Presenilins Modulate Cellular Activity of Ryanodine Receptors

Payne, Andrew J., Presenilins Modulate Cellular Activity of Ryanodine Receptors. Doctor of Philosophy (Biomedical Sciences), December, 2012, 160 pp., 7 tables, 39 figures, bibliography 241 titles. Ryanodine Receptors (RyRs) are large, endoplasmic reticulum (ER) intracellular calcium channels in excitable cells. RyRs are major cellular mediators of calcium-induced calcium release and crucial regulators of intracellular calcium homeostasis. Disruption of RyR function has been described in pathologies of dysregulated calcium such as Alzheimer’s disease (AD). Presenilins (PS1 and PS2) are ER transmembrane proteins expressed in the central nervous system mediating calcium homeostasis and Notch signaling, and act as the proteolytic core of γ-secretase in amyloid cleavage. Previous single channel electrophysiology studies described a direct interaction between RyR and the N-termini of presenilin 1 (PS1NTF) and presenilin 2 (PS2NTF) that resulted in differential modulation of the RyR open probability and mean Ca2+ current at the RyR single channel level. We herein tested the hypothesis that PS1NTF and PS2NTF functionally modulate RyRs in a physiologically relevant in vitro model resulting in changes to RyR-mediated intracellular calcium release. Confocal microscopy, microfluorimetry, and coimmunoprecipitation studies confirmed a physical interaction between RyRs and PS-NTFs in human neuroblastoma SH-SY5Y cells, an in vitro AD model. Live cell fluorescent calcium imaging was used to quantify the effects of overexpression of PS1NTF or PS2NTF on Ca2+ release from RyR mediated stores. PS1NTF was found to increase RyR gating to the full open state at physiologically normal calcium concentrations. Verifying the previous electrophysiology data, PS2NTF had no effect on RyR at physiological calcium concentrations. Mutagenesis of critical cysteine residues on the PSNTFs was applied to determine the effect of specific structure-function differences between PS1 and PS2 molecules that underlie the isoform specific modulation of RyR calcium release. Mutations to PS2NTF removing disulfide bridging cysteines recapitulated PS1NTF-like regulation of RyR Ca2+ release. Our findings indicate that PS1NTF and PS2NTF bind RyRs differentially. Our results indicate a novel mechanism of intracellular calcium regulation by the PS-RyR interaction and a novel target for the treatment of AD, neurodegenerative disorders, and diseases controlled by RyR and PS functions.