Characterization of Muscarinic M4 receptor‐G16a fusion protein in CHO‐K1 cells α

The muscarinic M4 receptor has been claimed to be involved in various diseases including schizophrenia, Alzheimer’s disease, and pain. In order to enhance in vitro M4 receptor signaling, we constructed a muscarinic M4‐G16a fusion protein and characterized the in vitro functional activity of muscarinic antagonists and agonists in CHO‐K1 cells. The M4‐G16a fusion protein exhibited the following radioligand binding affinity values for several antagonists (atropine Ki= 1.9 nM, scopolamine Ki= 1.3 nM, tiotropium Ki= 0.2 nM, ipratropium Ki=1.1 nM, darifenacin Ki= 71 nM, tolterodine Ki=15.2 nM, oxybutynin Ki=7.9 nM, 4‐DAMP Ki=72 nM and methoctramine Ki= 1834 nM ) and agonists (carbachol Ki= 26.5 nM, oxotremorine Ki= 8.0 nM and pilocarpine Ki= 11.7 nM ). These affinity values for both antagonists and agonists were similar to the non‐fused wild type M4 receptor clone in CHO‐K1 cells. The G‐protein coupling efficiency of this fusion protein, especially to Gia protein, was retained when tested in the stimulation of oxotremorine‐mediated [35S]GTPgS binding activity. Our data imply that physical tethering of G16a to M4 receptor cDNA does not disrupt the M4 receptor coupling to endogenous Gia proteins. This was further confirmed in oxotremorine competition with [3H]NMS in the presence or absence of a non‐hydrolyzable GTP analog GTPgS without altering agonist binding to the M4 receptor. Fusion of the M4 receptor to the G16a protein enhanced receptor activation of the G16a protein to generate a robust [Ca2+]i response upon carbachol challenge (EC50= 18.4 nM) and oxotremorine (EC50= 1.7 nM) using FLIPR [Ca2+]i release assays. These results demonstrate that the M4‐G16a fusion protein offers a robust signaling tool for a high throughput functional screening compared to conventional Gi‐mediated response for M4 receptor ligands.