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Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution
Published on: August 16, 2016
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Isolated pores dissected from human two-pore channel 2 are functional
Christopher J Penny1,2, Taufiq Rahman3, Altin Sula2
1Department of Cell and Developmental Biology, University College London, London, WC1E 6BT, UK.
Scientific Reports
|December 13, 2016
Summary
Researchers studied divergent pore regions of human TPC2, a voltage-gated ion channel. Both pore regions function independently, aiding in understanding complex ion channel evolution and function.
Area of Science:
- Ion channel biophysics
- Molecular evolution
- Cellular physiology
Background:
- Voltage-gated ion channels evolved from single-domain precursors via intragenic duplication.
- Modularity is understood in symmetrical channels, but not in complex, divergent asymmetrical channels.
- Human TPC2 is a two-domain channel, representing an evolutionary intermediate.
Purpose of the Study:
- To isolate and characterize the divergent pore regions of human TPC2.
- To investigate the independent function and properties of these pore regions.
- To explore their potential as tools for studying complex ion channel architecture.
Main Methods:
- Isolation and characterization of TPC2 pore regions in HeLa cells and E. coli.
- Localization studies using ER targeting.
- Functional assays with liposome reconstitution for ion uptake (Ca2+ and Na+).
- Inhibition studies with known channel blockers.
- Computational modeling of pore structure and function.
Main Results:
- Both isolated pore regions localized to the ER and caused Ca2+ depletion.
- A mutated ER-targeted pore region showed no effect, indicating functional relevance.
- One pore region expressed well in E. coli, purified, and formed a stable tetramer.
- Reconstituted liposomes showed Ca2+ and Na+ uptake, inhibited by known blockers.
- Computational modeling supported cationic permeability and drug interactions.
Conclusions:
- Despite divergence, both TPC2 pore regions are constitutively active and retain wild-type pore properties independently.
- These 'pore-only' proteins offer a tractable system for probing the functional architecture of complex ion channels.
- The findings provide insights into the evolution of multi-domain voltage-gated ion channels.
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