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Title: Studies on the Nav3.1 auxiliary subunit of the voltage gated sodium channel
Author: Havard, A.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2003
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The voltage gated sodium channel is of great importance to living organisms allowing neurotransmission and muscle contraction to occur with great speed. The channel in CNS comprises a large α subunit forming the pore, ion selectivity filter and voltage sensor. Coupled to this are the smaller Navβ2.1 (absent in PNS) and either Navβ1.1 or Navβ3.1 subunits. The extracellular domain of Navβ3.1 based on sequence homology to myelin P0, is predicted to form a V-type immunoglobulin fold and a short intracellular domain contains a potential YXXΦ signal for internalisation by clathrin coated pits. Recombinant Navβ3.1 extracellular domain was used in electrophysiological experiments in which Xenopus oocytes were injected with cRNA encoding Nav1.3 or Nav1.4 with or without Navβ3.1. Those expressing α alone were incubated with Navβ3.1 recombinant extracellular domain and the currents recorded. Recombinant extracellular domain was used as an antigen to make monoclonal antibodies which were epitope mapped using mutated recombinant protein. Circular Dichroism (CD) and Fourier Transform Infra-Red (FTIR) spectra were obtained to assess the secondary structure of the extracellular domain protein. The results showed a predominantly β-sheet secondary structure within the extracellular domain which was able to modify the gating kinetics of the alpha subunit. Antibody production was unsuccessful. Although an antibody was produced and mutagenised extracellular domain protein was used in epitope mapping studies, the antibody had poor specificity. The intracellular domain was investigated using the yeast-2-hybrid system with the m adaptor subunits of clathrin coated pits to ascertain which it interacted with. The YLAI signal of the intracellular domain was able to interact with the m3 subunits of the adaptor complexes, suggesting that this may act as a pathway for internalisation of this protein.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available