Roles of IP3R and RyR Ca2+ Channels in Endoplasmic Reticulum Stress and β-Cell Death

Roles of IP 3 R and RyR Ca 2+ Channels in Endoplasmic Reticulum Stress and β-Cell Death Dan S. Luciani , Kamila S. Gwiazda , Ting-Lin B. Yang , Tatyana B. Kalynyak , Yaryna Bychkivska , Matthew H.Z. Frey , Kristin D. Jeffrey , Arthur V. Sampaio , T. Michael Underhill and James D. Johnson From the Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Comlumbia, Canada Corresponding author: James D. Johnson, jimjohn{at}interchange.ubc.ca Abstract OBJECTIVE— Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of diabetes, but the roles of specific ER Ca 2+ release channels in the ER stress–associated apoptosis pathway remain unknown. Here, we examined the effects of stimulating or inhibiting the ER-resident inositol trisphosphate receptors (IP 3 Rs) and the ryanodine receptors (RyRs) on the induction of β-cell ER stress and apoptosis. RESEARCH DESIGN AND METHODS— Kinetics of β-cell death were tracked by imaging propidium iodide incorporation and caspase-3 activity in real time. ER stress and apoptosis were assessed by Western blot. Mitochondrial membrane potential was monitored by flow cytometry. Cytosolic Ca 2+ was imaged using fura-2, and genetically encoded fluorescence resonance energy transfer (FRET)–based probes were used to measure Ca 2+ in ER and mitochondria. RESULTS— Neither RyR nor IP 3 R inhibition, alone or in combination, caused robust death within 24 h. In contrast, blocking sarco/endoplasmic reticulum ATPase (SERCA) pumps depleted ER Ca 2+ and induced marked phosphorylation of PKR-like ER kinase (PERK) and eukaryotic initiation factor-2α (eIF2α), C/EBP homologous protein (CHOP)–associated ER stress, caspase-3 activation, and death. Notably, ER stress following SERCA inhibition was attenuated by blocking IP 3 Rs and RyRs. Conversely, stimulation of ER Ca 2+ release channels accelerated thapsigargin-induced ER depletion and apoptosis. SERCA block also activated caspase-9 and induced perturbations of the mitochondrial membrane potential, resulting eventually in the loss of mitochondrial polarization. CONCLUSIONS— This study demonstrates that the activity of ER Ca 2+ channels regulates the susceptibility of β-cells to ER stress resulting from impaired SERCA function. Our results also suggest the involvement of mitochondria in β-cell apoptosis associated with dysfunctional β-cell ER Ca 2+ homeostasis and ER stress. Footnotes Published ahead of print at http://diabetes.diabetesjou