Fig. 6.

Mechanism of action of ARN11391. (A) Effect of ARN11391 on ITPR1 channel activity. Left: representative single-channel currents recorded in on-nucleus patch-clamp experiments from HEK293 (HEKR1) cells stably expressing ITPR1-YFP (Vp = + 40-mV). Each trace is from a separate experiment. Right: open channel probability for experiments with vehicle or ARN11391 (20 µM) in the pipette solution. **P <0.01 vs. vehicle (Student’s t-test). (B) Effect of ARN11391 on HEK293 cells with inducible (tetracycline) expression of wild type or mutant ITPR1. Top: representative traces showing changes in Fluo-4 fluorescence elicited by UTP (5 µM). Cells were preincubated with vehicle (DMSO) or ARN11391 (10 µM). Where indicated, tetracycline (Tet) was added to cells to induce IPTR1 expression. Bottom: summary of data. The symbols report the maximal change in fluorescence. *P <0.05; **P <0.01. ***P <0.001 vs. vehicle (ANOVA with Dunnett’s post-hoc test). (C) Data from Fluo-4 experiments done on HEK293 cells expressing wild type (left), R269W (middle), and T267M (right) ITPR1. Cell were previously treated with tetracycline and then stimulated with 5 µM UTP as in (B). *P <0.05; ***P <0.001 (ANOVA with Dunnett's post-hoc test). (D) IP3 uncaging experiments carried out in HEK293 cells expressing the indicated mutant ITPR1. Cells were treated with tetracycline before experiments. Uncaging was done in the presence and absence of 10 µM ARN11391. **P <0.01 (Student’s t-test).