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Electrochemical impedance spectroscopy (EIS) was used to characterize voltage-dependent closure of PorB class II (PorBII) porin from Neisseria meningitidis incorporated in a tethered bilayer lipid membrane (tBLM). The tBLM's lower leaflet was fabricated by depositing a self assembled monolayer (SAM) of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) on a gold electrode, and the upper leaflet was formed by depositing1,2-dioleoyl-sn-glycero-3-phoshocholine (DOPC) liposomes. At 0mV bias DC potential, incorporation of PorBII decreased the membrane resistance (R(m)) from 2.5 MΩc m(2) to 0.6 MΩ cm(2), giving a ΔR(m) of 1.9 MΩ cm(2) and a normalized ΔR(m) (ΔR(m) divided by the R(m) of the tBLM without PorBII) of 76%. When the bias DC potential was increased to 200 mV, the normalized ΔR(m) value decreased to 20%. The effect of applied voltage on ΔR(m) was completely reversible, suggesting voltage-dependent closure of PorBII. The voltage dependence of PorBII was further studied in a planar bilayer lipid membrane made from 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhytPC). Following a single insertion event, PorBII exhibited multiple conductance states, with reversible, voltage-dependent closure of PorBII porin occurring at high transmembrane potentials. The trimetric porin closed in three discrete steps, each step corresponding to closure of one conducting monomer unit. The most probable single channel conductance was 4.2 nS. The agreement between results obtained with the tBLM and pBLM platforms demonstrates the utility of EIS to screen channel proteins immobilized in tBLM for voltage-gated behavior., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A tethered bilayer lipid membrane (tBLM) was fabricated on a gold electrode using 1,2-dipalmitoyl-sn-glycero-phosphothioethanol as a tethering lipid and the membrane fractions of Saccharomyces pombe yeast cells to deposit the upper leaflet. The membrane fractions were characterized using transmission electron microscopy and dynamic light scattering and found to be similar in size to small unilamellar vesicles of synthetic lipids. The dynamics of membrane-fraction deposition and rupture on the tethering-lipid layer were measured using quartz crystal microgravimetry. The electrochemical properties of the resulting tBLM were characterized using electrical impedance spectroscopy and cyclic voltammetry. The tBLM's electrical resistance was greater than 1 MOmegacm(2), suggesting a defect-free membrane. The suitability of tBLM produced using membrane fractions for measuring ion-channel activities was shown by a decrease in membrane resistance from 1.6 to 0.43 MOmegacm(2) following addition of gramicidin. The use of membrane fractions to form high-quality tBLM on gold electrodes suggests a new approach to characterize membrane proteins, in which the upper leaflet of the tBLM is deposited, and overexpressed membrane proteins are incorporated, in a single step. This approach would be especially useful for proteins whose activity is lost or altered during extraction, purification, and reconstitution, or whose activities are strongly influenced by the lipid composition of the bilayer.