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Gabapentin (Neurontin, Pfizer Global R & D) is a novel anticonvulsant, antihyperalgesic, and antinociceptive agent with a poorly understood mechanism of action. In this study, we show that gabapentin (EC50 2 microM) inhibited up to 70 to 80% of the total K+-evoked Ca2+ influx via voltage-dependent calcium channels (VD-CCs) in a mouse pituitary intermediate melanotrope clonal mIL-tsA58 (mIL) cell line. mIL cells endogenously express only gamma-aminobutyric acid type B (GABA(B)) gb1a-gb2 receptors. Moreover, activity of the agonist gabapentin was dose dependently and completely blocked with the GABA(B) antagonist CGP55845 and was nearly identical to the prototypic GABA(B) agonist baclofen in both extent and potency. Antisense knockdown of gb1a also completely blocked gabapentin activity, while gb1b antisense and control oligonucleotides had no effect, indicating that gabapentin inhibition of membrane Ca2+ mobilization in mIL cells was dependent on a functional GABA(B) (gb1a-gb2) heterodimer receptor. In addition, during combined whole cell recording and multiphoton Ca2+ imaging in hippocampal neurons in situ, gabapentin significantly inhibited in a dose-dependent manner subthreshold soma depolarizations and Ca2+ responses evoked by somatic current injection. Furthermore, gabapentin almost completely blocked Ca2+ action potentials and Ca2+ responses elicited by suprathreshold current injection. However, larger current injection overcame this inhibition of Ca2+ action potentials suggesting that gabapentin did not predominantly affect L-type Ca2+ channels. The depressant effect of gabapentin on Ca2+ responses was coupled to the activation of neuronal GABA(B) receptors since they were blocked by CGP55845, and baclofen produced similar effects. Thus gabapentin activation of neuronal GABA(B) gb1a-gb2 receptors negatively coupled to VD-CCs can be a potentially important therapeutic mechanism of action of gabapentin that may be linked to inhibition of neurotransmitter release in some systems.

Gamma-aminobutyric acid (GABA) activates two qualitatively different inhibitory mechanisms through ionotropic GABA(A) multisubunit chloride channel receptors and metabotropic GABA(B) G protein-coupled receptors. Evidence suggests that pharmacologically distinct GABA(B) receptor subtypes mediate presynaptic inhibition of neurotransmitter release by reducing Ca2+ conductance, and postsynaptic inhibition of neuronal excitability by activating inwardly rectifying K+ (Kir) conductance. However, the cloning of GABA(B) gb1 and gb2 receptor genes and identification of the functional GABA(B) gb1-gb2 receptor heterodimer have so far failed to substantiate the existence of pharmacologically distinct receptor subtypes. The anticonvulsant, antihyperalgesic, and anxiolytic agent gabapentin (Neurontin) is a 3-alkylated GABA analog with an unknown mechanism of action. Here we report that gabapentin is an agonist at the GABA(B) gb1a-gb2 heterodimer coupled to Kir 3.1/3.2 inwardly rectifying K+ channels in Xenopus laevis oocytes. Gabapentin was practically inactive at the human gb1b-gb2 heterodimer, a novel human gb1c-gb2 heterodimer and did not block GABA agonism at these heterodimer subtypes. Gabapentin was not an agonist at recombinant GABA(A) receptors as well. In CA1 pyramidal neurons of rat hippocampal slices, gabapentin activated postsynaptic K+ currents, probably via the gb1a-gb2 heterodimer coupled to inward rectifiers, but did not presynaptically depress monosynaptic GABA(A) inhibitory postsynaptic currents. Gabapentin is the first GABA(B) receptor subtype-selective agonist identified providing proof of pharmacologically and physiologically distinct receptor subtypes. This selective agonism of postsynaptic GABA(B) receptor subtypes by gabapentin in hippocampal neurons may be its key therapeutic advantage as an anticonvulsant.

Numerous mutant G protein-coupled receptors with diminished or no function have been described that are naturally occurring or that are the product of gene manipulation. It has largely been assumed that receptor mutants do not affect the function of the wild-type receptor; however, the occurrence of G protein-coupled receptor dimerization suggests the possibility that an intermolecular interaction between mutant and wild-type receptors can occur. We have shown previously that the D2 dopamine receptor (D2DR) exists as dimers in cell lines and brain tissue. In this study, we demonstrated that mutant D2DR can modulate the function of the wild-type D2DR. While attempting to elucidate the structure of the D2DR dimer, we demonstrated that nonfunctional D2DR substitution and truncation mutants antagonized wild-type D2DR function. Furthermore, from analyses of this interaction between the receptor mutants and the D2DR, using photoaffinity labeling, we provide evidence that the D2DR is oligomeric in the cell.

Direct evidence is lacking to show whether the gamma-aminobutyric acid (GABA)(B) gb1-gb2 heterodimer is the signaling form of the receptor. In this study, we tested whether gb1a or gb2 subunits when coexpressed with truncated receptors or metabotropic glutamate receptor mGluR4 could form functional GABA receptors. Coexpression of the ligand binding N-terminal domain of gb1a or the C-terminal portion of gb1a composing the seven-transmembrane segments and intracellular loops with gb2 could not reconstitute functional receptors. We next examined whether mGluR4, which forms homodimers and is structurally related to GABA(B), could act as a surrogate coreceptor for gb1 or gb2. The coexpression of mGluR4 and gb1a led to the expression of gb1a monomers on cell surface membranes as determined by immunoblot analysis and flow cytometry. However, mGluR4-gb1a heterodimers were not formed, and membrane-expressed gb1a monomers were not functionally coupled to adenylyl cyclase in human embryonic kidney 293 cells or activated inwardly rectifying potassium (Kir) channels in Xenopus oocytes. Similarly, the coexpression of mGluR4 and gb2 led to nonfunctional GABA receptors. GABA-activated distal signaling events resulted only after the coexpression and heterodimerization of gb1 and gb2. Taken together with the truncated receptor studies, the data suggest that a high degree of structural specificity is required to form the functional GABA(B) receptor that is a gb1-gb2 heterodimer.