Figure 6

Structural and functional consequences of mutations at the allosteric site of the M₂ receptor. The wild-type receptor (A–C) and a mutant in which allosteric Site 2 was eliminated by the substitution of alanine for tyrosine at positions 80 and 83 (D–F) were compared in molecular dynamics simulations and in binding assays on membranes from CHO cells. (A,D) Simulations of receptor-bound tacrine as viewed from a position above the extracellular surface. Two allosteric sites are available on an otherwise vacant protomer of the wild-type receptor (Sites 1 and 2) (A), whereas only one site is available on the mutant (i.e., Site 1) (D) or an NMS-occupied protomer (not shown). These computations were performed using PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC; http://www.pymol.org/). (B,E) Binding of tacrine. Membranes from CHO cells expressing the wild-type receptor (B) or the mutant (E) were mixed simultaneously with [³H]NMS (filled square, 1 nM; filled circle, 3 nM; filled triangle, 10 nM) and tacrine at the concentrations shown on the abscissa. Binding was measured after equilibration of the mixture for 21 h at 24 °C, and the data were analyzed in terms of Eq. (3) (n = 2). The lines in the figure depict the fitted curves, and the values of F₂ are shown in the insets. The fitted parametric values are listed in Table S10. (C,F) Depictions of the interaction between tacrine (open rhombus) and a dimeric receptor in which the orthosteric site of one protomer is occupied by NMS (open circle). In the wild-type receptor (C), the NMS-free protomer can bind two molecules of tacrine (Sites 1 and 2), whereas an NMS-occupied protomer can bind only one (Site 1). In the mutant (F), only one molecule of tacrine can bind (Site 1) irrespective of NMS.