Specific amino-terminal cysteine residues form cross-links. (A) Representative blots from HEK293F cells transfected with hP2X1 C349A, R25C, N26C, K27C, K28C, V29C, or G30C treated with 3.2 U/ml apyrase, then BMB or DMSO (apyrase only). Of the mutants tested, only R25C and G30C showed strong enhancement of the ∼110 kD band with BMB treatments. This suggests that only R25C and G30C can dimerize within 10.9 Å. Outlines show images were taken from the same blot, while the dotted line shows where lanes have been removed for clarity. (B) Histogram showing the average percentage dimer present for the above mutants tested with all three cross-linkers. This shows that only R25C and G30C show ∼60% dimerization with BMB or BM(PEG)2 treatments. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; n = 3–5).
