Pregabalin action at a model synapse: Binding to presynaptic calcium channel α2-δ subunit reduces neurotransmission in mice

European Journal of Pharmacology 553 (2006) 82 – 88 www.elsevier.com/locate/ejphar

Pregabalin action at a model synapse: Binding to presynaptic calcium channel α2-δ subunit reduces neurotransmission in mice☆ Indu Joshi, Charles P. Taylor ⁎ Department CNS Biology, Pfizer Global R&D, 2800 Plymouth Road, Ann Arbor, MI 48105, USA Received 13 April 2006; received in revised form 6 September 2006; accepted 8 September 2006 Available online 23 September 2006

Abstract Pregabalin, ((S)-3-(aminomethyl)-5-methylhexanoic acid, also known as (S)-3-isobutyl GABA, Lyrica™) is approved for treatment of certain types of peripheral neuropathic pain and as an adjunctive therapy for partial seizures of epilepsy both the EU and the USA and also for generalized anxiety disorder in the EU. Though pregabalin binds selectively to the alpha2-delta (α2–δ) auxiliary subunit of voltage-gated calcium channels, the cellular details of pregabalin action are unclear. The high density of α2−δ in skeletal muscle fibers raises the question of whether pregabalin alters excitation–contraction coupling. We used the mouse soleus neuromuscular junction from mice containing an artificially mutated α2–δ Type 1 protein (R217A) as a model to examine the effect of pregabalin. Pregabalin reduced nerve-evoked muscle contractions by 16% at a clinically relevant concentration of 10 μM in wildtype mice. When acetylcholine receptors were blocked with curare, pregabalin had no effect on contraction from direct stimulation of muscle, suggesting a lack of drug effects on contraction coupling. Our data are consistent with pregabalin having no effect on striated muscle L-type calcium channel function. However, in mice expressing mutant (R217A) α2–δ Type 1, there was no significant effect of pregabalin on nerve-evoked muscle contraction. We propose that pregabalin reduces presynaptic neurotransmitter release without altering postsynaptic receptors or contraction coupling and that these effects require high affinity binding to α2–δ Type 1 auxiliary subunit of presynaptic voltage-gated calcium channels. © 2006 Elsevier B.V. All rights reserved. Keywords: Presynaptic; Calcium channel; Analgesia; Anticonvulsant; Anxiolytic; Transmitter release

1. Introduction Pregabalin has recently been approved and marketed for the treatment of peripheral neuropathic pain and epilepsy in the European Union and in the USA for neuropathic pain from postherpetic neuralgia, diabetic neuropathy and as adjunctive treatment for partial seizures. It recently has received approval in EU for treatment of generalized anxiety disorder. It is effective and well tolerated in several large randomized and placebocontrolled clinical trials for each of these indications (Beydoun et al., 2005; Dworkin et al., 2003; Lesser et al., 2004; Pande et al., 2003; Pande et al., 2004; Pohl et al., 2005; Richter et al., 2005; Rickels et al., 2005; Rosenstock et al., 2004).

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Disclosure: Authors are full-time employees of Pfizer Global R&D. ⁎ Corresponding author. Tel.: +1 734 622 7017; fax: +1 734 622 7178. E-mail address: [email protected] (C.P. Taylor).

0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.09.019

Pregabalin (Lyrica™) is a close structural relative of gabapentin (Neurontin™), both being alkylated analogues of γaminobutyric acid (GABA), the primary inhibitory neurotransmitter in the central nervous system (Bryans and Wustrow, 1999). However, neither compound interacts with GABAA or GABAB receptors in radioligand binding assays, nor does pregabalin alter physiological responses to GABAA or GABAB ligands in cultured neurons or recombinant receptor systems, indicating that neither compound interacts with pre- or postsynaptic GABA receptors. Furthermore, pregabalin and gabapentin are not blockers or substrates for GABA transporters (Su et al., 2005) and neither compound alters rat whole-brain GABA concentrations (Errante and Petroff, 2003). The exact cellular mechanism of action of pregabalin is still unclear, although evidence from several studies suggests that it reduces excitatory neurotransmitter release in a manner similar to the structurally related compound, gabapentin (Cunningham et al., 2004; Dooley et al., 2002; Dooley et al., 2000a; Dooley et al., 2000b; Fehrenbacher et al., 2003; Fink et al., 2002;

