FIGURE 9.

Effect of CuPhen and atropine on the formation of disulfide cross-linked M3R dimers (i2 loop Cys mutants). Membranes prepared from COS-7 cells expressing the indicated mutant M3Rs containing Cys substitutions within the C-terminal segment of the i2 loop were incubated with (+) or without (−) 100 μm CuPhen for 10 min (at room temperature). CuPhen-treated samples were left either untreated or incubated with atropine (Atr; 1 μm), a muscarinic antagonist/inverse agonist. The L264C-M3′(3C)-Xa construct (16) served as a positive control. Membrane extracts were then subjected to SDS-PAGE under non-reducing conditions, and M3Rs were detected via Western blotting using the polyclonal anti-M3R antibody. The observed pattern of bands indicates that CuPhen is required for efficient M3R-M3R disulfide cross-linking displayed by some of the studied mutant receptors and that atropine has no significant effect on the efficiency of M3R dimer formation. The blots shown are representative of two independent experiments. Asterisk, putative receptor monomers; arrow, putative cross-linked receptor dimers.