Premium
Use of membrane permeable ligands as pharmacological chaperones to rescue the plasma membrane expression of a mutant muscarinic M 1 receptor
Author(s) -
Sawyer Gregory William,
Shults Crystal
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1173.12
Subject(s) - muscarinic acetylcholine receptor , receptor , allosteric regulation , muscarinic acetylcholine receptor m1 , muscarinic acetylcholine receptor m5 , chemistry , chinese hamster ovary cell , mutant , muscarinic acetylcholine receptor m2 , microbiology and biotechnology , muscarinic acetylcholine receptor m3 , ligand (biochemistry) , biophysics , biochemistry , biology , gene
In this investigation, we determined whether membrane permeable muscarinic receptor selective agonists, antagonists, and allosteric ligands cause a mutant M 1 muscarinic receptor to traffic from ER to the plasma membrane of CHO cells. This receptor, M 1LL/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 M 1LL/AA receptors transiently expressed in CHO cells using confocal microscopy and the ER marker DsRed‐ER. Ligands that caused GFP‐tagged M 1LL/AA receptors to redistribute from the ER were then used in intact, whole cell [ 3 H] N ‐methyscopolamine ([ 3 H]NMS) binding assays. In these assays, CHO cells transiently expressing M 1LL/AA receptors were incubated with equally spaced concentrations (0.5 log unit) of ligand and then washed and incubated with [ 3 H]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 M 1LL/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].