Familial Alzheimer's disease-associated presenilin 1 mutants promote γ-secretase cleavage of STIM1 to impair store-operated Ca2+ entry.

Some forms of familial Alzheimer's disease (FAD) are caused by mutations in presenilins (PSs), catalytic components of a γ-secretase complex that cleaves target proteins, including amyloid precursor protein (APP). Calcium (Ca(2+)) dysregulation in cells with these FAD-causing PS mutants has been attributed to attenuated store-operated Ca(2+) entry [SOCE; also called capacitative Ca(2+) entry (CCE)]. CCE occurs when STIM1 detects decreases in Ca(2+) in the endoplasmic reticulum (ER) and activates ORAI channels to replenish Ca(2+) stores in the ER. We showed that CCE was attenuated by PS1-associated γ-secretase activity. Endogenous PS1 and STIM1 interacted in human neuroblastoma SH-SY5Y cells, patient fibroblasts, and mouse primary cortical neurons. Forms of PS1 with FAD-associated mutations enhanced γ-secretase cleavage of the STIM1 transmembrane domain at a sequence that was similar to the γ-secretase cleavage sequence of APP. Cultured hippocampal neurons expressing mutant PS1 had attenuated CCE that was associated with destabilized dendritic spines, which were rescued by either γ-secretase inhibition or overexpression of STIM1. Our results indicate that γ-secretase activity may physiologically regulate CCE by targeting STIM1 and that restoring STIM1 may be a therapeutic approach in AD.

Characterization of Two-Pore Channel 2 by Nuclear Membrane Electrophysiology.

Lysosomal calcium (Ca(2+)) release mediated by NAADP triggers signalling cascades that regulate many cellular processes. The identification of two-pore channel 2 (TPC2) as the NAADP receptor advances our understanding of lysosomal Ca(2+) signalling, yet the lysosome is not amenable to traditional patch-clamp electrophysiology. Previous attempts to record TPC2 single-channel activity put TPC2 outside its native environment, which not reflect TPC2's true physiological properties. To test the feasibility of using nuclear membrane electrophysiology for TPC2 channel characterization, we constructed a stable human TPC2-expressing DT40TKO cell line that lacks endogenous InsP3R and RyR (DT40TKO-hTPC2). Immunostaining revealed hTPC2 expression on the ER and nuclear envelope. Intracellular dialysis of NAADP into Fura-2-loaded DT40TKO-hTPC2 cells elicited cytosolic Ca(2+) transients, suggesting that hTPC2 was functionally active. Using nuclear membrane electrophysiology, we detected a ~220 pS single-channel current activated by NAADP with K(+) as the permeant ion. The detected single-channel recordings displayed a linear current-voltage relationship, were sensitive to Ned-19 inhibition, were biphasically regulated by NAADP concentration, and regulated by PKA phosphorylation. In summary, we developed a cell model for the characterization of the TPC2 channel and the nuclear membrane patch-clamp technique provided an alternative approach to rigorously investigate the electrophysiological properties of TPC2 with minimal manipulation.