Channel activity of a phytotoxin of Clavibacter michiganense ssp. nebraskense in tethered membranes.

Solid-supported membranes immobilized on gold electrodes were used to detect and characterize the spontaneously inserting anion-selective protein channel (Clavibacter anion channel, CAC) present in the culture fluid of Clavibacter michiganense ssp. nebraskense. Three different membrane systems varying in the composition of the first chemisorbed monolayer were investigated by means of impedance spectroscopy. Conductance changes of the immobilized lipid membranes were sensitively detected after adding the culture fluid of the bacteria to the solid-supported membranes, indicating that the relative change in conductance is largest if the lipid layer is attached to the surface via a flexible lipid anchor. Variation in the d.c. potential revealed that CAC exhibits a voltage dependence in these tethered membranes which can be described by an exponential function in accordance with previous results obtained from patchclamp measurements and impedance analysis. The addition of an inhibitor that selectively blocks anion channels abolished the channel conductance almost completely, indicating that the increased conductivity can be attributed to the specific insertion of the CAC. A linear dependence of the channel conductance on the chloride concentration was found, which was modulated by the charges of the second lipid monolayer. The results demonstrate that tethered lipid membranes on gold surfaces in conjunction with impedance spectroscopy allows one to monitor and characterize water-soluble spontaneously inserting channels, providing an effective means to probe for bacterial toxins.