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Kaleta, Marta (2018) Development of blocking antibodies to the voltage-gated calcium channel cav3.2. Doctoral thesis, London Metropolitan University.
T-type voltage-dependent calcium channel Cav3.2 is a four-domain channel participating in various physiological and pathological processes principally in the nervous system but also in non-excitable cells. Upregulation of Cav3.2 or mutations in this channel are found in pain syndromes, certain cancers and epilepsy. This thesis describes the experiments aimed at generating antibodies directed to Cav3.2 that could alter the gating of these channels.
A structural analysis of Cav3.2 channel was performed in silico on the basis of the published protein sequence using bioinformatics tools. The analysis identified several putative extracellular loops of Cav3.2. Three of these loops were chosen as targets for antibodies due to their proximity to the channel’s pore.
Antibodies were chosen to target these loops due to the relative ease of their production, and were generated against synthetic peptides with the corresponding sequence attached to a carrier protein and injected into rabbits. The antibodies obtained were purified from the sera and analysed for their affinity to the corresponding peptides in vitro using ELISA and flow cytometry.
A stable cell line HEK/Cav3.2 was analysed to confirm the localisation and accurate expression of functional Cav3.2 in these cells. This cell line was used to assess the binding of the generated antibodies and their influence on Cav3.2 current.
In order that the influence of the antibodies on the gating of Cav3.2 channel could be measured, a calcium sensitive fluorescent dye-based assay was developed and validated. The generated antibodies proved to be partial inhibitors of Cav3.2 channel conductance, confirming that extracellular loops of Cav3.2 are a promising drug target which may pave the way to designing selective anti-Cav3.2 inhibitors.
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