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.
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.
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, Otto-Krayer-Haus, Albertstrasse 25, D-79104 Freiburg, Germany.
Dagmar
D
Blöcker
Christoph
C
Bachmeyer
Roland
R
Benz
Klaus
K
Aktories
Holger
H
Barth
D036002:ADP Ribose Transferases / Q000378:metabolism*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001665:Binding Sites; D001905:Botulinum Toxins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D016466:CHO Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002522:Chlorocebus aethiops; D002738:Chloroquine / Q000494:pharmacology; D003014:Clostridium botulinum / Q000737:chemistry*; D006224:Cricetinae; D004553:Electric Conductivity; D004926:Escherichia coli / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000187:drug effects* / Q000502:physiology; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology*; D008051:Lipid Bilayers; D018942:Macrolides; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000633:toxicity; D017384:Sequence Deletion; D014709:Vero Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism
D000900:Anti-Bacterial Agents; D007473:Ion Channels; D008051:Lipid Bilayers; D018942:Macrolides; D011993:Recombinant Fusion Proteins; D018698:Glutamic Acid; D002738:Chloroquine; C040929:bafilomycin A1; D036002:ADP Ribose Transferases; D011065:Poly(ADP-ribose) Polymerases; D001905:Botulinum Toxins; C096324:botulinum toxin type C