title: Characterization of Two-Pore Channel 2 by Nuclear Membrane Electrophysiology.
abstract: 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.
authors.affiliation: School of Biomedical Sciences, LKS Faculty of Medicine, University of Hong Kong, Hong Kong, China.
authors.forename: Claire Shuk-Kwan
authors.initials: CS
authors.lastname: Lee
authors.affiliation: School of Biomedical Sciences, LKS Faculty of Medicine, University of Hong Kong, Hong Kong, China.
authors.forename: Benjamin Chun-Kit
authors.initials: BC
authors.lastname: Tong
authors.affiliation: School of Biomedical Sciences, LKS Faculty of Medicine, University of Hong Kong, Hong Kong, China.
authors.forename: Cecily Wing-Hei
authors.initials: CW
authors.lastname: Cheng
authors.affiliation: School of Biomedical Sciences, LKS Faculty of Medicine, University of Hong Kong, Hong Kong, China.
authors.forename: Harry Chun-Hin
authors.initials: HC
authors.lastname: Hung
authors.affiliation: School of Biomedical Sciences, LKS Faculty of Medicine, University of Hong Kong, Hong Kong, China.|Research Centre of Heart, Brain, Hormone and Healthy Aging, LKS Faculty of Medicine, University of Hong Kong, Hong Kong, China.
authors.forename: King-Ho
authors.initials: KH
authors.lastname: Cheung
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D002460:Cell Line; D002645:Chickens; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D055724:Electrophysiological Phenomena; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D008954:Models, Biological; D009249:NADP / Q000031:analogs & derivatives / Q000378:metabolism; D009685:Nuclear Envelope / Q000502:physiology*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation
chemical_list: D015220:Calcium Channels; C530224:TPCN2 protein, human; D009249:NADP; C024376:NAADP; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium
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