title: Channel formation in model membranes by the adenylate cyclase toxin of Bordetella pertussis: effect of calcium.
abstract: Calmodulin-dependent adenylate cyclase toxin (ACT or CyaA) of Bordetella pertussis requires calcium ions for target cell binding, formation of hemolytic channels, and delivery of its enzyme component into cells. We examined the effect of calcium and calmodulin on toxin interaction with planar lipid bilayers. While calmodulin binding did not affect the properties of CyaA channels, addition of calcium ions and toxin to the same side of the membrane caused a steep increase of the channel-forming capacity of CyaA. The calcium effect was highly specific, since among other divalent cations only strontium caused some CyaA activity enhancement. The minimal stimulatory concentration of calcium ions ranged from 0.6 to 0.8 mM, depending on the ionic strength of the aqueous phase. Half-maximal channel activity of CyaA was observed at 2-4 mM, and saturation was reached at 10 mM calcium concentration, respectively. The unit size of single CyaA channels, assessed as single-channel conductance, was not affected by calcium ions, while the frequency of CyaA channel formation strongly depended on calcium concentration. The calcium effect was abrogated upon deletion of the RTX repeats of the toxin, suggesting that binding of calcium ions to the repeats modulates the propensity of CyaA to form membrane channels.
authors.affiliation: Lehrstuhl für Biotechnologie, Theodor-Boveri-Institut (Biozentrum) der Universität Würzburg, Am Hubland, D-97074 Würzburg, Federal Republic of Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Knapp
authors.forename: Elke
authors.initials: E
authors.lastname: Maier
authors.forename: Georg
authors.initials: G
authors.lastname: Polleichtner
authors.forename: Jirí
authors.initials: J
authors.lastname: Masín
authors.forename: Peter
authors.initials: P
authors.lastname: Sebo
authors.forename: Roland
authors.initials: R
authors.lastname: Benz
mesh_terms: D037361:Adenylate Cyclase Toxin / Q000494:pharmacology*; D001665:Binding Sites; D001886:Bordetella pertussis / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D008051:Lipid Bilayers; D008567:Membranes, Artificial; D008954:Models, Biological; D013056:Spectrophotometry, Ultraviolet
chemical_list: D037361:Adenylate Cyclase Toxin; D002147:Calmodulin; D007473:Ion Channels; D008051:Lipid Bilayers; D008567:Membranes, Artificial; D002118:Calcium