Subunit Of Voltage-gated Calcium Channels Research Articles

The Calcium Channel α2δ-2 Subunit Partitions with CaV2.1 into Lipid Rafts in Cerebellum: Implications for Localization and Function

The accessory alpha2delta subunits of voltage-gated calcium channels are highly glycosylated transmembrane proteins that interact with calcium channel alpha1 subunits to enhance calcium currents. We compared the membrane localization and processing of native cerebellar alpha2delta-2 subunits with alpha2delta-2 stably expressed in tsA-201 cells. We identified that alpha2delta-2 is completely concentrated in cholesterol-rich microdomains (lipid rafts) in cerebellum, in which it substantially colocalizes with the calcium channel alpha1 subunit CaV2.1, although CaV2.1 is also present in the Triton X-100-soluble fraction. In tsA-201 cells, unlike cerebellum, alpha2delta-2 is not completely proteolytically processed into alpha2-2 and delta-2. However, this processing is more complete in the lipid raft fraction of tsA-201 cells, in which alpha2delta-2 also colocalizes with CaV2.1. Cholesterol depletion of intact cells disrupted their lipid rafts and enhanced CaV2.1/alpha2delta-2/beta4 currents. Furthermore, alpha2delta-2 coimmunoprecipitates with lipid raft-associated proteins of the stomatin family. The apparent affinity of alpha2delta-2 for its ligand gabapentin is increased markedly in the cholesterol-rich microdomain fractions, in both cerebellum and the stable alpha2delta-2 cell line. In contrast, alpha2delta-2 containing a point mutation (R282A) has a much lower affinity for gabapentin, and this is not enhanced in the lipid raft fraction. This R282A mutant alpha2delta-2 shows reduced functionality in terms of enhancement of CaV2.1/beta4 calcium currents, suggesting that the integrity of the gabapentin binding site may be important for normal functioning of alpha2delta-2. Together, these results indicate that both alpha2delta-2 and CaV2.1 are normally associated with cholesterol-rich microdomains, and this influences their functionality.

The α1-β-Subunit Interaction That Modulates Calcium Channel Activity Is Reversible and Requires a Competent α-Interaction Domain

High voltage-gated calcium channels consist of a pore-forming subunit (alpha(1)) and three nonhomologous subunits (alpha(2)/delta, beta, and gamma). Although it is well established that the beta-subunit promotes traffic of channels to the plasma membrane and modifies their activity, the reversible nature of the interaction with the alpha(1)-subunit remains controversial. Here, we address this issue by examining the effect of purified beta(2a) protein on Ca(V)1.2 and Ca(V)2.3 channels expressed in Xenopus oocytes. The beta(2a)-subunit binds to the alpha(1)-interaction domain (AID) in vitro, and when injected into oocytes, it shifts the voltage dependence of activation and increases charge movement to ionic current coupling of Ca(V)1.2 channels. This increase depended on the integrity of AID but was not abolished by bafilomycin, demonstrating that the alpha(1)-beta interaction through the AID site can take place at the plasma membrane. Furthermore, injection of beta(2a) protein inhibited inactivation of Ca(V)2.3 channels and converted fast inactivating Ca(V)2.3/beta(1b) channels to slow inactivating channels. Inhibition of inactivation required larger concentration of beta(2a) in oocytes expressing Ca(V)2.3/beta(1b) channels than expressing Ca(V)2.3 alone but reached the same maximal level as expected for a competitive interaction through a single binding site. Together, our data show that the alpha(1)-beta interaction is reversible in intact cells and defines calcium channels beta-subunits as regulatory proteins rather than stoichiometric subunits.

The importance of occupancy rather than affinity of CaV(beta) subunits for the calcium channel I-II linker in relation to calcium channel function.

