Aim: To generate M2AChR-Gi1alpha fusion protein in baculovirus-Sf9 cells system and detect the effects of various muscarinic ligands and magnesium ion on the interaction of fused M2AChR and Gi1alpha.
Methods: M2AChR-Gi1alpha fused DNA was generated in a two step polymerase chain reaction (PCR) and then expressed in Sf9 cells to produce fusion protein. [3H] L-quinuclidinyl benzilate (QNB) and 35S GTPgammaS binding experiments were performed to study the function of M2AChR-Gi1alpha fusion protein.
Results: The expression level of M2AChR-Gi1alpha was (20.12 +/- 0.14) nmol/g protein. The affinity of GDP to Gi1alpha partner changed in the presence of different muscarinic ligands. IC50 values (95 % confidence limit) of GDP in the presence of acetylcholine (ACh), carbamylcholine, (4-hydroxy-2-butynyl)-1-trimethylammonium-m-chloro-carbanilate chloride (McN-A-343), pilocarpine, and atropine were 178 (148 - 214) micromol/L, 158 (126 - 199) micromol/L, 66 (56 - 78) micromol/L, 62 (55 -7 2) micromol/L, and 5.0 (4.6 - 5.5) micromol/L, respectively, and that in the absence of muscarinic ligand was 15.9 (14.3 - 17.6) micromol/L. Apparent affinity for GDP in the presence of carbamylcholine was markedly decreased with increasing MgCl2 concentrations, although the apparent affinity in the presence of atropine was not affected.
Conclusion: The M2AChR-Gi1alpha fusion protein has the pharmacological specificity of M2 receptor and the efficient signaling of the two partners. Affinity of GDP to ligand-bound fusion protein represents the species of muscarinic ligands. Mg2+ is necessary for the action of M2AChR on Gi1alpha.