Western blot of PS1 (A) or PS2 (B) immunoprecipitates from lysates of Sf9 cells co-expressing either PS1-WT or PS1-M146L (A) or PS2-WT or PS2-N141I (B) with rat InsP₃R isoforms 1 or 3; expressed proteins shown at top and probing antibodies shown on right. (C) Western blots of PS1 (top) and PS2 (bottom) immunoprecipitates from mouse brain lysate. Top: Lanes 1 and 2: total input; lane 3: IgG immunoprecipitate control; lane 4: PS1 immunoprecipitate. Bottom: Lane 1: total input; lane 2: PS2 immunoprecipitate; lane3: rabbit serum immunoprecipitate control.

(A) Stable expression of PS1-WT and PS1-M146L proteins in wild-type (WT) and InsP₃R-deficient (KO) DT40 cell lines that stably expressed APP_SWE. Actin probed as loading control. (B) ELISA measurements of Aβ₄₀ (top), Aβ₄₂ (middle) and Aβ₄₂/Aβ₄₀ ratio (bottom) secreted over 48 h by InsP₃R-expressing wild-type (WT; left) or InsP₃R-deficient (KO DT40 cells; right) DT40 cells stably expressing APP_SWE alone (blue) or APP_SWE with PS1-WT (red) or PS1-M146L (green). Asterisks: p < 0.01 compared with control WT cells. Cross: p < 0.01 compared with control WT cells; double cross: p < 0.01 compared with PS1-WT cells.

Representative InsP₃R single-channel current recordings in presence of saturating (10 μM; A) or sub-saturating (100 nM; B) InsP₃ in DT40 cells stably transfected with PS1 WT or M146L. Pipette [Ca²⁺] was 1 μM, optimal for channel activity; arrows: zero current level. Summary of effects of PS1 expression on InsP₃R P_o (C), mean open time (τ_o) (D) and mean closed time (τ_c) (E). Asterisks: p < 0.01, unpaired t-test. Data presented in Supplementary Table 2.

(A,B) Representative current recordings in isolated nuclei from Sf9 cells infected with PS2 WT or N141I baculovirus in the presence of saturating (10 μM; A) or sub-saturating (33 nM; B) InsP₃. Pipette [Ca²⁺] was 1 μM; arrows: zero current level. Summary of effects of PS2 expression on InsP₃R P_o (C), channel mean open time (τ_o) (D) and channel mean closed time (τ_c) (E). Asterisks: p < 0.01, unpaired t-test. Data presented in Supplementary Table 3.

(A,B) Responses to strong stimulation by BCR antibody of DT40 cell [Ca²⁺]_i. (A) Representative single cell responses to 5 μg/ml anti-IgM (added at arrow) in untransfected (blue) and PS1-WT (red) and PS1-M146L (green) stably-transfected DT40 cells. (B) Summary of peak [Ca²⁺]_i responses triggered by 5 μg/ml anti-IgM (n = 90). Asterisk: p < 0.01 compared with WT and PS1-WT (C-F) Responses to weak stimulation by BCR antibody of DT40 cell [Ca²⁺]_i. (C) Representative single cell [Ca²⁺]_i responses to 50 ng/ml anti-IgM (IgM; added at arrow) stimulation of BCR in control (blue), PS1-WT (red) and PS1-M146L (green and pink) stably-transfected DT40 cells. (D) Summary of percentage of cells responding to 50 ng/ml anti-IgM (n = 90). ~ 30% (purple) of PS1-M146L expressing cells exhibited a different, exaggerated [Ca²⁺]_i response. (E) [Ca²⁺]_i oscillation frequency triggered by anti-IgM in WT DT40, PS1-WT expressing and PS1-M146L expressing cells. (F) Summary of latencies to first response in WT DT40, PS1-WT expressing and PS1-M146L expressing cells. The 30% of PS1-M146L expressing cells that exhibited the exaggerated response had nearly no latency (purple). (G-I) Spontaneous [Ca²⁺]_i oscillations in PS1-expressing DT40 cells. (G) Representative spontaneous single cell [Ca²⁺]_i oscillations in control (blue) and PS1-WT (red) and PS1-M146L (green and purple) stably-transfected DT40 cells. Some PS1-M146L expressing cells (~ 4%, purple) displayed a distinct, exaggerated spontaneous [Ca²⁺]_i signal (bottom). (H) Percentage of cells displaying spontaneous [Ca²⁺]_i oscillations (n = 90). (I) Spontaneous [Ca²⁺]_i oscillation frequency observed in WT DT40 cells and PS1-WT- and PS1-M146L-expressing DT40 cells. Asterisks: p < 0.01 compared with WT DT40 cells. Asterisks with bars: p < 0.01 PS1-Wt vs PS1-M146L.

