Fig. 6: CISD1 directly binds to IP₃R and increases its calcium channel activity.

a The ___domain architecture of human CISD1. The transmembrane ___domain (TM) and the CDGSH ___domain are indicated. The FL represents the full-length protein of human CISD1. We generated the D1 construct which lacks the CDGSH ___domain. b, c HEK293T cells were transfected as indicated and cell lysates were subjected to anti-Myc immunoprecipitation followed by immunoblot analysis. Three independent experiments were repeated and the band intensity for the interaction between CISD1 and IP₃R1 shown by co-immunoprecipitation experiments was displayed in bar graphs (bottom). d Measurement of the IP₃R activity of WT (black, n = 63) and CISD1 KO (red, n = 68) cells when transfected with exogenous empty vector (vector, black and red), CISD1 WT (blue, n = 70), and CISD1 C74A mutant (green, n = 74). The bar graphs indicate the magnitude of the change during IP₃ treatment. e, f Measurement of ER (e) and cytosolic (f) calcium modulation in WT (black) and CISD1 KO (red) MEF cells with empty vector (vector). Similar experiments were also conducted for CISD1 KO MEF cells expressing exogenous CISD1 WT (blue) or C74A mutant (green). In all, 100 µM ATP was delivered to initiate IP₃R-mediated calcium release. The right side bar graphs indicate the quantification of the normalized calcium traces using AUC of calcium release during ATP treatment. n = 114–162 cells. One-way ANOVA with Tukey’s multiple comparisons test was used (b–f). ****p < 0.0001. ***p < 0.001. ns represents not significant. Source data, the exact p values, and n number of each experiment are included within the Source Data file. All data were presented as mean ± SD.