Go transduces GABAB-receptor modulation of N-type calcium channels in cultured dorsal root ganglion neurons

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Abstract
High-voltage-activated (HVA) calcium channel currents (I Ba) were recorded from acutely replated cultured dorsal root ganglion (DRG) neurons. I Ba was irreversibly inhibited by 56.9±2.7% by 1 μM ω-conotoxin-GVIA (ω-CTx-GVIA), whereas the 1,4-dihydropyridine antagonist nicardipine was ineffective. The selective γ-aminobutyric acidB (GABAB) agonist, (−)-baclofen (50 μM), inhibited the HVA I Ba by 30.7±5.4%. Prior application of ω-CTx-GVIA completely occluded inhibition of the HVA I Ba 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-Go antiserum, raised against the C-terminal decapeptide of the Gα o subunit, reduced (−)-baclofen inhibition of the HVA I Ba, whereas replating DRGs in the presence of the anti-Gi antiseram did not. Using anti-Gα o antisera (1∶2000) and confocal laser microscopy, Gα o localisation was investigated in both unreplated and replated neurons. Gα 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 Gα o immunoreactivity was completely blocked by preincubation with the immunising Gαo undecapeptide (1 μg · ml−1) for 1 h at 37° C. A similar treatment also blocked recognition of Gα o in brain membranes on immunoblots. These results provide evidence that GABAB inhibition of N-type calcium channels in acutely replated DRGs occurs via Gα o.

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