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Anthrax Toxin Protective Antigen: Inhibition of Channel Function by Chloroquine and Related Compounds and Study of Binding Kinetics Using the Current Noise Analysis

Lehrstuhl für Biotechnologie, Theodor-Boveri-Institut (Biozentrum) der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany

Correspondence: Address reprint requests to Roland Benz, Lehrstuhl für Biotechnologie, Theodor-Boveri-Institut (Biozentrum) der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany. Tel.: 49-0931-888-4501; Fax: 49-0931-888-4509; E-mail: roland.benz{at}mail.uni-wuerzburg.de.

Protective antigen (PA) of the tripartite anthrax toxin binds to a cell surface receptor and mediates the transport of two enzymatic components, edema factor and lethal factor, into the cytosol of host cells. Here recombinant PA₆₃ from Bacillus anthracis was reconstituted into artificial lipid bilayer membranes and formed ion permeable channels. The heptameric PA₆₃-channel contains a binding site for 4-aminoquinolones, which block ion transport through PA in vitro. This result allowed a detailed investigation of ligand binding and the stability constants for the binding of chloroquine, fluphenazine, and quinacrine to the binding site inside the PA₆₃-channel were determined using titration experiments. Open PA₆₃-channels exhibit 1/f noise in the frequency range between 1 and 100 Hz, whereas the spectral density of the ligand-induced current noise was of Lorentzian type. The analysis of the power density spectra allowed the evaluation of the on- and off-rate constants ( and ) of ligand binding. The on-rate constants of ligand binding were between 10⁶ and 10⁸ M^–1 s^–1 and were dependent on the ionic strength of the aqueous phase, sidedness of ligand addition, as well as the orientation and intensity of the applied electric field. The off-rates varied between 10 s^–1 and 2600 s^–1 and depended mainly on the structure of the ligand.

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