Fig. 4: Mutations in the xanthine-binding site of TRPC5 affect responses to the xanthine-based TRPC5 agonist AM237.

a, b, d, e, g, h, j, k Representative traces from single 96-well plates (N = 3; error bars show standard deviations over technical replicates) showing changes of [Ca²⁺]_i in response to 0.32 nM to 5 µM of EA (a, d, g, j) or AM237 (b, e, h, k) in HEK 293 cells transiently expressing wild-type TRPC5-SYFP2 (a, b), TRPC5-SYFP2_Q573T (d, e), TRPC5-SYFP2_F576A (g, h) or TRPC5-SYFP2_W577A (j, k). c Concentration–response data for experiments in a and b (scatter plots showing normalised data for three independent experiments each; n/N = 3/6). f Concentration–response data for experiments in d (scatter plot showing normalised data for three independent experiments; n/N = 3/6) and e (scatter plot showing normalised data for two independent experiments; n/N = 2/4). i Concentration–response data for experiments in g (scatter plot showing normalised data for two independent experiments; n/N = 2/4; because no upper limit of the EA response of F576A could be determined, the EC₅₀ for EA is unknown but can be estimated from the fitted curve to be at least 1400 nM; AM237 gave no response up to 5 µM). l Concentration–response data for experiments in j (scatter plot showing normalised data for two independent experiments; n/N = 2/5; AM237 gave no response up to 5 µM). Responses were calculated at 240–300 s compared to [Ca²⁺]_i at baseline (0–60 s). Averages of technical repeats of each individual experiment were normalised to the maximum response and vehicle control (0 nM) for the independent experiment, combined, and fit with GraphPad Prism 8 (variable slope, four-parameter logarithmic fit).