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Maneuf et al., 2001) by binding to the α2–δ auxiliary subunit (Gee et al., 1996) of voltage-gated calcium channels in brain and spinal cord. Calcium channels purified from native tissues contain the main calcium conducting pore protein (α1) and also several auxiliary proteins, including a cytosolic β subunit, and transmembrane α2−δ and γ subunits (see Arikkath and Campbell, 2003 for a review of calcium channel auxiliary subunits). Calcium channel α1 subunits are products of 10 separate genes, and these different channels are expressed in a heterogeneous manner in different brain and peripheral tissues (Yu and Catterall, 2004). Most of the various calcium channel α1 type proteins are thought to associate with a single α2−δ protein in vivo. There are two different genes (CACNA2D1, CACNA2D2 or α2−δ Types 1 and 2, respectively) that each encode closelyrelated α2−δ proteins that have high affinity binding sites for gabapentin and pregabalin (Hobom et al., 2000; Klugbauer et al., 2003). Two other α2−δ isoforms (CACNA2D3, CAC NA2D4) appear to lack these high affinity binding sites (Marais et al., 2001; Qin et al., 2002). Several studies suggest that the major isoform of α2−δ in neocortex, spinal cord dorsal horn and skeletal muscle is α2−δ Type 1 (Bian et al., 2006; Cole et al., 2005). Generation of a knockout mouse strain that lacks protein expression of α2−δ protein would be a good test of the contribution of this binding site to pregabalin pharmacology. However, knockouts of α2−δ Type 1 are lethal in mice at birth (J. Offord, unpublished observations) and spontaneous genetic mutant mice that are functional knockouts of α2−δ Type 2 have frequent seizures, ataxia, tremors and short lifespan (Brill et al., 2004), which compromises their use as an experimental tool. To avoid the difficulties with α2−δ knockout mice and to investigate the contribution of α2−δ drug binding to pharmacology of pregabalin, a genetically-modified mouse was generated with an arginine-to-alanine mutation at amino acid 217 in the α2–δ Type 1 protein gene sequence (GenBank accession no. NM_009784, Mus musculus, voltage-dependent calcium channel alpha2–delta subunit (Type 1)) (Bian et al., 2006; Bramwell et al., 2004; Li et al., 2003). This mutation reduced [3H]pregabalin binding in forebrain, without altering the abundance of α2−δ protein expression, or causing any obvious changes in mouse phenotype or behavior (Bian et al., 2006). The α2–δ Type 1 subunit is expressed at high levels in skeletal, cardiac and vascular smooth muscle and in the brain (Angelotti and Hofmann, 1996) and can associate with several different α1 subunit isoforms. Heterologous studies in which α2–δ subunits have been co-expressed with various calcium channel α1 subunits show that α2–δ increases the expression level of functional calcium channels in the membrane, and alters the voltage dependence and kinetics of calcium currents (Arikkath and Campbell, 2003). It has been reported that gabapentin binds to α2–δ protein from brain and skeletal muscle with similar kinetics and high affinity (Kd = 38 nM in brain and 29 nM in skeletal muscle (Gee et al., 1996; Suman-Chauhan et al., 1993)). However, the effects of high-affinity α2−δ drug ligands on skeletal muscle function have not been previously examined. The high density of α2−δ protein in skeletal muscle fibers (Gee et al., 1996) therefore raises questions of whether pregabalin alters skeletal muscle physiological processes such

