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Article: Characterization of Two-Pore Channel 2 by Nuclear Membrane Electrophysiology

TitleCharacterization of Two-Pore Channel 2 by Nuclear Membrane Electrophysiology
Authors
Issue Date2016
PublisherNature Publishing Group: Open Access Journals. The Journal's web site is located at http://www.nature.com/srep/index.html
Citation
Scientific Reports, 2016, v. 6, p. 20282 How to Cite?
AbstractLysosomal 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.
Persistent Identifierhttp://hdl.handle.net/10722/223815
ISSN
2015 Impact Factor: 5.228
2015 SCImago Journal Rankings: 2.073
PubMed Central ID

 

DC FieldValueLanguage
dc.contributor.authorLee, CSK-
dc.contributor.authorTong, BCK-
dc.contributor.authorCheng, CWH-
dc.contributor.authorHung, HCH-
dc.contributor.authorCheung, KH-
dc.date.accessioned2016-03-18T02:29:22Z-
dc.date.available2016-03-18T02:29:22Z-
dc.date.issued2016-
dc.identifier.citationScientific Reports, 2016, v. 6, p. 20282-
dc.identifier.issn2045-2322-
dc.identifier.urihttp://hdl.handle.net/10722/223815-
dc.description.abstractLysosomal 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.-
dc.languageeng-
dc.publisherNature Publishing Group: Open Access Journals. The Journal's web site is located at http://www.nature.com/srep/index.html-
dc.relation.ispartofScientific Reports-
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.titleCharacterization of Two-Pore Channel 2 by Nuclear Membrane Electrophysiology-
dc.typeArticle-
dc.identifier.emailCheung, KH: kingho.cheung@hku.hk-
dc.identifier.authorityCheung, KH=rp01463-
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.1038/srep20282-
dc.identifier.pmid26838264-
dc.identifier.pmcidPMC4738322-
dc.identifier.hkuros257127-
dc.identifier.volume6-
dc.identifier.spage20282-
dc.identifier.epage20282-
dc.publisher.placeUnited Kingdom-

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