The Ca(V)beta subunits of voltage-gated calcium channels regulate the trafficking and biophysical properties of these channels. We have taken advantage of mutations in the tyrosine residue within the alpha interaction domain (AID) in the I-II linker of Ca(V)2.2 which reduce, but do not abolish, the binding of beta1b to the AID of Ca(V)2.2. We have found that the mutation Y388S decreased the affinity of Ca(V)beta1b binding to the Ca(V)2.2 I-II linker from 14 to 329 nm. However, the Y388S mutation had no effect on current density and cell surface expression of Ca(V)2.2/alpha2delta-2/beta1b channels expressed in human embryonic kidney tsA-201 cells, when equivalent proportions of cDNA were used. Furthermore, despite the 24-fold reduced affinity of Ca(V)beta1b for the Y388S I-II linker of Ca(V)2.2, all the key features of modulation as well as trafficking by Ca(V)beta subunits remained intact. This is in contrast to the much more marked effect of the W391A mutation, which abolished interaction with the Ca(V)2.2 I-II linker, and very markedly affected the trafficking of the channels. However, using the Xenopus oocyte expression system, where expression levels can be accurately titrated, when Ca(V)beta1b cDNA was diluted 50-fold, all evidence of interaction with Ca(V)2.2 Y388S was lost, although wild-type Ca(V)2.2 was still normally modulated by the reduced concentration of beta1b. These results indicate that high affinity interaction with the alpha1 subunit is not necessary for any of the modulatory effects of Ca(V)beta subunits, but occupancy of the interaction site is important, and this will occur, despite the reduced affinity, if the Ca(V)beta subunit is present in sufficient excess.

Molecular basis of inherited calcium channelopathies: role of mutations in pore-forming subunits1

The pore-forming alpha subunits of voltage-gated calcium channels contain the essential biophysical machinery that underlies calcium influx in response to cell depolarization. In combination with requisite auxiliary subunits, these pore subunits form calcium channel complexes that are pivotal to the physiology and pharmacology of diverse cells ranging from sperm to neurons. Not surprisingly, mutations in the pore subunits generate diverse pathologies, termed channelopathies, that range from failures in excitation-contraction coupling to night blindness. Over the last decade, major insights into the mechanisms of pathogenesis have been derived from animals showing spontaneous or induced mutations. In parallel, there has been considerable growth in our understanding of the workings of voltage-gated ion channels from a structure-function, regulation and cell biology perspective. Here we document our current understanding of the mutations underlying channelopathies involving the voltage-gated calcium channel alpha subunits in humans and other species.

Pregabalin for neuropathic pain based on recent clinical trials

Pregabalin is a ligand for the alpha-2-delta subunit of voltage-gated calcium channels with anticonvulsant, analgesic, and anxiolytic properties. It has predictable absorption across the gastrointestinal tract, is neither metabolized nor protein-bound, and has minimal drug-drug interactions. It is effective with two or three-times daily dosing in a dose range of 150 to 600 mg daily. Seven published prospective, randomized clinical trials in postherpetic neuralgia (PHN) and painful diabetic peripheral neuropathy (DPN) demonstrate pain relief, decreased sleep interference, and improvements in several secondary outcome measures. The 50% responder rates for PHN and DPN compare favorably with other first-line agents for neuropathic pain. Pregabalin is well tolerated in most patients with infrequent severe adverse effects. Pregabalin is an important addition to the treatment armamentarium for neuropathic pain.

Pregabalin Exerts Oppositional Effects on Different Inhibitory Circuits in Human Motor Cortex: A Double‐blind, Placebo‐controlled Transcranial Magnetic Stimulation Study

To explore acute effects of pregabalin (PGB) on human motor cortex excitability with transcranial magnetic stimulation (TMS). PGB, 600 mg/day, was orally administered in 19 healthy subjects twice daily in a randomized, double-blind, placebo-controlled crossover design. Several measures of motor cortex excitability were tested with single- and paired-pulse TMS. Mean short-interval intracortical inhibition (SICI) was reduced after PGB (74 +/- 7% of unconditioned response) compared with placebo (60 +/- 6% of unconditioned response). In contrast, mean long-interval intracortical inhibition (LICI) was increased by PGB (26 +/- 4% of unconditioned response) compared with placebo (45 +/- 8% of unconditioned response), and mean cortical silent period (CSP) showed an increase from 139 +/- 8 ms or 145 +/- 8 ms after placebo to 162 +/- 7 ms or 161 +/- 10 ms after PGB. Motor thresholds, intracortical facilitation, and corticospinal excitability were unaffected. The observed excitability changes with oppositional effects on SICI and LICI or CSP suggest gamma-aminobutyric acid (GABA)B-receptor activation. They are markedly distinct from those induced by gabapentin, although both PGB and gabapentin are thought to mediate their function by binding to the alpha2-delta subunit of voltage-gated calcium channels. Conversely, the TMS profile of PGB shows striking similarities with the pattern evoked by the GABA-reuptake inhibitor tiagabine.

