The InsP₃R Ca²⁺ release channel. Cartoon depicting three of four InsP₃R molecules (in different colors) in a single tetrameric channel structure. Part of the lumenal loop connecting transmembrane helices 5 and 6 of each monomer dips into the fourfold symmetrical axis, creating the permeation pathway for Ca²⁺ efflux from the lumen of the endoplasmic reticulum. Reproduced from [15] with permission from the American Physiological Society

APP processing is dependent on InsP₃R. 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. From [10] with permission from Elsevier

Exaggerated [Ca²⁺]_i signaling in mutant PS-expressing DT40 cells. 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). Of PS1-M146L-expressing cells, ~30% (purple) exhibited a different, exaggerated [Ca²⁺]_i response. e [Ca²⁺]_i oscillation frequency triggered by anti-IgM in WT DT40, PS1-WT- and PS1-M146L-expressing cells. f Summary of latencies to first response in WT DT40, PS1-WT- and PS1-M146L-expressing cells. The 30% of PS1-M146L-expressing cells that exhibited the exaggerated response had nearly no latency (purple). Asterisks, p<0.01 compared with WT DT40 cells. Asterisks with bars, p<0.01 PS1-Wt vs PS1-M146L. From [10] with permission from Elsevier

Effects of PS1 expression on InsP₃R single channel activity in Sf9 cells. a–c Representative current recordings in isolated nuclei from Sf9 cells infected with PS1 WT or M146L baculoviruses in the 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 the absence of InsP₃ (a), whereas InsP₃R channels were activated in the 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. From [10] with permission from Elsevier

Modal gating analyses of InsP₃R channels under the influence of FAD-linked mutant PS. Distinct single channel InsP₃R gating behaviors from EVER1 (control) vs PS1-M146L-expressing Sf9 cells. Each section consists of a set of four traces of the same single channel current record: (top) unprocessed current trace, (second) idealized current trace generated using Qub software (third), idealized current trace after burst analysis (closing events <10 ms were filtered), and (bottom) modal assignment by analyzing channel burst (t_b) and gap (t_g) durations [28]. In EVER1-infected cells, low P_o is associated with prevalence of L gating mode. In PS1-M146L infected cells, enhanced P_o is manifested by increased t_b and decreased t_g. Channel occupancy of H mode is dominant, whereas occupancy of L gating mode is significantly decreased. From [9] with permission from the American Association for the Advancement of Science

Structural determinants of the InsP₃R. a Overall domain structure. The InsP₃R molecule depicted as a linear amino acid sequence, with the amino terminal InsP₃ binding region (red), coupling region (yellow), transmembrane region (green), and carboxyl tail (blue) depicted. b Linear amino acid sequence. Residues are numbered according to the rat type 1 SI+, SII+, SIII– sequence (protein accession number 121838). The structural features shown are: Arm sub-domain and β-trefoil in the InsP₃-binding suppressor domain; β-trefoil and armadillo repeats in InsP₃ binding core-domain; armadillo repeats in the coupling/regulatory domain; alternative splicing regions SI, SII, and SIII for type 1 InsP₃R; opt deletion in type 1 InsP₃Rmutant; Δ18 deletion in type 1 InsP₃R; ATP-binding site ATPA; transmembrane helices TM1–6, and pore-forming P region with selectivity filter; dimerizing region; tetramer forming region; S1589 and S1755 are PKA/PKG phosphorylation sites; mutation of Pro1059 to Leu in type 1 InsP₃R associated with SCA15; mutation of Val494 to Ile in type 1 InsP₃R associated with SCA15. The sequences involved in the interaction of InsP₃R channel with the following proteins are also depicted: CaBP1; IRBIT; CIB1; Na⁺/K⁺-ATPase; HAP1 and Htt^exp; poly-glutamine (poly-Q)-expanded ataxins 1 and 2; protein 4.1N; CA8; Bcl-2, Bcl-x_L, and Mcl-1. Modified from [15] with permission from the American Physiological Society

Hypothetical molecular mechanism of enhanced Aβ production due to Ca²⁺ disruption in FAD PS cells. 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 or other cell surface receptors by extracellular ligands activates phospholipase C (PLC), which cleaves phosphatidylinositol bisphosphate 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 cytoplasmic Ca²⁺ concentration promotes β-secretase activity [23] and Aβ production [19, 52] which, together with mutant PS-enhanced production of amyloidogenic Aβ, results in plaque formation. From [9] with permission from the American Association for the Advancement of Science