Mechanism of action of the anticonvulsant drug gabapentin
In this study, the whole-cell patch clamp technique and Fura-2 calcium (Ca2+) imaging techniques were employed to investigate the involvement of voltage activated calcium currents (VACCs) in the mechanism of action of the anticonvulsant drug gabapentin. Molecular biology studies were also carried out to assess which VACC subunits were required for drug sensitivity in particular cell cultures. Also investigated were the effects, if any, of gabapentin at GABA receptors and on cells of the F11 line. Voltage activated calcium currents were activated in cultured DRG neurones until a consistent current magnitude was achieved. Effects of gabapentin could then be measured by activation of subsequent currents in the presence of drug. The effects of various other compounds were also tested in this way, e.g. the effects of the related drug pregabalin, effects of the inhibitory neurotransmitter GABA, the effects of the GABAB antagonist saclofen and the effect of treating the cells with pertussis toxin. Gabapentin inhibited voltage activated calcium currents. This inhibition was voltage dependent but frequency independent. Gabapentin did not act at GABAA or GABAB receptors. Gabapentin effects were PTx sensitive suggesting a G-protein mechanism was involved. An experimental protocol for calcium fluorescence imaging was designed. This protocol allowed comparison of gabapentin and other drug effects against control responses, and is a protocol that could be used as an assay system to test the properties of compounds related to gabapentin. Gabapentin and pregabalin ((S)-isobutylgaba) both reduced the magnitude of evoked Ca2+ transients in both cultured DRG neurones and in F11 cells. The R-stereoisomer of pregabalin, PD-C, was shown to increase the magnitude of these transients. Both this increase and the decrease associated with gabapentin were PTx sensitive. These results indicate that gabapentin attenuates calcium currents at VACCs. The gabapentin binding site has been shown to be the α2δ subunit and it was observed that the activity of gabapentin is dependent on a G-protein linked mechanism and on the presence of modulatory VACC β subunits.