Super-resolution spatial, temporal and functional characterisation of voltage-gated calcium channels involved in exocytosis
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The process of information transfer between neurons or endocrine cells is one of the most important, intricate and temporally precise processes in the body. Exocytosis, which is central to the process of excitation-‐secretion coupling, is triggered by calcium signalling through voltage-‐gated calcium channels. Super-‐resolution imaging offers the possibility to fully understand the spatial relationship between the SNARE proteins involved in exocytosis, vesicles and the associated voltage-‐gated calcium channels. In this thesis the focus is on exploring the trigger for exocytosis, specifically the spatial and functional role that voltage-‐gated calcium channels play in this process. Super-‐ resolution imaging techniques have been applied to measure the interaction between Cav2.2 calcium channels and the syntaxin1a SNARE protein, where binding was found to affect the overall channel distribution. A novel method of caged dye conjugated ω-‐ conotoxin GVIA binding was developed for live cell single molecule imaging of Cav2.2 calcium channels. An innovative approach to analyse channel functionality and the distribution of calcium events at the plasma membrane was developed to create a temporal-‐spatial map of calcium activity across the cell. These developments, combined with newly developed techniques in optical patching and simultaneous calcium and vesicle imaging reveal the functional relationship of voltage-‐gated calcium channel and exocytosis at unprecedented spatial and temporal scales.