Diverse Calcium Channel Types are Present in the Human Placental Syncytiotrophoblast Basal Membrane

The functional expression of calcium channels has been scarcely studied in human placental syncytiotrophoblast. We have presently sought to characterize Ca 2+ currents of the healthy syncytiotrophoblast basal membrane using purified basal membranes reconstituted in giant liposomes subjected to patch-clamp recordings. We detected presence of channels with high permeability to Ca 2+ (relative PCa/PK up to 99.5) using K + solutions in symmetric conditions. Recordings performed in Ba 2+ gradients showed Ba 2+-conducting channels in 100% of experiments. Ba 2+ total patch currents were consistently blocked by addition of NiCl 2, Nifedipine (L-type voltage-gated calcium channel blocker) or Ruthenium Red (TRPV5–TRPV6 channel blocker); Nifedipine and Ruthenium Red exerted a synergic blocking effect on Ba 2+ total patch currents. Immunohistochemistry of placental villi sections evidenced presence of α 1 subunit of voltage-gated calcium channels and TRPV5–TRPV6 channels in basal and apical syncytiotrophoblast plasma membranes; these three calcium channels were also detected in purified basal and apical fractions using Western blot. These results show the presence of three types of calcium channels in the syncytiotrophoblast basal membrane by both functional and molecular means. These basal membrane calcium channels would not be directly involved in mother-to-fetus Ca 2+ transport, but could participate in other relevant trophoblast processes, such as exocytosis and Ca 2+ transport regulation.

Calcium channel alpha 2-delta type 1 subunit is the major binding protein for pregabalin in neocortex, hippocampus, amygdala, and spinal cord: An ex vivo autoradiographic study in alpha 2-delta type 1 genetically modified mice

Pregabalin is a synthetic amino acid compound effective in clinical trials for the treatment of post-herpetic neuralgia, diabetic peripheral neuropathy, generalized anxiety disorder and adjunctive therapy for partial seizures of epilepsy. However, the mechanisms by which pregabalin exerts its therapeutic effects are not yet completely understood. In vitro studies have shown that pregabalin binds with high affinity to the alpha 2-delta (α 2-δ) subunits (Type 1 and 2) of voltage-gated calcium channels. To assess whether α 2-δ Type 1 is the major central nervous system (CNS) binding protein for pregabalin in vivo, a mutant mouse with an arginine-to-alanine mutation at amino acid 217 of the α 2-δ Type 1 protein (R217A mutation) was generated. Previous site-directed mutagenesis studies revealed that the R217A mutation dramatically reduces α 2-δ 1 binding to pregabalin in vitro. In this autoradiographic analysis of R217A mice, we show that the mutation to α 2-δ Type 1 substantially reduces specific pregabalin binding in CNS regions that are known to preferentially express the α 2-δ Type 1 protein, notably the neocortex, hippocampus, basolateral amygdala and spinal cord. In mutant mice, pregabalin binding was robust throughout regions where the α 2-δ Type 2 subunit mRNA is abundant, such as cerebellum. These findings, in conjunction with prior in vitro binding data, provide evidence that the α 2-δ Type 1 subunit of voltage-gated calcium channels is the major binding protein for pregabalin in CNS. Moreover, the distinct localization of α 2-δ Type 1 and mutation-resistant binding (assumed to be α 2-δ Type 2) in brain areas subserving different functions suggests that identification of subunit-specific ligands could further enhance pharmacologic specificity.

Biology and therapy of fibromyalgia. New therapies in fibromyalgia

Fibromyalgia is a chronic, musculoskeletal pain condition that predominately affects women. Although fibromyalgia is common and associated with substantial morbidity and disability, there are no US Food and Drug Administration-approved treatments. However, progress has been made in identifying pharmacological and non-pharmacological treatments for fibromyalgia. Recent pharmacological treatment studies have focused on selective serotonin and norepinephrine reuptake inhibitors, which enhance serotonin and norepinephrine neurotransmission in the descending pain pathways and lack many of the adverse side effects associated with tricyclic medications. Promising results have also been reported for medications that bind to the α2δ subunit of voltage-gated calcium channels, resulting in decreased calcium influx at nerve terminals and subsequent reduction in the release of several neurotransmitters thought to play a role in pain processing. There is also evidence to support exercise, cognitive behavioral therapy, education, and social support in the management of fibromyalgia. It is likely that many patients would benefit from combinations of pharmacological and non-pharmacological treatments, but more study is needed.

