Document Detail

Channel formation by the binding component of Clostridium botulinum C2 toxin: glutamate 307 of C2II affects channel properties in vitro and pH-dependent C2I translocation in vivo.
MedLine Citation:
PMID:  12731878     Owner:  NLM     Status:  MEDLINE    
The binding component (C2II) of the binary Clostridium botulinum C2 toxin mediates transport of the actin ADP-ribosylating enzyme component (C2I) into the cytosol of target cells. C2II (80 kDa) is activated by trypsin cleavage, and proteolytically activated C2II (60 kDa) oligomerizes to heptamers in solution. Activated C2II forms channels in lipid bilayer membranes which are highly cation selective and voltage-gated. A role for this channel in C2I translocation across the cell membrane into the cytosol is discussed. Amino acid residues 303-331 of C2II contain a conserved pattern of alternating hydrophobic and hydrophilic residues, which likely facilitates membrane insertion and channel formation by creating two antiparallel beta-strands. Some of the residues are in strategic positions within the putative C2II channel, in particular, glutamate 307 (E307) localized in its center and glycine 316 (G316) localized on the trans side of the membrane. Here, single-lysine substitutions of these amino acids and the double mutant E307K/G316K of C2II were analyzed in vivo and in artificial lipid bilayer experiments. The pH dependence of C2I transport across cellular membranes was altered, and a pH of <or=5.2 was needed for C2I translocation into target cells; otherwise, no change in C2II-promoted entry of C2I into Vero cells was observed. The channel properties of C2II were substantially changed by the mutations, as evidenced by reduced cation selectivity. Interestingly, the voltage dependence of wild-type C2II was completely lost for the E307K mutant, which means that E307 is responsible for voltage gating. Chloroquine blocked the E307K mutant channel and intoxication of Vero cells by mutant C2II and C2I, indicating that chloroquine binding does not involve E307. Overall, the voltage gating and cation selectivity of the C2II channel do not play an important role in translocation of C2I into the cytosol.
Dagmar Blöcker; Christoph Bachmeyer; Roland Benz; Klaus Aktories; Holger Barth
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Publication Detail:
Type:  Comparative Study; In Vitro; Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Biochemistry     Volume:  42     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  2003 May 
Date Detail:
Created Date:  2003-05-06     Completed Date:  2003-06-24     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  5368-77     Citation Subset:  IM    
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, Otto-Krayer-Haus, Albertstrasse 25, D-79104 Freiburg, Germany.
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MeSH Terms
ADP Ribose Transferases / metabolism*
Anti-Bacterial Agents / pharmacology
Binding Sites
Botulinum Toxins / genetics,  metabolism*,  toxicity
CHO Cells / cytology,  drug effects,  metabolism
Cercopithecus aethiops
Chloroquine / pharmacology
Clostridium botulinum / chemistry*
Electric Conductivity
Escherichia coli / genetics
Glutamic Acid / genetics,  metabolism*
Hydrogen-Ion Concentration
Ion Channel Gating / drug effects*,  physiology
Ion Channels / drug effects,  physiology*
Lipid Bilayers*
Mutagenesis, Site-Directed
Mutation / genetics
Poly(ADP-ribose) Polymerases / metabolism
Protein Binding
Recombinant Fusion Proteins / metabolism,  toxicity
Sequence Deletion
Vero Cells / cytology,  drug effects,  metabolism
Reg. No./Substance:
0/Anti-Bacterial Agents; 0/Botulinum Toxins; 0/Ion Channels; 0/Lipid Bilayers; 0/Macrolides; 0/Recombinant Fusion Proteins; 0/botulinum toxin type C; 54-05-7/Chloroquine; 56-86-0/Glutamic Acid; 88899-55-2/bafilomycin A1; EC 2.4.2.-/ADP Ribose Transferases; EC Polymerases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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