title: Anthrax biosensor, protective antigen ion channel asymmetric blockade.
abstract: The significant threat posed by biological agents (e.g. anthrax, tetanus, botulinum, and diphtheria toxins) (Inglesby, T. V., O'Toole, T., Henderson, D. A., Bartlett, J. G., Ascher, M. S., Eitzen, E., Friedlander, A. M., Gerberding, J., Hauer, J., Hughes, J., McDade, J., Osterholm, M. T., Parker, G., Perl, T. M., Russell, P. K., and Tonat, K. (2002) J. Am. Med. Assoc. 287, 2236-2252) requires innovative technologies and approaches to understand the mechanisms of toxin action and to develop better therapies. Anthrax toxins are formed from three proteins secreted by fully virulent Bacillus anthracis, protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa), and edema factor (EF, 89 kDa). Here we present electrophysiological measurements demonstrating that full-length LF and EF convert the current-voltage relationship of the heptameric PA63 ion channel from slightly nonlinear to highly rectifying and diode-like at pH 6.6. This effect provides a novel method for characterizing functional toxin interactions. The method confirms that a previously well characterized PA63 monoclonal antibody, which neutralizes anthrax lethal toxin in animals in vivo and in vitro, prevents the binding of LF to the PA63 pore. The technique can also detect the presence of anthrax lethal toxin complex from plasma of infected animals. The latter two results suggest the potential application of PA63 nanopore-based biosensors in anthrax therapeutics and diagnostics.
authors.affiliation: United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21702-5011, USA.
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Halverson
authors.forename: Rekha G
authors.initials: RG
authors.lastname: Panchal
authors.forename: Tam L
authors.initials: TL
authors.lastname: Nguyen
authors.forename: Rick
authors.initials: R
authors.lastname: Gussio
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Little
authors.forename: Martin
authors.initials: M
authors.lastname: Misakian
authors.forename: Sina
authors.initials: S
authors.lastname: Bavari
authors.forename: John J
authors.initials: JJ
authors.lastname: Kasianowicz
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial / Q000032:analysis* / Q000494:pharmacology*; D001408:Bacillus anthracis / Q000472:pathogenicity; D001427:Bacterial Toxins / Q000032:analysis* / Q000494:pharmacology*; D015374:Biosensing Techniques / Q000379:methods*; D023482:Bioterrorism; D002462:Cell Membrane; D004594:Electrophysiology; D005260:Female; D006801:Humans; D006825:Hybridomas; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000187:drug effects* / Q000502:physiology; D051379:Mice; D008807:Mice, Inbred BALB C; D009101:Multiple Myeloma / Q000473:pathology; D036103:Nanotechnology; D016062:Porosity; D011956:Receptors, Cell Surface; D012680:Sensitivity and Specificity; D013154:Spleen / Q000166:cytology
chemical_list: D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D007473:Ion Channels; D011956:Receptors, Cell Surface; C030325:anthrax toxin