Channel formation by the binding component of XXXX C2 toxin: @entity2227 307 of C2II affects channel properties in vitro and pH-dependent C2I translocation in vivo.

@entity1522 @entity2227 @entity503 @entity2228 @entity535 @entity6940 @entity72534 @entity376 @entity857 @entity72535
The binding component (C2II) of the binary @entity6940 C2 toxin mediates transport of the actin @entity857 -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. @entity535 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, @entity2227 307 (E307) localized in its center and @entity2228 316 (G316) localized on the trans side of the membrane. Here, single- @entity376 substitutions of these @entity1522 and the double mutant @entity72534 / @entity72535 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 @entity72534 mutant, which means that E307 is responsible for voltage gating. @entity503 blocked the @entity72534 mutant channel and intoxication of Vero cells by mutant C2II and C2I, indicating that @entity503 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.
