Vol. 305, Issue 2, 786-795, May 2003

An Unusual Form of the Association Binding Kinetics of N-[³H]Methylscopolamine to the Split Muscarinic M_2trunk/M_2tail Receptor

Francesca Novi, Marco Scarselli, Giovanni U. Corsini and Roberto Maggio

Department of Neurosciences, University of Pisa, Pisa, Italy

The muscarinic M₂ receptor was split at the third cytoplasmic loop into two fragments: the one containing the first five transmembrane regions and the N-terminal part of the third cytoplasmic loop was named M_2trunk, while the other, which contained the last two transmembrane regions and the C-terminal part of the third cytoplasmic loop, was named M_2tail. As seen in many other G protein-coupled receptors, when these two fragments were transfected together in COS-7 cells they rescued the pharmacological profile and the functional activity of the wild-type M₂ receptor. Conversely, N-[³H]methylscopolamine ([³H]NMS) association binding experiments showed a substantial difference between the wild-type M₂ and the split M_2trunk/M_2tail receptors. The progression of the association binding kinetic of the M_2trunk/M_2tail receptor was strictly dependent upon the amount of the fragment DNA transfected. When the amount of transfected DNA was 4 µg/plate and the B_max of [³H]NMS at equilibrium was around 200 fmol/mg protein the form of the association was that of classical saturation, but when the amount of transfected DNA was lower the [³H]NMS association reached a maximum binding point and then declined to a lower equilibrium binding level. The form of the association was temperature-dependent: as the temperature was lowered, the maximum binding point tended to be higher. We suggest that this peculiar form of the [³H]NMS association binding to the muscarinic M_2trunk/M_2tail receptor is attributable to a less stable interaction between the trunk and the tail fragments of the split receptor.