Gabapentin is not a GABAB receptor agonist
Introduction
Type B gamma-aminobutyric acid (GABAB) receptors are present at pre- and post-synaptic loci throughout the central nervous system, where they inhibit adenylate cyclase activity, cause post-synaptic hyperpolarisation and inhibit neurotransmitter release (for review see Bowery, 1993; Couve et al., 2000; Billinton et al., 2001). The recent cloning of two GABAB receptor subunits, termed GABAB1 and GABAB2, which form functional receptors by heterodimerisation has raised the possibility that differences in GABAB receptor subunit composition may give rise to pharmacologically distinct receptor subtypes (Kaupmann et al., 1998a, Kaupmann et al., 1998b; White et al., 1998; Jones et al., 1998; Kuner et al., 1999). A further source of pharmacological diversity may also arise from splice variants of these GABAB receptor subunits.
Two splice variants of the GABAB1 subunit were originally described, termed GABAB1a and GABAB1b, which differ in the extreme amino terminal domain (Kaupmann et al., 1997). Thus, the GABAB1a receptor subunit contains an extra sequence of 130 residues which encode two Sushi domains (Hawrot et al., 1998). Additional splice variants have now been described such as the human GABAB1c receptor subunit which lacks 62 residues within one of the Sushi domains at the amino terminus, the rat GABAB1c receptor subunit which contains a 31-residue insertion in the second extracellular loop and the rat GABAB1d receptor subunit which contains a divergent carboxy terminal tail (Pfaff et al., 1999; Isomoto et al., 1998). Carboxy terminal splice variants have also been described for the GABAB2 receptor subunit although these remain uncharacterised as yet pharmacologically (Calver et al., 2000).
Thus far, the general consensus is that there are no differences in the pharmacological profile of GABAB receptor heterodimers containing alternatively spliced forms of the GABAB1 receptor subunit (Kaupmann et al., 1997; Galvez et al., 1999; Malitschek et al., 1999; Pfaff et al., 1999; Green et al., 2000). This is in contrast to the pharmacological characterisation of natively expressed GABAB receptors, which suggest diversity between pre-synaptic GABAB auto- and heteroreceptors, as well as between pre- and post-synaptic GABAB receptors (see Bonanno and Raiteri, 1993; Dutar and Nicoll, 1988; Yamada et al., 1999). However, recently, the 3-alkylated GABA analogue gabapentin (Neurontin) has been reported to be a GABAB receptor agonist which selectively activates post-synaptic receptors containing the GABAB1a receptor subunit but not the GABAB1b receptor subunit (Ng et al., 2001).
Simplistically, gabapentin might confer its selectivity by interacting with the 130 extra N-terminal amino acids that differentiate the GABAB1a subunit from the GABAB1b subunit. However, gabapentin activation of GABAB1a containing receptors is abolished by the competitive GABAB receptor antagonist CGP55845A (Ng et al., 2001), despite the fact that this and similar antagonists have identical affinities for GABAB1a- and GABAB1b-containing GABAB receptor heterodimers and do not themselves bind the GABAB2 receptor subunit (Green et al., 2000; Galvez et al., 1999; Malitschek et al., 1999). These observations raise doubts as to the validity of gabapentin being able to differentiate GABAB receptors comprised of different GABAB1 splice variants and, as such, we have re-evaluated its effects on different recombinant heterodimeric configurations of GABAB receptors, as well as native hippocampal GABAB receptors. This study is of particular importance as there is a widely held belief that development of compounds with this type of pharmacological profile will be important in developing new treatments for pathologies such as drug addiction, spasticity, pain and epilepsy that have limited side-effect profiles.
Section snippets
Materials
2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium (NBQX), d-2-amino-5-phosphonopentanoate (AP5), [1-(S)-3,4-dichlorophenyl)ethyl]amino-2-(S)-hydroxypropyl-p-benzyl-phosphonic acid (CGP55845A), (R)-baclofen, 1-(4,4-diphenyl-3-butenyl)-3-piperidine carboxylic acid (SKF89976A) and (40 Ci/mmol) were obtained from Tocris Cookson (Bristol, UK). Bicuculline and GABA were purchased from Sigma Aldrich, UK. 3-[1-(R)-[[2(S
Gabapentin does not directly compete with known GABAB receptor antagonists
The direct interaction of gabapentin with GABAB1a/B2 and GABAB1b/B2 receptors expressed in cell lines was investigated in competition binding experiments using the selective, high-affinity GABAB receptor antagonist, . Specific binding of this radioligand was inhibited by GABA with pKi values of 5.14±0.04 and 4.77±0.04 for the GABAB1a/B2 and GABAB1b/B2 receptors, respectively (Fig. 1). Baclofen showed similar affinity with pKi values of 4.72±0.03 and 4.36±0.03, respectively (Fig. 1
Discussion
Progress in the understanding of GABAB receptor function has been greatly advanced since the cloning of the GABAB1 and GABAB2 receptor subunits and the demonstration that heterodimerisation of these subunits is required for normal receptor activity (see Couve et al., 2000; Billinton et al., 2001; Prosser et al., 2001). The advent of these findings has allowed researchers to compare the pharmacological diversity of GABAB receptors expressed in brain (Bonanno and Raiteri, 1993; Dutar and Nicoll,
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