Characterization of Two-Pore Channel 2 by Nuclear Membrane Electrophysiology

Lysosomal calcium (Ca2+) 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 Ca2+ 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 Ca2+ 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.published_or_final_versio

Mechanism disrupted capacitative calcium entry familial alzheimer’s disease

The Conference program's website is located at http://www.grc.org/programs.aspx?year=2013&program=calsi

Lysosomal Ca2+ disruption induces autophagy impairment in familial Alzheimer’s disease

Poster Presentation: no. P15Autophagy is an evolutionary conserved cellular pathway for the cell to cope with stress and starvation. Deranged autophagy has been observed in Alzheimer’s disease (AD)[1] and it has been reported that presenilin-1(PS1) is essential for the maintenance of acidic lysosomal pH[2]. However, the underlying molecular mechanism for lysosomal alkalization due to PS1 mutation is still obscure. Two-pore channel 2 (TPC2) is a lysosomal Ca2+ release channel that was found to play a role in lysosomal alkalization[3]. Here we found that PS1 interacted with TPC2 channel in the human neuroblastoma SH-SY5Y. The lysosomal Ca2+ content was significantly lower while its pH was higher in PS1-M146L expressing SH-SY5Y than those of wild-type PS1 expressing cell and GFP-expressing control cell. Intriguingly, reduced lysosomal Ca2+ content and alkalization in PS1-M146L can be rescued by treating the cell with TPC2 specific inhibitor, NED-19. Consistent Ca2+ disruption and lysosomal alkalization were observed in fibroblasts isolated from AD patients with PS1 mutation and these derangements were corrected by NED-19. Together, our results suggested that lysosomal Ca2+ disruption due to PS1 mutation plays a role in the pathogenic mechanism for autophagy impairment in familial AD and this provided a novel target for therapeutic intervention of the disease