Potency of A-425619 is altered by co-application of capsaicin and acidic pH. A) A-425619 inhibits currents activated by half maximal concentrations of either capsaicin (1 μM) or protons (pH 5.5) with similar efficacy (20.1 vs. 25.9 nM). Co-application of these same concentrations of capsaicin and acidic pH results in a significant rightward shift in the IC50 of A-425619 (704 nM). Reducing the capsaicin concentration to 100 nM (near the EC75 under acidic conditions) or 30 nM (~EC35) shifts the IC50 of A-425619 back to the left (63.6 and 23.2 nM respectively). B) Schild plot of the effect of A-425619 at different capsaicin concentrations. The resulting data points were fit with a linear regression with a slope of 1.13 and a pA2 value of 47.9 nM. The linear relationship is consistent with the results that show acidic conditions increases the affinity of capsaicin for the TRPV1 channel and that A-425619 is a competitive antagonist at the capsaicin site.