Gabapentin-mediated inhibition of voltage-activated Ca2+ channel currents in cultured sensory neurones is dependent on culture conditions and channel subunit expression

We have used the whole cell patch clamp method and fura-2 fluorescence imaging to study the actions of gabapentin (1-(aminoethyl) cyclohexane acetic acid) on voltage-activated Ca2+ entry into neonatal cultured dorsal root ganglion (DRG) neurones and differentiated F-I I (embryonic rat DRGxneuroblastoma hybrid) cells. Gabapentin (2.5 muM) in contrast to GABA (10 muM) did not influence testing membrane potential or input resistance. In current clamp mode gabapentin failed to influence the properties of evoked single action potentials but did reduce the duration of action potentials prolonged by Ba2+. Gabapentin attenuated high voltage-activated Ca2+ channel currents in a dose- and voltage- dependent manner in DRG neurones and reduced Ca2+ influx evoked by K-1 depolarisation in differentiated F-I I cells loaded with fura-2. The sensitivity of DRG neurones to gabapentin was not changed by the GABA(B) receptor antagonist saclofen but pertussis toxin pre-treatment reduced the inhibitory effects of gabapentin. Experiments following pre-treatment of DRG neurones with a PKA-activator and a PKA-inhibitor implicated change in phosphorylation state as a mechanism, which influenced gabapentin action. Sp- and Rp-analogues of cAMP significantly increased or decreased gabapentin-mediatcd inhibition of voltage-activated Ca2+ channel currents. Culture conditions used to maintain DRG neurones and passage number of differentiated F-I I cells also influenced the sensitivity of Ca2+ channels to gabapentin. We analysed the Ca2+ channel subunits expressed in populations of DRG neurones and F-I I cells that responded to gabapentin had low sensitivity to gabapentin or were insensitive to gabapentin, by Quantitative TaqMan PCR. The data obtained from this analysis suggested that the relative abundance of the Ca2+ channel beta(2) and alpha(2)delta subunit expressed was a key determinant of gabapentin sensitivity of both cultured DRG neurones and differentiated F-I I cells. In conclusion, gabapentin inhibited part of the high voltage-activated Ca2+ current in neonatal rat cultured DRG neurones via a mechanism that was independent of GABA receptor activation, but was sensitive to pertussis toxin. Gabapentin responses identified in this study implicated Ca2+ channel beta(2) subunit type as critically important to drug sensitivity and interactions with alpha(1) and alpha(2)delta subunits may be implicated in antihyperalgesic therapeutic action for this compound. (C) 2002 Elsevier Science Ltd. All rights reserved.