Abstract
The fusion construct pEGFP-PTXS1 was assembled by ligating cDNA encoding the S1 subunit of Bordetella pertussis toxin (PTX) into the plasmid pEGFP-C1 (which codes for enhanced green fluorescent protein). Microinjection of pEGFP-PTXS1 (1–100 ng/μl) into the nucleus of dissociated rat sympathetic ganglion neurons resulted in functional expression as determined from the diffuse green fluorescence and disruption of norepinephrine-mediated N-type Ca 2+ channel modulation. The heterologously expressed toxin retained specificity for G α i/o-dependent pathways as VIP-mediated modulation of N-type Ca 2+ channels and muscarine-mediated inhibition of M-type K + channels persisted in pEGFP-PTXS1 expressing neurons. These data demonstrate that the S1 subunit of PTX is readily expressed in mammalian neurons and remains functional following fusion to the C-terminus of another protein.
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