Abstract
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.
Highlights
Modulation of the cytoplasmic Ca2+ concentration elicits universal signals that regulate processes involved in life and death[1]
The single-channel properties of human TPC2 (hTPC2) were successfully revealed by the use of lipid bilayer electrophysiology and retargeting channel proteins to the plasma membrane[10,12,13]; these approaches, have major drawbacks that might not truly reflect the biophysical properties of hTPC2 in its native membrane environment
Human two-pore channel 2 (TPC2) is predominantly expressed in the late endosome, lysosome, and endoplasmic reticulum (ER) membrane[11]; we tested whether nuclear membrane electrophysiology[18,19] could be employed to characterize the electrophysiological properties of the hTPC2 channel
Summary
Modulation of the cytoplasmic Ca2+ concentration elicits universal signals that regulate processes involved in life and death[1]. Single-channel currents of human TPC2 (hTPC2) have been successfully recorded by reconstituting purified protein into an artificial lipid bilayer[10,13] or by retargeting hTPC2 protein to the plasma membrane by mutating the dileucine lysosomal signalling motif[12] These electrophysiological studies provided examples of the biophysical properties of the hTPC2 channel mediated by NAADP, these approaches have their drawbacks. TPC2 channel is not mediated by NAADP but phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2]14,15; this finding prompts the need for the development of a novel methodology for characterizing the hTPC2 channel in its native membrane In this regard, we generated a stable DT40 cell line expressing hTPC2 (DT40TKO-hTPC2) that lacked both functional InsP3R and RyR (DT40TKO)[16,17] to eliminate the influences by these intracellular Ca2+ release channels. We developed a cell model with minimal manipulation that, combined with nuclear membrane electrophysiology[18], enabled us to rigorously investigate the biophysical properties of the TPC2 channel in its native membrane
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