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as excitation–contraction coupling (ECC) or if its actions are limited within the neuromuscular junction and other synapses. Using the mouse neuromuscular junction as a model system, this study tests the hypotheses that pregabalin-induced reduction in neurotransmission requires high-affinity binding to the α2–δ protein and that pregabalin does not alter skeletal muscle excitation–contraction coupling. 2. Materials and methods 2.1. R217A mutant mice A strain of mutant mice with the R217A mutation to α2−δ Type 1 that was previously shown to reduce [3H]gabapentin binding in a recombinant system (Wang et al., 1999), has been fully described in a separate communication (Bian et al., 2006). Briefly, a polymerase chain reaction fragment with the R217A mutation to the α2−δ Type 1 gene was generated from a mouse genomic DNA library. This fragment was introduced, through homologous recombination, into a yeast strain carrying a mouse genomic library. A clone containing a portion of the α2−δ Type 1 genomic DNA with the mutation was placed into a neo cassette to generate the targeting construct used to create the mutant mice. This targeting vector was introduced into 129/SvEvBRD embryonic stem cells via electroporation and homologous recombination. Selected clones were implanted into blastocytes derived from C57Bl/6 albino mice and transferred into C57Bl/6 pseudo-pregnant dams. The offspring chimeras were bred with C57Bl/6 mice. The pups with the mutation were then bred with 129/SvEv mice to generate a hybrid heterozygous colony that was inbred to obtain individually genotyped mice (either homozygous R217A mutant or homozygous wildtype) used in this study. Animal care and surgical procedures were conducted in accordance with the Declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals as adopted by the National Institutes of Health. The American Association for the Accreditation of Laboratory Animal Care accredited housing facilities. An internal animal use committee approved all experimental procedures. 2.2. In vitro isometric tension measurements Mice (male about 2–3 months) were anesthetized and killed by decapitation. The soleus muscle and nerve were removed and placed into oxygenated (95% O2 and 5% CO2) Tyrode's solution (in mM; NaCl 126, KCl 3.5, MgSO4 1, CaCl2 2, NaHCO3 26, Na2HPO4 1.25, glucose 10) at 35 °C for 1 h. The distal muscle tendon was securely fastened with silk thread and tied to an isometric force transducer (Radnoti). The nerve was taken up into a polyethylene suction pipette for nerve electrical stimulation (0.5 ms, 10–20 μA). Bipolar wire electrodes were used for direct muscle stimulation (0.5 ms, 100–1000 μA) in the presence of the cholinergic receptor blocker, curare. Indicated drugs were added to oxygenated superfused solutions. Tension responses (in mg) were digitized and recorded using Clampex 9 software (Axon Instruments).

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Fig. 1. Pregabalin reduced contraction strength in wild type and not R217A muscle preparations. A. Soleus muscle tension measurements were made in response to a 5 Hz, 10 s train of stimuli to the nerve in wildtype and R217A mice before and after a 15 min bath application of 100 μM pregabalin. B. Representative graph of normalized first peak amplitudes to control demonstrates that 100 μM pregabalin reduced responses by 35% in wildtype mouse tissues with little effect in tissues from R217A mutant mice. C. Responses to a 15 min bath application of pregabalin were concentration-dependent in wildtypes (n = 8) while higher pregabalin concentrations had only nonsignificant effects in muscle preparations from R217A (n = 8) mice.

2.3. Statistical analysis Data were expressed as a percent change from pre-treatment (baseline) and represented as mean ± standard error. The Sigma Stat® software package (version 3.1) was employed for statistical analysis. The mean response of each strain was compared with a one-way ANOVA followed by the Dunnett's test to compare mean response of each group to the mean response of the R217A group. The level of statistical significance for all tests was p = 0.05. 3. Results 3.1. Pregabalin reduces muscle contraction only with high-affinity binding to α2–δ In order to measure muscle tension consistently in soleus muscle preparations, the soleus nerve was directly stimulated with a 5 Hz stimulus train of constant-current pulses (0.05 to 1 μA) for 10 s (each pulse was 0.5 ms duration). This stimulus protocol was applied every 5 min for about 20 min to establish a steady baseline after which 100 μM pregabalin was bath applied. In six wildtype muscles studied, pregabalin reduced muscle contraction by 30% (example in Fig. 1A) as indicated by a decrease in peak amplitudes of all responses within the train. In six muscles taken from R217A mice where the α2–δ Type 1 protein was mutated, pregabalin caused no apparent change in muscle contraction (example in Fig. 1A). As all peak responses in the train were affected in the wildtype muscles, the first peak amplitude of each train was measured during control (15 min) and pregabalin application (20 min) and plotted as a percent of

