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Title: Molecular and cell biological studies of a novel intracellular rat brain ion channel protein
Author: Duncan, Rory R.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1997
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A cDNA clone, p64H1, was isolated from rat brain. This encoded a homologue of p64, a putative chloride channel protein. The work provided the first direct evidence of a p64-related gene in neural tissue, and evidence for the existence of a p64-related gene family. p64H1 was demonstrated to be an integral membrane protein of approximately 30 kDa. It contained a single transmembrane domain, a short luminal domain and a large cytoplasmic domain. The cytoplasmic domain was predicted to contain multiple consensus phosphorylation sites, and phosphorylation by Protein Kinase C was demonstrated in vitro. A polyclonal antiserum raised against a recombinant p64H1 fusion protein was used to investigate the cellular localisation of native and heterologously-expressed p64H1. The protein was expressed in a wide variety of tissues. Within rat brain, p64H1 mRNA was found to be enriched in the cerebellum and hippocampus, and immunohistochemistry showed that the protein expression within the cerebellum was confined to the Purkinje cell layer. Recombinant p64H1 expressed in cultured cells was confirmed to be an endoplasmic reticulum membrane protein using indirect immunofluorescence. Finally, the interactions of p64H1 with other cellular proteins were investigated using both classical biochemical techniques and a yeast two-hybrid interaction assay. These varied approaches provided data which helped to characterise a previously undescribed protein, and may help in the elucidation of its precise cellular function.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available