Pregabalina. Aportaciones de los ligandos 2 de canales de calcio en el tratamiento de la epilepsia y el dolor neuropático

To review and update the contributions of a new class of drugs, named calcium channel alpha2delta protein ligands, on the treatment of epilepsy and neuropathic pain. A novel class of anticonvulsants are ligands for the auxiliary-associated protein alpha2delta subunit of voltage-gated calcium channels in the central nervous system. Gabapentin and pregabalin are members of this group. Pregabalin is a higher-potency and higher-effective analogue of gabapentin that act as a potent ligand for this site. The anticonvulsant action of pregabalin is probably due to its ability to reduce neurotransmitter release from activated epileptogenic neurons, without demonstrated effects on GABAergic receptors or mechanisms. In well-done clinical trials, pregabalin 150-600 mg/day has been shown to be highly effective and well tolerated as adjunctive therapy in patients with partial seizures. In several randomized, double-blind, clinical trials, oral pregabalin 150-600 mg/day was superior to placebo in relieving neuropathic pain associated with diabetic neuropathy and postherpetic neuralgia. Pregabalin demonstrates in humans an extensive and rapid absorption and a highly predictable and linear pharmacokinetics, a profile that makes it easy to use in clinical practice. The pharmacological activity of pregabalin is similar but not identical to that of gabapentin, and pregabalin shows possible advantages. Pregabalin and calcium channel alpha2delta protein ligands showed relevant advances on epilepsy and neuropathic pain treatment. In peripheral neuropathic pain conditions, if the criteria for efficacy are based on both pain relief and quality of life measures, pregabalin/gabapentin are suggested as choice treatment.

Increase in Expression of α1 and α2/δ1 Subunits of L-Type High Voltage-Gated Calcium Channels After Sustained Ethanol Exposure in Cerebral Cortical Neurons

Previous reports revealed up-regulation of L-type high voltage-gated calcium channels (HVCCs) in mouse brains with ethanol physical dependence. We investigated mechanisms of enhancement of L-type HVCC function using mouse cerebrocortical neurons exposed to 50 mM ethanol for 3 days and the brains of mouse physically dependent on ethanol. Ethanol facilitated 30 mM KCl-stimulated 45Ca2+ influx in dose- and duration-dependent manners, which was abolished by nifedipine, an inhibitor specific to L-type HVCCs, but not by inhibitors for other types of HVCCs. Increase in [3H]PN200-110 binding to the particulate fractions from the ethanol-treated neurons was due to increased Bmax value with no changes in Kd value. Western blot analysis showed the increased expression of α1C, α1D, and α2/δ1 subunits with decreased β4 subunit expression and no changes in expressions of α1A, α1B, α1F, and α2 subunits. A similar pattern of the changes in the expression of these subunits of L-type HVCCs were observed in the cerebral cortex from mouse with ethanol physical dependence. These results indicate that sustained ethanol exposure to the neurons induces up-regulation of L-type HVCCs, which is due to increased expressions of α1C, α1D, and α2/δ1 subunits, and produces no alterations in P/Q- and N-type HVCC functions.

Targeting alpha2-delta calcium channel subunit for pain therapeutics

Voltage-gated calcium channels (VGCCs) play an important role in the transmission of nociceptive information. Previous studies have focused on targeting the α1 subunit of VGCCs, specifically the N-type and T-type VGCCs, for the development of analgesic drugs. New research has highlighted an underlying role for the auxiliary α2-δ subunit in in vivo models of neuropathic pain. Further evidence that the marketed pain therapies gabapentin and pregabalin target α2-δ subunits within the VGCC to pharmacologically modulate calcium channel signaling and attenuate nociceptive transmission, thus alleviating pain symptoms, has prompted the development of novel α2-δ ligands as candidate analgesics. This article will highlight promising novel compounds emerging in the recent literature.

Structure–activity relationships of α-amino acid ligands for the α 2δ subunit of voltage-gated calcium channels

A series of α-amino acids were identified as ligands which compete with gabapentin for binding to the α 2δ subunit of voltage-dependent Ca 2+ channels. Potent analogs were identified. Their activity in an in vivo pain assay is described.

Expedited SAR study of high-affinity ligands to the α 2δ subunit of voltage-gated calcium channels: Generation of a focused library using a solution-phase Sn2Ar coupling methodology

The SAR of the lead compound 3, a novel ligand for the α 2δ subunit of voltage-gated calcium channels, was rapidly explored. Utilizing a parallel solution-phase Sn2Ar coupling approach, a focused library was obtained. The library was evaluated in vitro and afforded a series of analogues with improved potencies. The SAR trends of the library are also described.