the first control response (Fig. 1B). Reduction in muscle contraction observed in wildtype mice and not in the R217A mice is therefore likely due to high affinity binding of pregabalin to α2–δ, which is selectively reduced in R217A mice. To delineate the efficacy of pregabalin, varying doses were bath applied to the soleus muscle. In wild type mice, 10 and 100 μM concentrations caused incremental decreases in muscle contraction as depicted by the average of first peak responses (n = 8) as seen in Fig. 1C. This concentration range is within exposures of pregabalin at therapeutic dosages, which are 1–6 μg/ml in plasma or 6.3–37.5 μM. Even at higher concentrations (100 μM to 1 mM), pregabalin did not produce significant reductions in muscle contraction in nerve-muscle preparations from R217A mice (Fig. 1C). Such high concentrations of pregabalin did not further reduce muscle contraction in wildtype muscles indicating that 100 μM was a saturating concentration, sufficient for assessing the maximal action of pregabalin (data not shown). 3.2. Lack of response to pregabalin in R217A muscle is not due to genetic contributions from mouse parent strains As described in the METHODS, the R217A mice were generated from three different parent strains (129S6/SvEV, C57BL/6J-Tyr c-2J/J albino, and C57Bl/65 wildtype). Because the mice are hybrid, the mutation at the α2−δ site in the R217A mice is the only genetic component that is uniform within individual homozygous mutant hybrid mice. Therefore genetic heterogeneity between individual hybrid mice could potentially contribute to the lack of effect of pregabalin in hybrid R217A mice. To test this, 100 μM pregabalin was bath applied to soleus muscles prepared from each of the three parent strains (n = 4 each) as described in the previous section. To monitor responses, a stimulation protocol of 2 Hz for 4 s at 2-min intervals between stimuli was given without drug for 14 min, followed by pregabalin for 16 min and then drug washout. This was repeated for all parent strains, the original wildtype hybrid mice and R217A hybrid mice. When the first peak amplitude of the train exposed to pregabalin (after 16 min) was compared to the first peak amplitude of the train in control, a similar reduction in muscle contraction was observed in all three parental strains, and reductions in all other strains were significantly different from results observed in R217A mice (Fig. 2, significance based on ANOVA p b 0.001 and Dunnett's test, see Materials and

Fig. 2. Pregabalin induced reductions in muscle contraction in all mouse strains except the R217A mutant. Pregabalin (100 μM) caused similar reductions in nerve stimulated muscle contraction in tissues from all three parent strains of wildtype mice, but not in R217A mutant mice (n = 5 for all types; ANOVA, p b 0.05 for comparison of each strain to R217A group).

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methods). This suggests that the lack of response of pregabalin in the R217A tissues is not due to the differences in drug sensitivity caused by genetic contributions from any of the parent strains. The extent of reduced muscle contraction in wildtype muscles was 80% as opposed to the 65% in Fig. 1B. Though this is likely due to a shorter interval between stimuli, this data further support pregabalin's action to reduce muscle contraction in wildtype mice but not in the R2176A mice. 3.3. Pregabalin does not alter excitation–contraction coupling in skeletal muscle (drug action is presumed at α2–δ located within the neuromuscular junction) The effect of pregabalin on nerve-evoked muscle contractions was unlikely to be postsynaptic, since pregabalin was inactive at a concentration of 100 μM to displace radioligand binding in an assay of nicotinic acetylcholine receptor function (data not shown). Furthermore, pregabalin had no effect on nerve-evoked contractions in mutant R217A mice (that possess wildtype acetylcholine receptors) confirming that pregabalin has no effect on cholinergic receptor function. However, voltagegated calcium channels are also associated with the skeletal muscle excitation–contraction coupling complex. The skeletal muscle dihydropyridine receptor serves dual functions, as a voltage sensor for excitation−contraction coupling through the directly associated ryanodine receptor proteins and also as an Ltype calcium channel (Rios and Brum, 1987; Tanabe et al., 1987). To determine whether pregabalin altered this postsynaptic coupling complex, curare (5 μM) was applied to the muscle to block postsynaptic acetylcholine receptors and disable neuromuscular synaptic junctions. In curare, excitation–contraction coupling could be monitored by measuring muscle contraction when the muscle fibers were directly stimulated with bipolar wire electrodes. Muscles were directly stimulated at 5 Hz (10 s train) at a superthreshold intensity for contraction (approximately 500 μA) every 5 min without (control) and with pregabalin application as above. Pregabalin did not alter muscle contraction in wildtype mice (Fig. 3A; ANOVA p b 0.001). This suggests that pregabalin induced reduction in muscle contraction as seen when the nerve was directly stimulated requires synaptic transmission and that the pregabalin effect on nerve-stimulated contractions was not caused by an action on excitation–

Fig. 3. Pregabalin has little effect when the muscle was directly stimulated. Pregabalin (in the presence of the acetylcholine receptor blocker curare, 5 μM) did not alter muscle contraction when the muscle was directly stimulated in wildtype mice. This suggests that synaptic transmission is required for pregabalin effect and that pregabalin does not alter excitation–contraction coupling.

