Cysteine reactive cross-linkers dimerize subunits. (A) Model of the hP2X1R showing the location of the G30C mutation (black spheres). The three subunits are shown in gray, light blue, and light pink. (B) Chemical structures of BMB and BM(PEG)2. The lower panel shows how BM(PEG)2 can cross-link two cysteine residues. (C and D) Representative blots from HEK293F cells transfected with hP2X1 C349A (C) and G30C C349A (D), treated with 3.2 U/ml−1 apyrase, then cysteine-reactive cross-linker or DMSO (apyrase only). Cells transfected with C349A (C) showed intense bands at ∼55 kD but did not show any distinct bands at ∼110 kD in any condition, indicating no dimerization of these subunits. Cells transfected with G30C C349A (D) showed robust bands at ∼55 kD and faint bands at ∼110 kD in apyrase only. The intensity of the ∼55-kD band decreased and the intensity of the ∼110-kD band increased with the addition of the cysteine cross-linkers, suggesting dimerization of the subunits.