Interaction via a Key Tryptophan in the I-II Linker of N-Type Calcium Channels Is Required for β1 But Not for Palmitoylated β2, Implicating an Additional Binding Site in the Regulation of Channel Voltage-Dependent Properties

The CaVbeta subunits of voltage-gated calcium channels regulate these channels in several ways. Here we investigate the role of these auxiliary subunits in the expression of functional N-type channels at the plasma membrane and in the modulation by G-protein-coupled receptors of this neuronal channel. To do so, we mutated tryptophan 391 to an alanine within the alpha-interacting domain (AID) in the I-II linker of CaV2.2. We showed that the mutation W391 virtually abolishes the binding of CaVbeta1b and CaVbeta2a to the CaV2.2 I-II linker and strongly reduced current density and cell surface expression of both CaV2.2/alpha2delta-2/beta1b and/beta2a channels. When associated with CaVbeta1b, the W391A mutation also prevented the CaVbeta1b-mediated hyperpolarization of CaV2.2 channel activation and steady-state inactivation. However, the mutated CaV2.2W391A/beta1b channels were still inhibited to a similar extent by activation of the D2 dopamine receptor with the agonist quinpirole. Nevertheless, key hallmarks of G-protein modulation of N-type currents, such as slowed activation kinetics and prepulse facilitation, were not observed for the mutated channel. In contrast, CaVbeta2a was still able to completely modulate the biophysical properties of CaV2.2W391A channel and allow voltage-dependent G-protein modulation of CaV2.2W391A. Additional data suggest that the concentration of CaVbeta2a in the proximity of the channel is enhanced independently of its binding to the AID by its palmitoylation. This is essentially sufficient for all of the functional effects of CaVbeta2a, which may occur via a second lower-affinity binding site, except trafficking the channel to the plasma membrane, which requires interaction with the AID region.

Tuning of RNA editing by ADAR is required in Drosophila

RNA editing increases during development in more than 20 transcripts encoding proteins involved in rapid synaptic neurotransmission in Drosophila central nervous system and muscle. Adar (adenosine deaminase acting on RNA) mutant flies expressing only genome-encoded, unedited isoforms of ion-channel subunits are viable but show severe locomotion defects. The Adar transcript itself is edited in adult wild-type flies to generate an isoform with a serine to glycine substitution close to the ADAR active site. We show that editing restricts ADAR function since the edited isoform of ADAR is less active in vitro and in vivo than the genome-encoded, unedited isoform. Ubiquitous expression in embryos and larvae of an Adar transcript that is resistant to editing is lethal. Expression of this transcript in embryonic muscle is also lethal, with above-normal, adult-like levels of editing at sites in a transcript encoding a muscle voltage-gated calcium channel.

Efficacy of Pregabalin in the Treatment of Generalized Anxiety Disorder

Pregabalin is a new anxiolytic that acts as a presynaptic inhibitor of the release of excessive levels of excitatory neurotransmitters by selectively binding to the alpha2-delta subunit of voltage-gated calcium channels. The current study evaluated the anxiolytic efficacy of BID versus TID dosing of pregabalin in patients with generalized anxiety disorder. Outpatients with Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition generalized anxiety disorder and having baseline Hamilton Anxiety (HAM-A) total scores > or =20 were randomized to 6 weeks of double-blind treatment with pregabalin 200 mg/d (BID; N = 78), 400 mg/d (BID; N = 89), or 450 mg/d (TID; N = 88) or placebo (N = 86). Mean improvement in HAM-A total score at last observation carried forward end point was significantly greater on pregabalin 200 (P = 0.006), 400 (P = 0.001), and 450 mg/d (P = 0.005) compared with placebo. Pairwise comparisons of BID versus TID dosing found no difference in HAM-A change score at end point. All 3 pregabalin dosage groups showed significantly greater efficacy versus placebo at end point on the HAM-A psychic and somatic anxiety factor scores. Improvement on both factors was rapid: significance versus placebo was achieved as early as the first assessment at week 1, with > or =30% reduction in HAM-A severity and equal or greater improvement for every subsequent visit in > or =38% of patients in all 3 pregabalin dosage groups (P < or = 0.001). Pregabalin was well tolerated, and despite the fixed-dose study design, discontinuations caused by adverse events ranged from 9% to 13%--comparable with that observed with placebo (8%). This study demonstrates that pregabalin is an effective treatment of generalized anxiety disorder, with BID dosing showing similar efficacy and comparable tolerability with TID dosing.

