Effects of various muscarinic ligands on the expression and function of m_5 muscarinic acetylcholine receptor-G_(11) fusion protein

AIM To test the couping function, the interreaction and the influence factors between m 5 muscarinic acetylcholine receptor(m 5AChR) and G 11 protein by generating m 5AChR and G 11 fusion protein(m 5AChR G 11 ) in baculovirus Sf9 cells system. METHODS m 5AChR G 11 Fused DNA was generated in a two step PCR and then expressed in Sf9 cells to produce fusion protein. QNB and [γ 35 S]GTP binding experiments were used to study the function of m 5AChR G 11 fusion protein. RESULTS The expression level of m 5AChR G 11 was (60.4± 2.0) nmol·g -1 protein. The affinity of GDP to G 11 partner changed in the presence of different muscarinic ligands. IC 50 values of GDP in the presence of acetylcholine(ACh), carbachol, pilocarpine, isopropamide, promethazine and atropine were 135, 63, 182, 4.1, 8.9 and 5.9 μmol·L -1 , respectively, and that in the absence of muscarinic ligand was 23.4 μmol·L -1 . CONCLUSION The m 5AChR G 11 fusion protein expressed in baculovirus sf9 cells system has pharmacological specificity for m 5 receptor and the efficient coupling function between the two partners. Affinity of GDP to ligand bound fusion protein represents the species of muscarinic ligands. This is helpful to screen and detect new specific ligands of the muscarinic receptors subtype.