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