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Title: Purification, functional and structural studies of the E.Electricus voltage-gated sodium ion channel
Author: O'Reilly, Andrias Oliver
Awarding Body: Birkbeck (University of London)
Current Institution: Birkbeck (University of London)
Date of Award: 2005
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The voltage-gated sodium ion channel regulates the transport of Na+ across the plasma membrane of electrically-excitable cells in a voltage-dependent manner. Its activity mediates the rising phase of action potentials. The sodium channel's essential role in the function of the nervous system has made it a target for a diverse array of toxins and insecticides during evolution. It is also the receptor for local anesthetics and drugs used in the treatment of disorders such as epilepsy and cardiovascular disease. Recently-solved crystal structures of bacterial potassium channels have revealed the structural basis of ion selectivity and channel gating that underlie their controlled, ionspecific conductance. These potassium channel structures were used to generate homology models for the sodium channel. Docking studies of an homology model with an insecticide, deltamethrin, were undertaken to identify potential binding contacts. Channel modeling studies were also undertaken of a simpler structure, antiamoebin, an antibiotic peptide that polymerizes in lipid bilayers to form channels. Comparisons of this channel model with bacterial potassium channel structures demonstrated common functionally-related structural features. The mam goals of this project were to structurally and functionally characterize purified voltage-gated sodium channels and to prepare homogeneous preparations suitable for crystallization. Sodium channels were purified from electric eel electroplax membranes, which are a rich natural source of this protein. Purification of channels from detergentsolubilized membranes was achieved using immunoaffinity chromatography or alternatively using a succession of chromatography steps, including ion exchange, lectin affinity and gel filtration chromatographies. Sodium channels are heavily glycosylated molecules and purified channels were enzymatically deglycosylated for some structural studies. The function of purified sodium channels was assessed using electrical studies in planar lipid bilayers. Channels displayed toxin-sensitivity and a single-channel conductance, 17.5 picoSiemens, that corresponds well with values reported in the literature for electric eel sodium channels (20-25 picoSiemens). Circular dichroism spectroscopy was used to determine the effect of a toxin, batrachotoxin, and a drug, lamotrigine, on the secondary structure of sodium channels. The channels' helicity increased in the presence of either ligand. This suggests that the ligands stabilize conformations associated with specific functional states, thus shifting the equilibrium between the mixture of functional states present in the ligand-free channels. The ability to detect sodium channels' toxin-sensitivity using circular dichroism spectroscopy was used as an assay for determining the effectiveness of different purification methods to yield functional protein for 2D crystallization trials. 2D crystallization is a technique of special application for membrane proteins as their structures are stabilised in a lipid bilayer. Electron crystallography and 3D reconstruction o can yield structures with resolutions that extend to 7-8 A, at which point elements of secondary structure can become distinguishable. A structure of the sodium channel at this resolution would enable more accurate modeling of this pharmacologically-important protein. 2D crystallization of sodium channels was attempted using a dialysis method and the lipid layer strategy. The latter technique sought to facilitate 2D crystallization by exploiting properties of the sodium channel, namely the significant amount of negative charges localized to the extracellular domain and the asymmetry in the sizes of the intra- and extra-cellular domains.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available