November 8-12, 2015
Transmembrane ion channels can be used in biomaterials as a way to selectively allow only certain molecules to pass through a given membrane. To produce these types of membranes predictably and reliably for commercial activity, its characterization is crucial. We have characterized the conductivity and ion selectivity of NaChBac, a voltage-gated sodium channel from Bacillus haloduran, using a planar bilayer membrane device. Furthermore, we have devised a dye-free flux assay to measure the channel function using stopped flow spectrometer.. Planar bilayer membrane is a common technique for ion channels function characterization. We first incorporated purified NaChBac protein into lipid vesicle. The proteoliposome is then fused to a lipid bilayer. By applying a controllable potential across the protein-embedded bilayer and measuring the current, the conductivity and permeability of the protein can be studied. We have tested NaChBac’s relative permeability for different ions using bi-ionic conditions. We found that the single channel conductivity of NaChBac to be 16.2 pS, which is similar to 12 pS, the value acquired from patch clamp experiment on live cell overexpressing NaChBac. We also found that the ion selectivity of NaChBac of K+ and Ca2+ to that of similar Na+, which was not the case in the same patch clamp study.
In addition, we have devised a flux assay for ion channels that uses a stopped flow spectrometer. Conventional flux assay for ion channels requires the use of ion-specific dyes. However the availability of the dyes and their selectivity limits the scope of ion channels that can be tested in a fluorescence flux assay. In our method, NaChBac-incorporating liposomes were diluted into a hypotonic solution, and the proteoliposome swells as a result of sodium ion influx through the protein. Mie scattering is measured and used to determine the size change of proteoliposome that is caused by solute flux. With this method, we have been able to characterize the ion conducting function of purified NaChBac protein.
Andrew Jo, Hiofan Hoi, and C. Montemagno, "Characterizing the functionality of transmembrane ion channels using planar bilayer membranes device and stopped flow spectrometer" in "Composites at Lake Louise (CALL 2015)", Dr. Jim Smay, Oklahoma State University, USA Eds, ECI Symposium Series, (2016). https://dc.engconfintl.org/composites_all/107