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Isolated pores dissected from human two-pore channel 2 are functional.

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cam.issuedOnline2016-12-12
dc.contributor.authorPenny, Christopher J
dc.contributor.authorRahman, Taufiq
dc.contributor.authorSula, Altin
dc.contributor.authorMiles, Andrew J
dc.contributor.authorWallace, BA
dc.contributor.authorPatel, Sandip
dc.contributor.orcidRahman, Md Taufiq [0000-0001-7247-2013]
dc.date.accessioned2018-07-24T13:29:46Z
dc.date.available2018-07-24T13:29:46Z
dc.date.issued2016-12-12
dc.description.abstractMulti-domain voltage-gated ion channels appear to have evolved through sequential rounds of intragenic duplication from a primordial one-domain precursor. Whereas modularity within one-domain symmetrical channels is established, little is known about the roles of individual regions within more complex asymmetrical channels where the domains have undergone substantial divergence. Here we isolated and characterised both of the divergent pore regions from human TPC2, a two-domain channel that holds a key intermediate position in the evolution of voltage-gated ion channels. In HeLa cells, each pore localised to the ER and caused Ca2+ depletion, whereas an ER-targeted pore mutated at a residue that inactivates full-length TPC2 did not. Additionally, one of the pores expressed at high levels in E. coli. When purified, it formed a stable, folded tetramer. Liposomes reconstituted with the pore supported Ca2+ and Na+ uptake that was inhibited by known blockers of full-length channels. Computational modelling of the pore corroborated cationic permeability and drug interaction. Therefore, despite divergence, both pores are constitutively active in the absence of their partners and retain several properties of the wild-type pore. Such symmetrical 'pore-only' proteins derived from divergent channel domains may therefore provide tractable tools for probing the functional architecture of complex ion channels.
dc.description.sponsorshipThis work was supported by BBSRC studentship BB/J014567 (CJP) and BBSRC grants BB/L006790 (BAW), BB/J019135 (BAW), BB/N01524X (SP) and BB/K000942 (SP). TR was supported by Royal Society grants RG69132 and RG65196. The SRCD studies were enabled by beamtime grants from the Soleil Synchrotron, France (to BAW).
dc.identifier.doi10.17863/CAM.25768
dc.identifier.eissn2045-2322
dc.identifier.issn2045-2322
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/278425
dc.languageeng
dc.language.isoeng
dc.publisherNature Publishing Group
dc.publisher.urlhttp://dx.doi.org/10.1038/srep38426
dc.rightsAttribution 4.0 International
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/
dc.subjectAmino Acid Sequence
dc.subjectCalcium Channels
dc.subjectCell Survival
dc.subjectHeLa Cells
dc.subjectHumans
dc.titleIsolated pores dissected from human two-pore channel 2 are functional.
dc.typeArticle
dcterms.dateAccepted2016-11-08
prism.number38426
prism.publicationDate2016
prism.publicationNameScientific Reports
prism.volume6
pubs.funder-project-idThe Royal Society (uf110479)
rioxxterms.licenseref.startdate2016-12-12
rioxxterms.licenseref.urihttp://creativecommons.org/licenses/by/4.0/
rioxxterms.typeJournal Article/Review
rioxxterms.versionVoR
rioxxterms.versionofrecord10.1038/srep38426

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