Fig. 6: Functional validation of gating residues in ion conduction pathway.
From: Conformational motions and ligand-binding underlying gating and regulation in IP3R channel
a Immunoblots of lysates prepared from the indicated HEK cell lines. HEK-3KO and endogenous hIP3R1 (Endo. hIP3R1) were generated by CRISPR/Cas 9 technology, the former is null for all IP3R subtypes while the latter expresses only IP3R1. Mutations and exogenously expressed IP3R1 (Exo. hR1) were stably expressed in HEK-3KO cells. All stable clonal cell lines result in expression levels above that of Endo. hIP3R1, as quantified in b. Data are presented as mean values + /- SEM (n = 4 independent experiments). HEK cell lines are color coded: blue—HEK-3KO, purple—endo hIP3R1, green—exo hIP3R1, pink—hIP3R1 F2546K #25, magenta—hIP3R1 F2546K #47, orange—hIP3R1 F2550T #116, red—hIP3R1 F2550N #615. brown—hIP3R1 F2550N #616. c Representative single cell Ca2+ traces in fura-2 loaded HEK cell lines stimulated with increasing concentrations of the muscarinic agonist carbachol (CCh). d Basal fluorescence, prior to stimulation in HEK cell lines. e Pooled data depicting the response of cell lines to CCh stimulation. Data in d-e are presented as mean values + /- SEM (n = at least 60 cells over three independent experiments). *significantly different from Endo. hIP3R1. #significantly different from Exo. hIP3R1. For 3 µM CCh: ###p < 0.0001; ##p = 0.0007, #p = 0.0395; for 30 µM CCh and 100 µM CCh: ###***p < 0.0001 One-way ANOVA with Tukey’s post-hoc test. Source data are provided as Source Data file.
