Figure 3.
Insensitivity of TGNs to BoNT/E is attributable to a paucity of its acceptors and can be overcome by using chimera EA, which binds to SV2C–L4, enters TGNs, and blocks CGRP release evoked by all stimuli. Rat cultured TGNs at 7 d in vitro were exposed to BoNT/E (A, B) or EA (C, D) at 37°C for 24 and 12 h, respectively. Release of CGRP (B, D) evoked over 30 min at 37°C by bradykinin (▴), 60 mm K+ (□), or 1 μm capsaicin (●) was measured before subjecting the cells to SDS-PAGE and Western blotting of intact and truncated SNAP-25 (A, C). The proportions of intact substrate remaining (B, D, ▿) were calculated from digital images of the gels; note that some symbols are overlapped. Inset in B, Western blots of rat cultured TGNs and CGNs after 7 d in vitro using SV2 isoform-specific antibodies. E, GST–SV2C–L4 protein immobilized on glutathione–Sepharose beads was incubated with EA (SC or DC), BoNT/A or /E (DC) at 4°C for 4 h; after washing, the bound protein eluted was analyzed by SDS-PAGE without heating (see Materials and Methods), followed by Western blotting with the antibodies indicated. F, A model substrate [GFP–SNAP-25 (134–206)–His6; 13.5 μm] (Wang et al., 2008) was incubated with 25 nm EA or its parental toxins for 30 min at 37°C and subjected to SDS-PAGE, followed by Coomassie staining. Data plotted are means ± SEM; n ≥ 3.