Programme Contents

GABA_B receptor gene knockdown impairs inhibition of N-type calcium channels in rat sensory neurons by α-conotoxin Vc1.1

G protein coupled GABA_B receptors are receptors for γ-aminobutyric acid (GABA) and are widely expressed in the central and peripheral nervous system. They regulate synaptic transmission and signal propagation by modulating the activity of voltage-gated calcium channels and inwardly rectifying potassium channels. Functional GABA_B receptors are heterodimers consisting of GABA_B1 and GABA_B2 subunits. The analgesic α-conotoxin Vc1.1, a peptide from the venom of the marine cone snail Conus victoriae, inhibits N-type calcium channels in sensory neurons via activation of G protein coupled GABA_B receptor (Callaghan et al., 2008). Although there is unambiguous pharmacological evidence demonstrating that Vc1.1 does not interact directly with N-type calcium channels but inhibits them via a voltage-independent mechanism involving the GABA_B receptor, to date, there are no molecular studies confirming the interplay between Vc1.1 and the GABA_B receptor. The aim of the present study was to examine the effect of the GABA_B agonist baclofen and Vc1.1 on N-type calcium channel currents recorded in sensory neurons following transient knock-down of the GABA_B receptor using RNA interference (RNAi). Dorsal root ganglion (DRG) neurons isolated from 3-12 days old rats were transfected with small interfering RNAs (siRNAs) targeting both GABA_B subunits. One day after transfection a reduction of over 50% in mRNA levels for GABA_B subunits was observed compared to control cells (mock cells transfected without siRNA and scrambled non-targeting siRNA transfected cells), as demonstrated in quantitative real-time PCR analysis of a minimum of 4 independent experiments. Moreover, suppression of GABA_B protein expression was evaluated immunocytochemically using a high-content imaging system and confocal laser scanning microscopy 2-3 days after transfection. Quantitative analysis of immunofluorescence-labeled GABA_B proteins in DRG neurons revealed significantly reduced GABA_B expression in cells transfected with GABA_B-targeting siRNAs compared to control cells. Whole-cell patch-clamp studies conducted 1-3 days after transfection demonstrated that knock-down of functional GABA_B receptor expression significantly reduced the inhibition of N-type calcium channels in response to both baclofen (30 μM) and Vc1.1 (100 nM) in isolated DRG neurons. This is in contrast to neurons transfected with a non-targeting siRNA which were not distinguishable from untransfected neurons. Taken together, these results confirm that α-conotoxin Vc1.1 modulates N-type calcium channels via activation of the GABA_B receptor in DRG neurons.

Callaghan, B., Haythornthwaite, A., Berecki, G., Clark, R.J., Craik, D.J., Adams, D.J. (2008), Journal of Neuroscience, 28, 10943–10951.