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

Solid-supported membranes immobilized on gold electrodes were used to detec t and characterize the spontaneously inserting anion-selective protein chan nel (Clavibacter anion channel, CAC) present in the culture fluid of Clavib acter michiganense ssp. nebraskense. Three different membrane systems varyi ng 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 relativ e change in conductance is largest if the lipid layer is attached to the su rface via a flexible lipid anchor. Variation in the d.c. potential revealed that CAC exhibits a voltage dependence in these tethered membranes which c an be described by an exponential function in accordance with previous resu lts obtained from patchclamp measurements and impedance analysis. The addit ion of an inhibitor that selectively blocks anion channels abolished the ch annel conductance almost completely, indicating that the increased conducti vity can be attributed to the specific insertion of the CAC. A linear depen dence of the channel conductance on the chloride concentration was found, w hich was modulated by the charges of the second lipid monolayer. The result s demonstrate that tethered lipid membranes on gold surfaces in conjunction with impedance spectroscopy allows one to monitor and characterize water-s oluble spontaneously inserting channels, providing an effective means to pr obe for bacterial toxins.