G(o) transduces GABAB-receptor modulation of N-type calcium channels in cultured dorsal root ganglion neurons.
High-voltage-activated (HVA) calcium channel currents (IBa) were recorded from acutely replated cultured dorsal root ganglion (DRG) neurons. IBa was irreversibly inhibited by 56.9 +/- 2.7% by 1 microM omega-conotoxin-GVIA (omega-CTx-GVIA), whereas the 1,4-dihydropyridine antagonist nicardipine was ineffective. The selective gamma-aminobutyric acidB (GABAB) agonist, (-)-baclofen (50 microM), inhibited the HVA IBa by 30.7 +/- 5.4%. Prior application of omega-CTx-GVIA completely occluded inhibition of the HVA IBa by (-)-baclofen, indicating that in this preparation (-)-baclofen inhibits N-type current. To investigate which G protein subtype was involved, cells were replated in the presence of anti-G protein antisera. Under these conditions the antibodies were shown to enter the cells through transient pores created during the replating procedure. Replating DRGs in the presence of anti-G(o) antiserum, raised against the C-terminal decapeptide of the G alpha o subunit, reduced (-)-baclofen inhibition of the HVA IBa, whereas replating DRGs in the presence of the anti-Gi antiserum did not. Using anti-G alpha o antisera (1:2000) and confocal laser microscopy, G alpha o localisation was investigated in both unreplated and replated neurons. G alpha o immunoreactivity was observed at the plasma membrane, neurites, attachment plaques and perinuclear region, and was particularly pronounced at points of cell-to-cell contact. The plasma membrane G alpha o immunoreactivity was completely blocked by preincubation with the immunising G alpha o undecapeptide (1 microgram.ml-1) for 1 h at 37 degrees C. A similar treatment also blocked recognition of G alpha o in brain membranes on immunoblots.(ABSTRACT TRUNCATED AT 250 WORDS)
Menon-Johansson
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stabliDepartment of Pharmacology, Royal Free Hospital School of Medicine, London, UK.
Berrow
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N
Dolphin
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D000595:Amino Acid Sequence; D000818:Animals; D001369:Axons / Q000502:physiology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism*; D002478:Cells, Cultured; D004594:Electrophysiology; D019204:GTP-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D005727:Ganglia, Spinal / Q000166:cytology / Q000378:metabolism*; D007150:Immunohistochemistry; D007834:Lasers; D008297:Male; D008853:Microscopy; D008969:Molecular Sequence Data; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D018080:Receptors, GABA-B / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D018080:Receptors, GABA-B; D010566:Virulence Factors, Bordetella; D019204:GTP-Binding Proteins