Use of membrane permeable ligands as pharmacological chaperones to rescue the plasma membrane expression of a mutant muscarinic M1 receptor

Abstract

In this investigation, we determined whether membrane permeable muscarinic receptor selective agonists, antagonists, and allosteric ligands cause a mutant M1 muscarinic receptor to traffic from ER to the plasma membrane of CHO cells. This receptor, M1LL/AA, possesses a L430A/L431A double point mutation and these mutations caused the receptor to be retained in the ER. We characterized the effect of membrane permeable ligands on the cellular distribution of GFP‐tagged M1LL/AA receptors transiently expressed in CHO cells using confocal microscopy and the ER marker DsRed‐ER. Ligands that caused GFP‐tagged M1LL/AA receptors to redistribute from the ER were then used in intact, whole cell [3H]N‐methyscopolamine ([3H]NMS) binding assays. In these assays, CHO cells transiently expressing M1LL/AA receptors were incubated with equally spaced concentrations (0.5 log unit) of ligand and then washed and incubated with [3H]NMS. Using this approach, we found that membrane permeable agonists (oxotremorine and pilocarpine), antagonists (atropine and scopolamine) and allosteric ligands (TBPB, VU0364572, and VU0357017) cause M1LL/AA receptors to traffic from the ER to the plasma membrane of CHO cells. Overall, we have developed a model system to learn more about using ligands as pharmacological chaperones to rescue the expression of mutant GCPRs. This work was supported by NINDS [Grant 1R15‐NS57742].