Effects of various muscarinic ligands on M2AChR-Gi1alpha fusion protein expressed in Sf9 insect cells.
OBJECTIVE
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.


CONCLUSIONS
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.
Wang
Hao
H
Department of Pharmacology, China Medical University, Shenyang 110001, China.
Guo
Zheng-Dong
ZD
Li
Zhi
Z
Liu
Hong-Rui
HR
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002417:Cattle; D002478:Cells, Cultured; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000096:biosynthesis* / Q000235:genetics; D020962:Heterotrimeric GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007313:Insecta / Q000166:cytology / Q000378:metabolism; D008024:Ligands; D015636:Magnesium Chloride / Q000494:pharmacology; D011869:Radioligand Assay; D043643:Receptor, Muscarinic M1; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
C086088:CHRM1 protein, human; D008024:Ligands; D043643:Receptor, Muscarinic M1; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic; D011993:Recombinant Fusion Proteins; D015636:Magnesium Chloride; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins