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Characterization of NaChBac in Artificial Lipid Bilayer Membrane

  • Author / Creator
    Jo, Andrew
  • NaChBac is a voltage-gated Na+ channel from the bacterium Bacillus haloduran. Studying this protein channel can help researchers discover potential pharmaceuticals, innovative strategies in eliminating biofilms, and could be used to improve desalinization technologies. This thesis will discuss the charaterization of NaChBac in artifical lipid membrane, including single molecule study on the conductance and ion selectivity using a planar bilayer membrane device and the development of a scattered light based stopped-flow flux assay. Planar lipid bilayer membrane is a common technique to characterize ion channel function. In this thesis, purified NaChBac protein was incorporated into lipid vesicles which were then fused to a planar lipid bilayer. By applying a controllable electrical potential (i.e. voltage) across the protein-embedded bilayer and measuring the resulting current, the conductance, gating behaviour, and ion selectivity of the open channel were studied. NaChBac was found to have three conductance levels at 268.5 ± 45.2 pS (mean ± SEM, n=2), 92.8 ± 18.0 pS (n=3), and 26.4 ± 5.6 pS (n=4), respectively. Its activation gating voltage (V1/2) is -28 mV and its apparent activation gating charge is 15.9 elementary charge. NaChBac tested under asymmetric saline buffer solution conditions was shown to have a relative permeability PNa+/PK+ ranging from 0.86 to 1.35 and PNa+/PCa2+ = 1.43 ± 0.20 (n=3, ±SEM). Conventional flux assay for ion channels requires the use of ion-specific dyes. However, the availability of the dyes and their selectivity limits the spectrum of ion channels that can be tested in a fluorescence flux assay. Instead, stopped-flow spectrometry can be used to monitor the change in size of the protein-incorporating vesicles that is caused by ion flux, which is in turn triggered by the transmembrane electrochemical gradient of the target ion species. With this method, the ion conducting function of purified NaChBac protein was shown. The method can potentially be used to test the functionality of other ion channels.

  • Subjects / Keywords
  • Graduation date
    Spring 2016
  • Type of Item
    Thesis
  • Degree
    Master of Science
  • DOI
    https://doi.org/10.7939/R3C53FH45
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.