Figure 4.

The effect of σ1R ligands on the σ1R–TRP interactions. Agarose-TRP was incubated with the σ1R and the agarose–TRP–σ1R complexes were separated from the free σ1R through three cycles of washing/resuspension. The agarose–TRP–σ1R complexes were then incubated for 30 min with rotation at room temperature (RT) in the presence of increasing concentrations of the σ1R ligands in a final volume of 300 µL (50 mM Tris–HCl [pH 7.5], 3 mM CaCl₂, and 0.2% CHAPS). Finally, the σ1Rs that remained attached to the TRP were resolved by SDS-PAGE and evaluated in immunoblots. Agonists dampened on the association of the σ1Rs with TRPA1, while antagonist induced different effects on σ1Rs binding to the TRPA1 N- or C-terminal domains. The σ1R agonists produced different effects on the TRPV1-σ1R associations, and while the antagonists did not alter these associations at TRPV1 N-terminus, they did diminish these complexes at the TRPV1 C-terminal domain. The association of TRPM8 with the σ1R was enhanced by agonists and dampened by antagonists. The assays were performed twice, and each point was duplicated. Representative blots are shown. PregS, pregnenolone sulfate; ProG, progesterone. Details as in Figure 1 .