Heterologous expression of a green fluorescent protein–pertussis toxin S1 subunit fusion construct disrupts calcium channel modulation in rat superior cervical ganglion neurons
The fusion construct pEGFP-PTXS1 was assembled by ligating cDNA encoding the S1 subunit of Bordetellapertussis 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 Ca2+ channel modulation. The heterologously expressed toxin retained specificity for Gαi/o-dependent pathways as VIP-mediated modulation of N-type Ca2+ 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.