The Two-pore channel (TPC1-3) family represents a recently identified class of endolysosomal ion channels. TPCs were originally proposed to be promising candidate channels for NAADP-induced Ca<sup>2+</sup> release. However, subsequent studies have emerged to propose an alternative view where TPCs may be Na+-selective channels regulated by the lysosome-specific phosphoinositide PI(3,5)P2 or voltage in an isoform-dependent manner. This thesis asks the question of whether pharmacological and ion permeation properties of TPCs, in particular the human TPC2, may satisfy or may be consistent with the requirement of a potential NAADP-sensitive Ca<sup>2+</sup>-release channel. These fundamental properties of hTPC2 were approached using patch-clamp electrophysiology and confocal fluorescence microscopy, and were analysed quantitatively to extract relevant physical parameters important to our understanding of their physiological and functional significance. Chapter 2 presents the basic electrophysiological characterisation of hTPC2. It follows a logical way by first determining the ion permeation properties, followed by the investigation of its physical relation with fractional Ca<sup>2+</sup> current and Ca<sup>2+</sup> nanodomains to rigorously prove that this Na<sup>+</sup> selectivity is sufficient to ensure negligible Ca<sup>2+</sup> leakage both experimentally and theoretically. This follows the logic that matter must not be created nor destroyed so that a Na+-selective channel that poses a physiologically significant energy barrier to Ca<sup>2+</sup> permeation from one side would not lead to the creation of Ca<sup>2+</sup> on the other side. Chapter 3 represents a natural progression from Chapter 2 and is aimed at investigating the underlying mechanisms responsible for the electrophysiological ion selectivity observed. This chapter also follows a logical way by first identifying spermine as a high valence intracellular blocker, its mutual antagonism with different external ionic species that allows the determination of ion-binding affinity, followed by the determination of the concentration dependence of ion conduction to identify possible lower affinity binding. By considering all the above qualities, the outcome is a coherent description and connection of ion binding selectivity, kinetic selectivity and ion binding configuration with the observed electrophysiological selectivity. Chapter 4 discusses the missing puzzles and how these questions might be addressed.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:730519 |
| Date | January 2015 |
| Creators | Lam, Andy Ka Ming |
| Contributors | Galione, Antony |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://ora.ox.ac.uk/objects/uuid:3a16d16e-f692-40d7-87f7-920151896038 |
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