing CB1 receptor GIRK channel responses to receptor internalization using a kinetic imaging assay"],"prefix":"10.1038","volume":"14","author":[{"given":"Haley K.","family":"Andersen","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Duncan G.","family":"Vardakas","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julie A.","family":"Lamothe","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tannis E. 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Neither Shackleford Pharma Inc. nor the medico-legal proceedings had any influence on the present study. All other authors of this paper declare no financial or non-financial competing interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"18314"}}