In this study, we explored the calcium-induced formation of phosphatidylserine-enriched lipid domains in Langmuir−Blodgett (LB) monolayers composed of the binary mixture of the two saturated lipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS) in a molar ratio of 4:1 by means of surface analysis tools. We employed fluorescence microscopy in conjunction with pressure−area isotherms, time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging, scanning electron microscopy (SEM), and lateral force microscopy to visualize and analyze lipid domains by their chemical and physical properties. TOF-SIMS and SEM images of DPPC/DPPS LB monolayers transferred onto gold surfaces clearly reveal that small amounts of calcium ions in the aqueous phase are sufficient to trigger the formation of circular DPPS-enriched domains, but domain formation is abolished in the presence of ethylene glycol-bis(β-aminoethyl) ether-N,N,N ‘ ,N ‘-tetraacetic acid (EGTA). Lateral force microscopy of solid supported lipid bilayers transferred from the air/water interface to mica surfaces and imaged in water also allowed visualization of these DPPS-enriched domains despite the absence of a topographic contrast.

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 lipid N,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 dc 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.