Membrane activity of an anion channel from Clavibacter michiganense ssp nebraskense

Abstract

The spontaneous insertion of an anion selective protein channel(Clavibacter anion channel, CAC) present in the culture fluid of Clavibacter michiganense ssp. nebraskense was monitored by using solid supported lipid membranes (SSMs) composed of the positively charged lipidN,N-dimethyl-N,N-dioctadecylammonium bromide immobilized via electrostatic interaction on a negatively charged monolayer of 3-mercaptopropionic acid chemisorbed on gold. 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 SSMs. Variation in the de potential revealed that CAC exhibits its unique voltage dependence in SSMs described by an exponential function in accordance to previous results obtained from patch clamp measurements. Protease treatment and the addition of an anion selective channel inhibitor diminished the increased conductivity of the membrane after CAC addition clearly indicating that the increased conductivity is attributed to the specific insertion of the channel protein. The results demonstrate the potential of this kind of electrostatically attached solid supported membrane combined with impedance spectroscopy allowing one to monitor and characterize water-soluble spontaneously inserting channel forming bacterial toxins.