Position of extracellular flanks of β2 relative to the α S1–S6 TM helices. (A) Immunoblots illustrating endogenous cross-linking of Cys-substituted α and Cys-substituted β2. Portions of each sample were reduced with DTT and oxidized for 20 min with 40 µM QPD. At the bottom of each lane is the fraction of the cross-linked α–β2 complex. (B and C) Extents of endogenous disulfide bond formation between Cys-substituted extracellular flanks of α S0–S6 with β2 TM1 (B) and β2 TM2 (C) flanks. The α residues substituted by Cys are color coded as shown. The extent of disulfide bond formation is represented by bars in the horizontal direction. In the cases in which the mean extent of disulfide bond formation was zero, the value 0.5% was plotted to identify these pairs as tested. The residues closest to the membrane are underlined. (D) Locations of the extracellular ends of β2 TM1 and TM2 relative to the extracellular ends of α S0–S6. See Fig. 7 legend for details.