G protein modulation of omega-conotoxin binding sites in neuroblastoma x glioma NG 108-15 hybrid cells.
Electrophysiological evidence shows that voltage-dependent calcium channel (VDCC) activity can be regulated by a large number of neurotransmitters. In particular, guanine nucleotide binding regulatory protein (G protein)-mediated inhibitory modulation of the channel activity has been deduced from evidence that GTP analogues and purified G proteins are able to mimic this effect. The G proteins involved are pertussis toxin (PTx) sensitive. The purpose of the present study was to investigate, using biochemical techniques, whether G protein activation modulates the recognition site for omega-conotoxin GVIA (CgTx), a peptide neurotoxin that selectively labels a population of high-threshold VDCC. Undifferentiated and differentiated (1 mM dibutyryl cyclic AMP, 4 days) NG 108-15 cells were used. In both crude cellular extracts specific binding of 125I-CgTx was characterized. Differentiation induced a sixfold increase in the number of binding sites and doubled the KD value. The in vitro addition of guanylylimidodiphosphate (GMP-PNP; a nonhydrolyzable analogue of GTP) to extracts prepared from differentiated cells reduced the 125I-CgTx binding by 48%. This effect, observed in undifferentiated cells as well, was also caused by other triphosphate guanine nucleotides, such as GTP, but not by guanosine 5'-O-(2-thiodiphosphate) or adenine nucleotides. Treatment of the cells with PTx prevented the GMP-PNP effect. Moreover, the results obtained after preincubation with specific antisera raised against the alpha subunits of Gi1-2 and Go suggest that Go is the G protein responsible for the observed effect.
Bergamaschi
S
S
Institute of Pharmacological Sciences, University of Milan, Italy.
Govoni
S
S
Battaini
F
F
Trabucchi
M
M
Del Monaco
S
S
Parenti
M
M
D000818:Animals; D001665:Binding Sites; D001932:Brain Neoplasms / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure; D015220:Calcium Channels; D019204:GTP-Binding Proteins / Q000502:physiology*; D005910:Glioma / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D006822:Hybrid Cells / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure; D015151:Immunoblotting; D007457:Iodine Radioisotopes; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure; D010456:Peptides, Cyclic / Q000378:metabolism*; D037342:Pertussis Toxin; D011233:Precipitin Tests; D011485:Protein Binding; D017981:Receptors, Neurotransmitter / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D020866:omega-Conotoxin GVIA
D015220:Calcium Channels; D007457:Iodine Radioisotopes; D010456:Peptides, Cyclic; D017981:Receptors, Neurotransmitter; D010566:Virulence Factors, Bordetella; C053157:omega-conotoxin receptor; D006165:Guanylyl Imidodiphosphate; D020866:omega-Conotoxin GVIA; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins