Menon-Johansson, Anatole Sebastian; (1993) An investigation into G protein modulation of high voltage activated calcium currents in acutely replated cultured sensory neurones of the rat. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Cultured rat dorsal root ganglion (DRG) neurones are a model of their presynaptic terminals in vivo. Acutely replating DRGs before use provides an axotomised cell body ideal for space clamp in electrophysiological recordings. In addition macromolecules can be introduced through transient pores created by the replating procedure. High voltage activated (HVA) calcium channel currents (IBa) were recorded in the whole cell configuration of the patch clamp technique. The HVA IBa in acutely replated DRGs were irreversibly inhibited 57% by ω-CTx-GVIA (1?M). The selective GABAB agonist, (-)-baclofen (50)μM), inhibited the HVA IBa by 30%. Prior application of ω-CTx-GVIA completely blocked baclofen inhibition of the HVA IBa. The modulation of the HVA IBa by baclofen is reduced by pre-incubation with pertussis toxin (PTX). This indicates that a GTP binding (G) protein is involved. PTX ADP-ribosylates the G proteins, Gαi and GαO. TO investigate which G protein subtype was involved, cells were replated in the presence of anti-Gα antisera raised against the C-terminal decapeptide of the Gα subunits, Gαi (SG1) and Gαo (OC1/2) at dilutions of 1:100-1:25. Only replating DRGs in the presence of OC1/2 reduced baclofen inhibition of the HVA IBa. DRGs replated in the presence of the Gαo decapeptide (80μg/ml), also significantly reduced baclofen inhibition of IBa. Pre-incubation of OC2 with the Gαo decapeptide for one hour at 37°C reversed the effect of both treatments alone. Using anti-Gα antisera and confocal laser microscopy, Gα localisation was investigated. Using OC1/2 (1:2000), immunoreactivity was observed at the plasma membrane, neurites, attachment plaques, perinuclear region and at points of cell-cell contact. This was blocked by pre-incubation with the Gαo decapeptide (1μg/ml) for one hour at 37°C. Immunoreactivity with SGI (1:2000-1:500) was also observed in the plasma membrane, cytoplasmic and perinuclear regions. To conclude, GABAB inhibition of ω-CTx-GVIA sensitive calcium channels in acutely replated DRGs is via Gαo.

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