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

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Penny, Christopher J 
Rahman, Taufiq 
Sula, Altin 
Miles, Andrew J 
Wallace, BA 


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.



Amino Acid Sequence, Calcium Channels, Cell Survival, HeLa Cells, Humans

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Scientific Reports

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Nature Publishing Group
The Royal Society (uf110479)
This 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).