[View Larger Version of this Image]

Figure S2. Effects of IP₃R-LBD_224-605 on cytoplasmic Ca²⁺ responses and ER Ca²⁺ homeostasis. (A) Single-cell [Ca²⁺]_c measurement in fura-2-loaded control (top, nontransfected) and IP₃R-LBD_224-605-mRFP1-transfected cells (bottom) stimulated with increasing doses of histamine (1-100 µM). Two groups of IP₃R-LBD_224-605-mRFP1-expressing cells were separately plotted according to their mRFP1 intensity (horizontal histogram). Normalized fura-2 ratio (340/380 nm excitation) changes (ΔR/R) from the three groups of cells are shown on the right. For experimental details see the Supplementary materials and methods. Representative traces from three separate experiments are shown on the figure. (B) Population measurement of [Ca²⁺]_c responses to increasing doses of histamine (1-100 µM). HeLa cells were transfected with cytosolic aequorin (control; black) or cotransfected with OMM-IP₃R-LBD_224-605; they were then placed in a luminometer chamber and stimulated with the Ca²⁺ agonist histamine, as described in the Materials and methods. [Ca²⁺] values were obtained by converting luminescence values into [Ca²⁺]. Traces are representative of seven experiments from three cell preparations. (C) [Ca²⁺]_er steady-state levels of control and ER-IP₃R-LBD_224-605-transfected HeLa cells. Cells were transfected with erAEQmut (control) and with ER-IP₃R-LBD_224-605 expression vector. Mean ± the SEM of steady state [Ca²⁺]_er is shown, after refilling of the ER in the presence of 1 mM CaCl₂ in the extracellular medium (n = 10, from 4 separate experiments). Before measurements, erAEQmut-transfected cells were reconstituted with coelenterazine n, after ER Ca²⁺ depletion in a solution containing 0 [Ca²⁺], 600 µM EGTA, and 1 µM ionomycin.