(A) Continuous single InsP₃R channel current traces recorded from outer membranes of nuclei isolated from PS deficient MEF (PSDKO) and PSDKO stably expressing human PS1-WT, FAD PS1-M146L or secretase-dead PS1-D257A (+40 mV; 10 µM InsP₃ and 1 µM free Ca²⁺ in pipette solution). Arrows indicate closed channel current levels. (B) Western blot forPS1 in PS-deficient and stably transduced MEF cells. Summary of InsP₃R channel P_o (C), τ_o (open circle) and τ_c (filled circle) (D) and modal gating analysis (E). Asterisks: p < 0.05 by ANOVA compared with PSDKO.

(A) Continuous single InsP₃R channel current trace (300 s) recorded from outer membrane of nucleus isolated from human B lymphoblaste at +20 mV with 10 µM InsP₃ and 1 µM free Ca²⁺ in pipette solution. Arrows indicate closed channel current levels. (B) Representative current traces (+20 mV) in nuclei isolated from human B lymphoblasts. Channel activity required presence of InsP₃ (n = 20) and was inhibited by heparin (n = 15). (C) I/V relationship obtained by ramping holding potential from −60 to +60 mV. (D) Summary of InsP₃R channel P_o, and mean open τ_o and closed τ_c durations.

(A) Representative current recordings (+20 mV) in outer membrane patches of Sf9 cell nuclei infected with different recombinant PS baculoviruses. EVER1 served as an ER membrane protein infection control. Pipette solution contained 1µM free Ca²⁺ and 100 nM InsP₃. Arrows indicate closed channel current level in this and all subsequent Figs. Summary of effects of PS on InsP₃R channel P_o (B), and mean open time τ_o (open circle) and mean closed time τ_c (filled circle) (C). (D) Summary of effects of PS on InsP₃R modal gating. Bars: mean ± SEM. Asterisks: p < 0.05 by ANOVA compared with EVER1-infected cells.

(A) Representative single cell Ca²⁺ responses to strong IgM stimulation (5µg/ml; arrow) in control human B lymphoblasts (CTL) or FAD lymphoblasts carrying PS1-A246E mutation. Dark lines below and to the left of each trace indicate zero Ca²⁺. (B) Responses to IgM stimulation. Percentages responding with Ca²⁺ oscillations (red) or large amplitude Ca²⁺ transients (blue). (C) Summary of peak amplitudes of high-amplitude transient Ca²⁺ responses triggered by 5 µg/ml anti-IgM. (D) Ca²⁺ oscillation frequency in response to anti-IgM. N = 3 experiments with 30 cells in each. Asterisk: p < 0.05, Student’s t-test. (E) Representative single cell Ca²⁺ responses to weak IgM stimulation (50 ng/ml; arrow) and (F) spontaneous oscillations during perfusion with serum-containing medium in lymphoblasts from unaffected (CTL) and FAD individuals. Dark lines: zero Ca²⁺ level. (G) Percentage of cells responding to IgM (black) or undergoing spontaneous Ca²⁺ oscillations in complete medium (blue). (H) Summaries of Ca²⁺ oscillation frequency in response to IgM (black) or spontaneous Ca²⁺ oscillations observed in complete medium (blue). Data in each group summarized from 4 experiments with 30 cells in each. Asterisks or #: p < 0.05 by ANOVA as compared with respective controls.

(A) Representative InsP₃R currents (+20 mV) in nuclei isolated from human FAD B lymphoblasts and control lymphoblasts from age-matched individuals without FAD activated with 10 µM InsP₃ and 1 µM Ca²⁺ in pipette solution. Summary of channel P_o (B), τ_o (open circles) and τ_c (filled circles) (C) and modal gating analysis (D). Asterisks: p < 0.05, ANOVA compared with CTL1. (E) Modal gating analyses. Each section shows continuous recording with gating mode assignment in color code below. In cells from normal individuals, low P_o is associated with switching between L and I modes. In cells from all three individuals with FAD, enhanced gating is manifested by increased occupancy of H mode at expense of L mode. F-H. Single InsP₃R channel current traces from human B cells activated by sub-optimal InsP₃. (F) Representative currents (+20 mV) in isolated nuclei from human FAD lymphoblasts and age-matched control B lymphoblastss activated by sub-optimal 100 nM InsP₃ and 1 µM Ca²⁺. Summary of InsP₃R P_o (G), and τ_o (open circle) and τ_c (filled circle) (H) from aged-matched control and FAD human B-lymphocblasts. Asterisks: p < 0.05 by student’s t-test.

APP is processed by either α-secretase or β-secretase, the latter leading to Aβ generation after subsequent cleavage by γ-secretase. Stimulation of G-protein coupled receptors (GPCR) or other cell surface receptors by extracellular ligands activates phospholipase C (PLC), which cleaves phosphatidylinositol bisphosphate (PIP₂) to produce InsP₃. InsP₃ binds to and activates the InsP₃R to release Ca²⁺ from ER stores, increasing cytoplasmic Ca²⁺ concentration. In normal cells, these Ca²⁺ signals are tightly regulated in time, space, and amplitude. In FAD cells, mutant PS exerts stimulatory effects on InsP₃R gating by modal switching to the H mode associated with prolonged channel openings. H mode gating generates exaggerated Ca²⁺ signaling by promoting additional release channel recruitment by CICR. Increased cytosolic Ca²⁺ concentration promotes β-secretase activity (52) and Aβ production (51, 54), which, together with mutant PS-enhanced production of amyloidogenic Aβ, results in plaque formation.

(A) Continuous single InsP₃R current trace (200 s) in outer membrane of nucleus isolated from embryonic cortical neuron (+40 mV with 10 µM InsP₃ and 1 µM free Ca²⁺ in pipette solution). Arrows: closed channel current level. (B) Representative current traces (+40 mV) in nuclei from C57BL/6 (wild type) or 3xTg-AD mice (E14 to E16). Channel activities in both mouse lines required InsP₃ and were inhibited by heparin. (C) InsP₃-activated currents from C57BL/6 (blue) or 3xTg-AD (red) mice were linear with 375 pS slope conductance. (D) Summary of InsP₃R channel P_o, τ_o and τ_c in cortical neuron nuclei. Bars: mean ± SEM. Asterisks: p < 0.05 by unpaired Student’s t-test. (E) Summary of InsP₃R modal gating analysis. Colors for gating modes same as Figs 1 and 3. (F) Modal gating analysis of InsP₃R from cortical neurons. Each section is a continuous single channel current record with modal assignment indicated by color code. In cells from C57BL/6 mouse, channel gating is alternates between L and I modes, whereas in 3xTg-AD mouse, InsP₃R gating alternates between H and I modes.