title: Quantification of potassium levels in cells treated with Bordetella adenylate cyclase toxin.
abstract: The aim of this study was to compare two methods for quantification of changes in intracellular potassium concentration (decrease from ∼140 to ∼20mM) due to the action of a pore-forming toxin, the adenylate cyclase toxin (CyaA) from the pathogenic bacterium Bordetella pertussis. CyaA was incubated with stably transfected K1 Chinese hamster ovary cells expressing the toxin receptor CD11b/CD18 and the decrease in potassium concentration in the cells was followed by inductively coupled plasma mass spectrometry (ICP-MS). It is shown that this method is superior in terms of sensitivity, accuracy, and temporal resolution over the method employing the potassium-binding benzofuran isophthalate-acetoxymethyl ester fluorescent indicator. The ICP-MS procedure was found to be a reliable and straightforward analytical approach enabling kinetic studies of CyaA action at physiologically relevant toxin concentrations (<1000ng/ml) in biological microsamples.
authors.affiliation: Laboratory of Molecular Biology of Bacterial Pathogens, Cell and Molecular Microbiology Division, Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czech Republic.
authors.forename: Tomas
authors.initials: T
authors.lastname: Wald
authors.affiliation: Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, 60200 Brno, Czech Republic.
authors.forename: Inga
authors.initials: I
authors.lastname: Petry-Podgorska
authors.affiliation: Laboratory of Molecular Biology of Bacterial Pathogens, Cell and Molecular Microbiology Division, Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czech Republic; Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Prague 2, Czech Republic.
authors.forename: Radovan
authors.initials: R
authors.lastname: Fiser
authors.affiliation: Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, 60200 Brno, Czech Republic.
authors.forename: Tomas
authors.initials: T
authors.lastname: Matousek
authors.affiliation: Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, 60200 Brno, Czech Republic.
authors.forename: Jiri
authors.initials: J
authors.lastname: Dedina
authors.affiliation: Laboratory of Molecular Biology of Bacterial Pathogens, Cell and Molecular Microbiology Division, Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czech Republic.
authors.forename: Radim
authors.initials: R
authors.lastname: Osicka
authors.affiliation: Laboratory of Molecular Biology of Bacterial Pathogens, Cell and Molecular Microbiology Division, Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czech Republic.
authors.forename: Peter
authors.initials: P
authors.lastname: Sebo
authors.affiliation: Laboratory of Molecular Biology of Bacterial Pathogens, Cell and Molecular Microbiology Division, Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czech Republic. Electronic address: masin@biomed.cas.cz.
authors.forename: Jiri
authors.initials: J
authors.lastname: Masin
mesh_terms: D037361:Adenylate Cyclase Toxin / Q000633:toxicity*; D000818:Animals; D001886:Bordetella pertussis / Q000201:enzymology*; D039481:CD11b Antigen / Q000235:genetics; D018821:CD18 Antigens / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D011188:Potassium / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection
chemical_list: D037361:Adenylate Cyclase Toxin; D039481:CD11b Antigen; D018821:CD18 Antigens; D005456:Fluorescent Dyes; D011188:Potassium
keywords: Adenylate cyclase toxin; Bordetella pertussis; ICP–MS; PBFI–AM; Potassium; RTX