Novel Cyclopropyl β-Amino Acid Analogues of Pregabalin and Gabapentin That Target the α2-δ Protein

As part of a program aimed at generating compounds with affinity for the alpha(2)-delta subunit of voltage-gated calcium channels, several novel beta-amino acids were prepared using an efficient nitroalkane-mediated cyclopropanation as a key step. Depending on the ester that was chosen, the target amino acids could be prepared in as few as three steps. The cyclopropyl amino acids derived from ketones proved to be potent binders of the alpha(2)-delta subunit of voltage-gated calcium channels, but did not interact with the large neutral amino acid system L (leucine) transporter. Anticonvulsant effects were observed in vivo with compound 34 but only after intracerebroventricular (icv) administration, presumably due to inadequate brain concentrations of the drug being achieved following oral dosing. However, pregabalin 1 was active in the DBA/2 model after oral (and icv) dosing, supporting a hypothesis that active transport is a prerequisite for such zwitterionic species to cross the blood-brain barrier.

Comparison of the Antinociceptive Profiles of Gabapentin and 3-Methylgabapentin in Rat Models of Acute and Persistent Pain: Implications for Mechanism of Action

The anticonvulsant gabapentin (GBP) has been shown effective for the treatment of neuropathic pain, although its mechanism of action remains unclear. A recent report has suggested that binding to the alpha(2)delta subunit of voltage-gated calcium channels contributes to its antinociceptive effect, based on the stereoselective efficacy of two analogs: (1S,3R)3-methylgabapentin (3-MeGBP) (IC(50) = 42 nM), which is effective in neuropathic pain models; and (1R,3R)3-MeGBP (IC(50) > 10,000 nM), which is ineffective (Field et al., 2000). The present study was designed to further examine the profiles of GBP and 3-MeGBP in rat models of acute and persistent pain. Systemic administration of GBP or (1S,3R)3-MeGBP inhibited tactile allodynia in the spinal nerve ligation model of neuropathic pain, whereas (1R,3R)3-MeGBP was ineffective. The antiallodynic effect of GBP, but not (1S,3R)3-MeGBP, was blocked by i.t. injection of the GABA(B) receptor antagonist [3-[[(3,4-dichlorophenyl)methyl]amino]propyl](diethoxymethyl)phosphinic acid (CGP52432). Systemic GBP or (1S,3R)3-MeGBP also inhibited the second phase of formalin-evoked nociceptive behaviors, whereas (1R,3R)3-MeGBP was ineffective. However, both (1S,3R)3-MeGBP and (1R,3R)3-MeGBP, but not GBP, inhibited first phase behaviors. In the carrageenan model of inflammatory pain, systemic GBP or (1R,3R)3-MeGBP failed to inhibit thermal hyperalgesia, whereas (1S,3R)3-MeGBP had a significant, albeit transient, effect. Systemic (1S,3R)3-MeGBP, but not GBP or (1R,3R)3-MeGBP, also produced an antinociceptive effect in the warm water tail withdrawal test of acute pain. These data demonstrate that GBP and 3-MeGBP display different antinociceptive profiles, suggesting dissimilar mechanisms of antinociceptive action. Thus, the stereoselective efficacy of 3-MeGBP, presumably related to alpha(2)delta binding, likely does not completely account for the mechanism of action of GBP.

Structure−Activity Relationships of Pregabalin and Analogues That Target the α2-δ Protein

Pregabalin exhibits robust activity in preclinical assays indicative of potential antiepileptic, anxiolytic, and antihyperalgesic clinical efficacy. It binds with high affinity to the alpha(2)-delta subunit of voltage-gated calcium channels and is a substrate of the system L neutral amino acid transporter. A series of pregabalin analogues were prepared and evaluated for their alpha(2)-delta binding affinity as demonstrated by their ability to inhibit binding of [(3)H]gabapentin to pig brain membranes and for their potency to inhibit the uptake of [(3)H]leucine into CHO cells, a measure of their ability to compete with the endogenous substrate at the system L transporter. Compounds were also assessed in vivo for their ability to promote anxiolytic, analgesic, and anticonvulsant actions. These studies suggest that distinct structure activity relationships exist for alpha(2)-delta binding and system L transport inhibition. However, both interactions appear to play an important role in the in vivo profile of these compounds.