Functional Incorporation of KcsA into Tethered Lipid Bilayer Membranes

Abstract

Tethered lipid bilayer membranes (tBLMs) are solid-supported lipid bilayers separated by a ∼ 2 nm thick hydrated layer from the solid interface. In comparison to cell membranes, they are simpler in their chemical composition and can therefore be quantitatively studied with a variety of experimental techniques. In comparison to free-standing or vesicle membranes, they are much more long-term stable. tBLMs can be formed by rapid solvent exchange,1 which leads to highly electrically insulating, defect-free bilayers,2 or by vesicle fusion, which results in membranes with higher residual conductance but makes protein reconstitution more straightforward. While we studied the structure and function of tBLMs produced by rapid solvent exchange extensively in the past,2 we have more recently optimized vesicle fusion protocols for tBLM formation and observed with neutron reflectometry that the membranes resulting from the two preparation methods are very similar in their molecular structure. Fluorescence correlation spectroscopy shows that lipid-label diffusion is identical in both cases. In this work, we incorporate the tetrameric potassium-selective channel KcsA from Streptomyces lividans into tBLMs through vesicle fusion. KcsA is reconstituted into POPE/POPG lipid vesicles that are spread onto the solid support to form the membranes. The functionality of the reconstituted channel is confirmed by electrochemical impedance spectroscopy (EIS), where we observe that the resistance of tBLMs with KcsA is 3 to 10 times smaller than the resistance of neat tBLMs in the presence of K+ ions, and with various blockers of the potassium channel.Supported by the NIH (1P01AG032131) and the AHAF (A2008-307).1Cornell, B.A, et al. 1997. Nature 387:580-583.2Valincius, G., et al. 2008. Biophys. J. 95:4845-4861.