The spontaneous insertion of an anion selective protein channel(Clavibacter
anion channel, CAC) present in the culture fluid of Clavibacter michiganen
se ssp. nebraskense was monitored by using solid supported lipid membranes
(SSMs) composed of the positively charged lipidN,N-dimethyl-N,N-dioctadecyl
ammonium 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 me
mbranes were sensitively detected after adding the culture fluid of the bac
teria 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 c
learly indicating that the increased conductivity is attributed to the spec
ific insertion of the channel protein. The results demonstrate the potentia
l of this kind of electrostatically attached solid supported membrane combi
ned with impedance spectroscopy allowing one to monitor and characterize wa
ter-soluble spontaneously inserting channel forming bacterial toxins.