Anthrax toxin protective antigen: inhibition of channel function by chloroquine and related compounds and study of binding kinetics using the current noise analysis

¹ Lehrstuhl für Biotechnologie

^* To whom correspondence should be addressed. E-mail: roland.benz{at}mail.uni-wuerzburg.de.

Submitted on July 23, 2004
Revised on August 13, 2004
Accepted on 24 November 2004

	Abstract

Protective antigen (PA) of the tripartite anthrax toxin binds to a cell surface receptor and mediates the transport of two enzymatic components edema factor (EF) and lethal factor (LF) 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, while 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 (k₁ and k_-1 ) 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 about 10 s^-1 and 2600 s ^-1 and depended mainly on the structure of the ligand.

Key Words: Bacillus anthracis, black lipid bilayer, channel, protective antigen, toxin

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