(A-C) Representative current recordings in isolated nuclei from Sf9 cells infected with PS1 WT or M146L baculoviruses in absence (A) or presence of saturating (10 μM; B) or sub-saturating (33 nM; C) InsP₃ in pipette solution. Channel activity was not evoked by PS1 alone in absence of InsP₃ (A) whereas InsP₃R channels were activated in presence of InsP₃ (B,C). Pipette [Ca²⁺] was 1 μM; arrows: zero current level. Summary of effects of PS1 expression on InsP₃R channel open probability P_o (D) mean open time (τ_o) (E) and mean closed time (τ_c) (F). Asterisks: p < 0.01, unpaired t-test. Data presented in Supplementary Table 1.

(A) ER [Ca²⁺], expressed as Mag-Fura2 ratio normalized to ratio at t=0, before and then during filling upon addition of MgATP (1.5 mM), and subsequent exposure to 1 μM thapsigargin in control (blue) and PS1-WT (red) and PS1-M146L (green) transfected mouse cortical neurons. (B) Summary of steady state ER [Ca²⁺] following MgATP induced loading in (A). (C) Summary of Ca²⁺ leak rate measured in presence of thapsigargin in (A). Asterisk: p = 0.01. (D) ER [Ca²⁺] during filling upon addition of MgATP (1.5 mM) in control (black) and EGFP (blue), PS1-WT (red) and PS1-M146L (green) transfected mouse cortical neurons. (E) Response of ER [Ca²⁺] to 33 nM InsP₃ in cells from (D) and in control cells in response to 10 μM InsP₃ (black trace). (F) Summary of initial Ca²⁺ release rate, expressed as Mag-Fura2 ratio, from (E). Asterisks: p < 0.01 compared with control and PS1-WT.

(A) ER [Ca²⁺], expressed as Mag-Fura2 ratio normalized to ratio at t=0 before and then during filling upon addition of MgATP (1.5 mM) and subsequent emptying by addition of InsP₃ (10 μM) in control (blue) and PS1-WT (red) and PS1-M146L (green) stable DT40 cells. Insert: Steady state ER [Ca²⁺] in presence of MgATP. Asterisks: p <0.01 compared with control cells. Cross: p<0.01 compared with PS1-WT. (B) Similar experiment with ER Ca²⁺ loading performed in presence of heparin (100 μg/ml). Asterisk: p < 0.01 and < 0.05 compared with control and PS1-WT cells, respectively. (C) ER [Ca²⁺] during filling upon addition of MgATP (1.5 mM) in presence of heparin (100 μg/ml), and then after removal of MgATP and addition of thapsigargin (1 μM), in control (blue) and PS1-WT (red) and PS1-M146L (green) stable DT40 cells. (D) Summary of ER Ca²⁺ leak rate, calculated as initial rate of decline of the Mag-Fura2 ratio. Asterisk: enhanced leak observed in M146L-expressing cells compared with control and PS1-WT (p < 0.01). (E) Similar experiments as in (D), except that heparin (100 μg/ml) was present during measurements of the Ca²⁺ leak rate. (F). Summary of ER Ca²⁺ leak rate in presence of heparin. (G) Similar experiment as in (D) performed in cells lacking InsP₃R expression (KO). Insert: steady-state ER [Ca²⁺] after MgATP-induced loading. (H) Summary of ER Ca²⁺ leak rate in PS1 expressing InsP₃R KO cells.