Thesis
The role of human Two-pore channel 1 in membrane protein trafficking
- Abstract:
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Two-pore channels (TPCs) are recognised as Ca2+/Na+ -permeable ion channels localised to endolysosomal organelles. Although the lysosomal isoform TPC2 has been extensively studied, the physiological processes that require TPC1 and how the channel properties of TPC1 are involved in these processes, remains less clearly defined.
Chapter 1 introduces the TPC family in the context of Ca2+ signalling and endo-lysosomal membrane protein trafficking. Chapter 2 details the methodologies and techniques used throughout this thesis.
Chapter 3 investigates the regulatory role of TPC1 in the intracellular trafficking of iron-loaded transferrin for cellular iron homeostasis. Trafficking and recycling experiments in CRISPR-edited TPC-deficient HeLa cells revealed that TPC1 specifically regulates transferrin uptake by influencing cell surface levels of the transferrin receptor (TfR), through regulation of endosomal trafficking and recycling. Impaired iron-loaded transferrin uptake reduces intracellular labile iron and increases the prevalence of disease-like iron-deficiency phenotypes in mammalian cells and mice, thereby concluding that TPC1 expression is critical for iron homeostasis.
Chapter 4 characterises the TPC1 ionic fluxes integral for cell-membrane protein trafficking. Through organelle-targeted Ca2+ buffering and depletion, endosomal Ca2+ stores (10-12 μM) were identified as the source of Ca2+ required for efficient TfR trafficking, whose localised endosomal TPC1-mediated Ca2+ nanodomains are segregated from global ER-derived Ca2+ transients. Generation and expression of a Na+ -deficient Ca2+ -permeable TPC1 mutant revealed that it is the Ca2+ modality, not Na+ fluxes, or endosomal pH changes, that regulate TfR membrane trafficking.
Chapter 5 expands upon the previous chapters and evaluates the selective or generic role of TPC1 in cell membrane trafficking and how TPC1 activity is gated. Three discrete membrane protein trafficking pathways, the transferrin cycle, LDL cycle, and pinocytosis, were examined using fluorescently-labelled ligands. TPC1 was shown to selectively regulate transferrin trafficking and pinocytosis through distinct channel ion modalities, indicating that there is specificity for the membrane trafficking pathways TPC1 mediates. TPC1 activity depended on the lipid PI(3,5)P2, since trafficking was impaired by a lipid-insensitive TPC1 and inhibitors of lipid synthesis.
Chapter 6 discusses the impact of the results obtained and future prospects for TPC1 research.
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(Preview, Dissemination version, pdf, 8.0MB, Terms of use)
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Authors
Contributors
+ Galione, A
- Institution:
- University of Oxford
- Division:
- MSD
- Department:
- Pharmacology
- Role:
- Supervisor
- ORCID:
- 0000-0002-4132-7646
+ Tammaro, P
- Institution:
- University of Oxford
- Division:
- MSD
- Department:
- Pharmacology
- Role:
- Supervisor
- ORCID:
- 0000-0002-6183-8187
+ Engineering and Physical Sciences Research Council
More from this funder
- Funder identifier:
- https://ror.org/0439y7842
- Funding agency for:
- Burton, WJ
- Grant:
- 2885841
- DOI:
- Type of award:
- DPhil
- Level of award:
- Doctoral
- Awarding institution:
- University of Oxford
- Language:
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English
- Keywords:
- Subjects:
- Deposit date:
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2026-08-27
- ARK identifier:
Terms of use
- Copyright holder:
- William John Burton
- Copyright date:
- 2026
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