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contraction coupling or the muscle contraction apparatus (Dulhunty et al., 2002). Furthermore, these data in combination with evidence that pregabalin does not alter muscle contraction in R217A mice suggest that pregabalin is not acting postsynaptically either on cholinergic receptors or on muscle excitation–contraction coupling. Rather, pregabalin is likely to be acting presynaptically. A recent study using dysgenic myotubes has shown that α2–δ Type 1 is associated with L-type calcium channels (located in muscle t-tubules) but is not essential for skeletal muscle excitation contraction coupling (Obermair et al., 2005). When the L-type calcium channel blocker isradipine (10 μM to allow both functional channel block (Triggle, 2006) and muscle tissue penetration) was applied to soleus muscles of both wildtype and R217A mice while following a similar direct nerve stimulation protocol, the extent of contraction reduction following 30 min of isradipine exposure was similar in both wildtype and R217A muscles (data not shown). This suggests that the mutation to α2–δ Type 1 on L-type calcium channel within muscle fibers does not have any significant effects on muscle contraction per se. 4. Discussion Null-mutant mice are available for all skeletal muscle isoforms of calcium channel subunits except for α2–δ Type 1 (Arikkath and Campbell, 2003). α2–δ Type 1 null-mutants die during early embryonic development, indicating the importance of α2–δ Type 1 for normal development. The generation of R217A mice, where a point mutation renders decreased drug binding affinity at α2–δ Type 1, has therefore been invaluable in understanding the pharmacology and physiology of high affinity α2–δ Type 1 ligand drugs. The results in this study are novel as the effect of pregabalin on α2–δ Type 1 at skeletal muscle neuromuscular junctions and in excitation–contraction coupling have been assessed for the first time. Our results indicate that pregabalin reduces contractions slightly but significantly in wildtype neuromuscular preparations by binding with high affinity to the α2–δ Type 1 subunit. Although the extent of reduced muscle contraction in response to pregabalin is quite small at a saturating concentration (100 μM), this is not surprising since there have been no reports that pregabalin and gabapentin cause muscle weakness as side effects (Arroyo et al., 2004; Dworkin et al., 2003). Our finding that 100 μM pregabalin is required for a saturating (maximum) effect agree with findings from other assays of neurotransmitter release, where 50% maximal effects occurred near 10 μM pregabalin (Dooley et al., 2000a). Although 100 μM is near the maximal pregabalin clinical plasma drug concentration (10 μg/mL or 67 μM), it is higher than the KI for radioligand binding of 20 nM (Taylor et al., 1993). This pronounced difference may be explained in part by the lack of divalent cations and endogenous ligands (e.g. L-leucine) in radioligand binding experiments (Brown et al., 1998; Dissanayake et al., 1997). Our results indicate a presynaptic neuromuscular action of pregabalin since only presynaptically triggered responses were reduced by pregabalin (contractions from transmural muscle

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stimulation in curare were not altered). The fact that pregabalin was unable to reduce muscle contraction in R217A skeletal muscles not only confirms the requirement of α2–δ Type 1 for pregabalin binding in skeletal muscles but also supports this preparation as a convenient and robust model to study α2-δ function. In addition, the inability of pregabalin to reduce muscle contraction in tissues from R217A mice cannot be attributed to unknown genetic factors since each of 3 parent strains show similar results to the wildtype hybrid mice. Though our results implicate a presynaptic site of action for pregabalin, we also explored whether pregabalin had any effects on excitation–contraction coupling. The dihydropyridine receptor or calcium channel CaV1.1 protein is one of the key constituents of the excitation–contraction coupling apparatus in skeletal muscle. The dihydropyridine receptor, which supports the L-type calcium current within T-tubules is a high voltageactivated calcium channel which functions as a voltage sensor in excitation–contraction coupling and possesses α2–δ Type 1 as an obligate auxiliary subunit (Wolf et al., 2003). Though one study has implicated a role for α2–δ Type 1 in excitation– contraction coupling (Alden and Garcia, 2002), a more recent study using dysgenic myotubes where the α2–δ Type 1 subunit was genetically silenced showed that the α2–δ Type 1 subunit is not required for skeletal muscle targeting of calcium channels or for the functional role of CaV1.1 calcium channels in activating skeletal muscle excitation–contraction coupling (Obermair et al., 2005). Results in the latter study are consistent with reports that use of gabapentin and pregabalin is not associated with side effects of muscle weakness when used to treat epilepsy or neuropathic pain (Arroyo et al., 2004; Dworkin et al., 2003). Our findings of a slight (15%) reduction of nerveinduced contractions by pregabalin suggest that spinal reflexes and other compensatory mechanisms are sufficient to overcome the relatively small decreases in skeletal muscle contraction strength that might be expected in vivo with pregabalin treatment. Our results showed that pregabalin has no effect on striated muscle excitation–contraction coupling, since muscle contraction in the presence of curare was not altered by pregabalin. Additionally, the L-type calcium channel blocker, isradipine, had equal effects on excitation–contraction coupling in wildtype and R217A muscles. Based on our results in this study we propose that pregabalin reduces presynaptic neurotransmitter release without altering postsynaptic receptor or muscle function and that these presynaptic effects require high affinity binding to α2–δ Type 1. A number of studies have used electrophysiology and neurotransmitter overflow measurements to show that gabapentin and pregabalin reduce the release of neurotransmitter in neuronal synapses. The neurotransmitters that have been studied to date include glutamate (Brown and Randall, 2005; Cunningham et al., 2004; Dooley et al., 2000b; Maneuf et al., 2001; Micheva et al., 2006), substance P and calcitonin gene-related peptide (Fehrenbacher et al., 2003), noradrenaline (Dooley et al., 2002; Dooley et al., 2000a), serotonin (Schlicker et al., 1985) inhibitory glycine (Bayer et al., 2004) and GABA (Micheva et al., 2006). No studies have examined the role of pregabalin on acetylcholine release in the neuromuscular junction prior to this

