Characterization of Two-Pore Channel 2 by Nuclear Membrane Electrophysiology

• Published in: Scientific reports Vol. 6; no. 1; p. 20282
• Main Authors: Lee, Claire Shuk-Kwan, Tong, Benjamin Chun-Kit, Cheng, Cecily Wing-Hei, Hung, Harry Chun-Hin, Cheung, King-Ho
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 03-02-2016; Nature Publishing Group
• Subjects: 13; 14/34; 631/443; 631/80; Animals; Calcium - metabolism; Calcium Channels - genetics; Calcium Channels - metabolism; Calcium Signaling - physiology; Calcium signalling; Cell Line; Channel gating; Chickens; Cyclic AMP-Dependent Protein Kinases - metabolism; Dialysis; Electrophysiological Phenomena; Electrophysiology; Endoplasmic Reticulum - metabolism; Fura-2; Gene Expression Regulation; HEK293 Cells; Humanities and Social Sciences; Humans; Lipids; Lysosomes - metabolism; Membranes; Models, Biological; multidisciplinary; NAADP; NADP - analogs & derivatives; NADP - metabolism; Nuclear Envelope - physiology; Patch-Clamp Techniques; Phosphorylation; Protein kinase A; Ryanodine receptors; Science
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep20282

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 InsP
3
R 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.