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

Christopher J Penny,Taufiq Rahman,Sandip Patel,Altin Sula,Andrew J Miles,B A Wallace

doi:10.1038/srep38426

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.

Highlights

Voltage-gated ion channels selective for Ca2+ (CaV), Na+ (NaV) and K+ (KV) perform a plethora of functions in both excitable and non-excitable cells
We designed constructs encoding each of the pore regions of human TPC2 (S5-S6 and S11-S12) (Fig. 1B)
To define the pore boundaries, a multiple sequence alignment was performed with the pore regions of previously characterised pore-only and full-length bacterial Na+ channels (Fig. 1C)

Summary

Introduction

Voltage-gated ion channels selective for Ca2+ (CaV), Na+ (NaV) and K+ (KV) perform a plethora of functions in both excitable and non-excitable cells. Mutations in these channels are the causal basis of numerous diseases, thereby rendering them clinically-relevant drug targets[1]. Two-pore channels (TPCs) are less well characterised members of the voltage-gated ion channel superfamily that, unusually, localise to intracellular acidic Ca2+ stores[5]. Recent crystal structures of a plant TPC12–14 have confirmed earlier biochemical reports that they form dimers from two-domain (DI and DII) subunits[15,16] This structural organisation identifies TPCs as a key intermediate in the evolution of voltage-gated ion channels from one-domain to four-domain channels (Fig. 1A). TPC2 DI and NsVBa pores (expected sizes: 16 kDa for TPC2 and 15 kDa for NsVBa). (E) Mass distribution of the purified pore using dynamic light scattering. (F) SRCD spectrum of purified TPC2 DI pore. (G) Thermal denaturation of purified TPC2 DI pore monitored using SRCD spectroscopy at 222 nm

Methods

Results

Conclusion