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 Bordetella pertussis toxin (PTX) into the plasmid pEGFP-C1 (which codes for enhanced green fluorescent protein). Microinjection of pEGFP-PTXS1 (1-100 ng/microl) 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 G alpha(i/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.
Ikeda
S R
SR
Laboratory of Molecular Physiology, Guthrie Research Institute, Sayre, PA 18840-1692, USA. sikeda@inet.guthrie.org
Jeong
S W
SW
Kammermeier
P J
PJ
Ruiz-Velasco
V
V
King
M M
MM
D000818:Animals; D020864:Calcium Channels, N-Type / Q000502:physiology*; D017931:DNA Primers; D018076:DNA, Complementary; D019204:GTP-Binding Proteins / Q000502:physiology; D015870:Gene Expression / Q000502:physiology; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000235:genetics*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009116:Muscarine / Q000494:pharmacology; D018721:Muscarinic Agonists / Q000494:pharmacology; D009474:Neurons / Q000737:chemistry / Q000502:physiology*; D018408:Patch-Clamp Techniques; D037342:Pertussis Toxin; D015221:Potassium Channels / Q000502:physiology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017783:Superior Cervical Ganglion / Q000166:cytology*; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000235:genetics*
D020864:Calcium Channels, N-Type; D017931:DNA Primers; D018076:DNA, Complementary; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D018721:Muscarinic Agonists; D015221:Potassium Channels; D011993:Recombinant Fusion Proteins; D010566:Virulence Factors, Bordetella; C083388:pertussis toxin, S1 subunit; D049452:Green Fluorescent Proteins; D014660:Vasoactive Intestinal Peptide; D009116:Muscarine; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins