GABAB receptor modulation of Ca2+ currents in rat sensory neurones by the G protein G(0): antisense oligonucleotide studies.
1. Calcium channel currents (IBa) were recorded in cultured dorsal root ganglion neurones (DRGs), 24-32 h after microinjection with 20-mer phosphorothioate antisense oligonucleotides complementary either to a G alpha o or a G alpha i unique sequence, or with a nonsense sequence. 2. The ability of the GABAB agonist (-)-baclofen (50 microM) to inhibit IBa was examined. The maximum peak current was inhibited by 35.3 +/- 4.0% (n = 11) in control non-injected cells, and by 38.1 +/- 2.6% (n = 11) and 34.8 +/- 4.2% (n = 5) in nonsense- and G alpha i oligonucleotide-injected cells. Following G alpha o oligonucleotide injection, (-)-baclofen inhibited IBa by 21.0 +/- 3.2% (n = 19). 3. Confocal immunocytochemical localization of G alpha o showed prominent staining at the plasma membrane in control DRGs, and this was also present in G alpha i and nonsense oligonucleotide-injected cells. The G alpha o staining at the plasma membrane was reduced by 76% in G alpha o oligonucleotide-injected cells. In contrast, confocal immunocytochemical localization of G alpha i showed immunostaining in the membrane and cytoplasm of control, G alpha o- and nonsense-injected DRGs, whereas this was depleted by 68% in G alpha i oligonucleotide-injected cells. 4. These results indicate that the GABAB receptor couples to voltage-sensitive calcium channels via the G protein G(o) and not Gi, and that antisense oligonucleotides can be used to deplete G proteins in DRGs.
Campbell
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Department of Pharmacology, Royal Free Hospital School of Medicine, London.
Berrow
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N
Dolphin
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D000818:Animals; D001464:Barium / Q000494:pharmacology; D001483:Base Sequence; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology*; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008845:Microinjections; D008969:Molecular Sequence Data; D009475:Neurons, Afferent / Q000187:drug effects / Q000378:metabolism*; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D018080:Receptors, GABA-B / Q000502:physiology*; D013873:Thionucleotides
D015220:Calcium Channels; D018076:DNA, Complementary; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D018080:Receptors, GABA-B; D013873:Thionucleotides; D001464:Barium; D019204:GTP-Binding Proteins