Cross-linking can be prevented by cysteine residue blockade. (A) Representative blots from HEK293F cells transfected with hP2X1 C349A and G30C C349A, treated with 3.2 U/ml apyrase only, apyrase then BMB, or apyrase then NEM followed by BMB. Cells transfected with C349A 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 showed the intensity of the ∼55 kD band decreased and the intensity of the ∼110 kD band increased with the addition of BMB, suggesting dimerization of the subunits. Prior treatment with NEM showed no enhancement of the ∼110 kD band in the presence of BMB. This shows that NEM treatment prevents BMB from accessing the introduced cysteine residue and inhibits dimerization. The outline shows lanes taken from different membranes. (B) Example blots from cells transfected with either G30C C349A or N284C C349A, and treated with either apyrase only or apyrase with BM(PEG)2. The mutation N284C did not show an enhancement of the ∼110 kD band with BM(PEG)2 treatment, suggesting these residues cannot be cross-linked. The dotted line indicates where lanes have been removed for clarity. (C) Homology models of two hP2X1 N284C receptors, showing that N284C (gray sticks) from two separate receptors could be within ∼13 Å of each other without the receptors clashing, as shown by line, while G30C were ∼51 Å apart. The two models were arranged manually to minimize the N284-N284 and G30-G30 distances while keeping the central axes of both models perpendicular to the membrane plane.
