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Title: Investigating ion channels of the nervous system
Author: Higgins, M.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2001
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In this thesis, I present data concerning several stages in synaptic transmission. I have investigated some of the channel proteins involved in the initiation and the propagation of changes of membrane potential in nerve cells. I have also studied the process of clathrin mediated endocytosis, which is essential in the propagation of messages across the synapses between cells. Firstly, the Shaker voltage-gated potassium channel has been expressed using the baculovirus-mediated expression system. The expressed protein binds to charybdotoxin. The expression of correctly folded protein was increased by calnexin co-expression, or by using a 'weaker' baculovirus promoter. The protein has been purified using a Ni2+-NTA column and consists of tetrameric particles when analysed by electron microscopy and image processing. The cyclic nucleotide-gated channel has been purified from bovine retina and studied by image processing and electron microscopy to yield a 35Å-resolution model. This shows a large domain, which I propose to be the membrane-spanning domain. Attached to this are two smaller domains. I suggest that these are the cyclic nucleotide-binding domains and that they hang below the channel in the cytosol where they act as an independently functioning pair of dimers. Preliminary expression and purification of the α-subunit of the olfactory CNG channel suggests that similar analyses of this protein will soon be possible. Finally, the role of AP180 and AP2 in the formation of clathrin coated vesicles has been investigated.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available