study. Our preliminary analyses indicate that pregabalin decreases endplate potential amplitudes recorded from neuromuscular junctions in wildtype muscles (unpublished observations). Additional experiments will be needed however, to verify that pregabalin has no effect on postsynaptic cholinergic receptor responses, and also to more fully characterize the nature of the presynaptic reduction of transmitter release. Several reports have indicated that pregabalin and gabapentin act to reduce cellular calcium influx or calcium currents mediated by voltage-gated calcium channels in neurons (Fink et al., 2002; Fink et al., 2000; Li et al., 2006; Martin et al., 2002; van Hooft et al., 2002), while other studies suggest that gabapentin has no effect on calcium currents measured at cell bodies (Canti et al., 2004; Schumacher et al., 1997; van Hooft et al., 2002). This contradiction, which also remains to be resolved, could be studied using microscopic imaging of calcium fluorescent probes at the presynaptic terminal of neuromuscular junctions. Such studies might determine whether the presynaptic mechanism of pregabalin (mediated by binding to α2–δ Type 1) involves reduced influx of calcium through channels, or alternatively, whether other intracellular signalling mechanisms are involved. The present experiments show that the mouse skeletal muscle neuromuscular synapse is a suitable model for further studies of α2−δ mediated pharmacological function. Our results are consistent with a presynaptic action of pregabalin at α2–δ Type 1 binding sites that are functionally coupled to subtle decreases in release of neurotransmitters. We propose that pregabalin is likely to have similar presynaptic effects at synapses in the brain and spinal cord, where anticonvulsant and analgesic actions are thought to be mediated. In addition, our results indicate that pregabalin has no effect on skeletal muscle excitation–contraction coupling mediated by L-type calcium channels within muscle fibers. Acknowledgements Indu Joshi is the recipient of a Pfizer Global R&D postdoctoral fellowship. Thanks to James Offord, Ti-Zhi Su, and Susan Lotarski for the generation, production and assistance in obtaining the mutant R217A mice. References Alden, K.J., Garcia, J., 2002. Dissociation of charge movement from calcium release and calcium current in skeletal myotubes by gabapentin. Am. J. Physiol., Cell Physiol. 283, C941–C949. Angelotti, T., Hofmann, F., 1996. Tissue-specific expression of splice variants of the mouse voltage-gated calcium channel alpha2/delta subunit. FEBS Lett. 397, 331–337. Arikkath, J., Campbell, K.P., 2003. Auxiliary subunits: essential components of the voltage-gated calcium channel complex. Curr. Opin. Neurobiol. 13, 298–307. Arroyo, S., Anhut, H., Kugler, A.R., Lee, C.M., Knapp, L.E., Garofalo, E.A., Messmer, S., 2004. Pregabalin add-on treatment: a randomized, doubleblind, placebo-controlled, dose-response study in adults with partial seizures. Epilepsia 45, 20–27. Bayer, K., Seifollah, A., Zeilhofer, H.U., 2004. Gabapentin may inhibit synaptic transmission in the mouse spinal cord dorsal horn through a preferential block of P/Q-type Ca2+ channels. Neuropharmacology 46, 743–749.

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