GABAB receptor modulation of Ca2+ currents in rat sensory neurones by the G protein G(0): antisense oligonucleotide studies.

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Abstract
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.