Identification of some specific drugs for M2 receptor using m2-Gilα fusion protein

OBJECTIVE To identify the speeific ligands for M2 using m2-Gilα fusion protein by detecting the signaling transduction of its two partners.METHODS m2-Gilα fused cDNAs was generated via a two-step PCR protocol and then expressed with baculovirus-Sf9 cell system. [3H]QNB saturation binding in Sf9 membrane was performed to determine the expression level of m2 protein or m2-Gilα fusion protein. High affinity [35S] GTPγS binding detected the effect of various M2 ligands on [35S] GTPγS binding in the coexistence of GDP, GTP or ATP.RESULTS The expression level of m2-Gilα was (18.14 ±0.17) pmol·mg(pro)-1 .In the presence of 10 μmol·L-1GTPγS, [3H]QNB binding with m2-Gilα fusion protein decreased while [3H]QNB binding with m2 protein was not influenced.The curves shifted right on the existence of muscarinic agonists comparing with their absence,and the left shift was observed with muscarinic antagonist. IC50 values of GDP for Ach.Pilo, ligand-free, alcuronium, AF-DX116, Iso and atropine were 168.4,152.3,14.7,6.84,6.12,5.59 and 4.52 μmol·L-1 respectively. CONCLUSION m2-Cilα fusion protein has the pharmacological specificity of muscarinic receptor and the efficient signal of the two partners. The affinity of GDP to ligand-bound fusion protein represents the species of muscarinic ligands. Ach and Pilo are agonists for m2-Gilα fusion protein, but atropine, alcuronium, AF-DX116 and Iso are antagonists or superantagonists.