title: Isolated pores dissected from human two-pore channel 2 are functional.
abstract: Multi-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.
authors.affiliation: Department of Cell and Developmental Biology, University College London, London, WC1E 6BT, UK.|Institute of Structural and Molecular Biology, Birkbeck College, University of London, London, WC1E 7HX, UK.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Penny
authors.affiliation: Department of Pharmacology, University of Cambridge, Cambridge, CB2 1PD, UK.
authors.forename: Taufiq
authors.initials: T
authors.lastname: Rahman
authors.affiliation: Institute of Structural and Molecular Biology, Birkbeck College, University of London, London, WC1E 7HX, UK.
authors.forename: Altin
authors.initials: A
authors.lastname: Sula
authors.affiliation: Institute of Structural and Molecular Biology, Birkbeck College, University of London, London, WC1E 7HX, UK.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Miles
authors.affiliation: Institute of Structural and Molecular Biology, Birkbeck College, University of London, London, WC1E 7HX, UK.
authors.forename: B A
authors.initials: BA
authors.lastname: Wallace
authors.affiliation: Department of Cell and Developmental Biology, University College London, London, WC1E 6BT, UK.
authors.forename: Sandip
authors.initials: S
authors.lastname: Patel
mesh_terms: D000595:Amino Acid Sequence; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism*; D002470:Cell Survival; D006367:HeLa Cells; D006801:Humans
chemical_list: D015220:Calcium Channels; C530224:TPCN2 protein, human