Figure 2.

(a) Current–voltage relationships of cyclase toxin (CyaA) pores initiated by positive potential (+ 50 mV) applied to the cis side. The membranes were formed from asolectin/n-decane. The aqueous phase contained 150 mM KCl and 10 mM 2-(4-(2-Hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid-potassium hydroxide (HEPES-KOH) pH 7; the temperature was 20 °C. The cis side contained in addition 320 ng/mL wild-type CyaA (full squares), 320 ng/mL ΔAC-CyaA (open squares), or 320 ng/mL CyaA 1008 (full circles) (adapted from [78]). (b) Current–voltage relationships of CyaA pores initiated by negative potential (−50 mV) applied to the cis side. The membranes were formed from asolectin/n-decane. The aqueous phase contained 150 mM KCl and 10 mM HEPES-KOH pH 7; the temperature was 20 °C. The cis side contained either 20 ng/mL wild-type CyaA (full circles), 160 ng/mL wild-type CyaA (full squares), or 320 ng/mL wild-type CyaA (full triangles) (adapted from [78]). (c) Increase of membrane conductance after the addition of 300 ng/mL CyaA to the cis side of a black asolectin/n-decane membrane (left side arrow) and of 0.8 mM Ca²⁺ first to the trans side of the membrane (middle arrow) and then to the cis side of the membrane (right side arrow). The aqueous phase contained 1 M KCl, pH 7 on both sides of the membrane. The addition of 300 ng/mL and CyaA added to the cis side of the membrane (left-hand side arrow) resulted in an increase of the membrane conductance (full squares) by a factor of about 100 over control in the absence of CyaA (full points). The addition of 0.8 mM CaCl₂ to the trans side (middle arrow) had no impact on membrane conductance, whereas the addition of 0.8 mM CaCl₂ to the cis side (right-hand side arrow) led to a dramatic increase of membrane conductance by many orders of magnitude (full squares). The temperature was 20 °C and 50 mV were applied to the cis side (adapted from [18]). (d) Single-pore recordings of asolectin/n-decane membranes in the presence of a 13 ng/mL concentration of the purified CyaA-E581K protein (lower line) or intact recombinant CyaA (upper line). Mutant E581K showed an increased pore lifetime, which seems to be linked to an increased hemolytic activity. The applied membrane potential was 50 mV, and the temperature was 20 °C (adapted from [72]).