Introduction

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Voltage gated calcium channels are multisubunit complexes that permit the influx of calcium ions into cells after changes in the plasma membrane potential. A number of voltage gated calcium channels exist, which can be broadly grouped into high-voltage (HVA) and low-voltage activated (LVA) channels. The HVA channels can be further subgrouped as L-, R-, P/Q-, and N-type, depending on their biophysical characteristics. The channels consist minimally of an ₁ pore protein that conducts current, contains the voltage sensor, and is the target of several drugs. Seven genes have been identified for the ₁ subunits of HVA channels and three for LVA channels (for reviews, see Hofmann et al., 1999; Lacinová et al., 2000). Although it is known that HVA channels are modulated by accessory subunits, it remains to be unequivocally established whether the LVA channels are associated with other proteins. The HVA auxiliary subunits are  (four genes), ₂ (three), and  (six). Most of these subunits have splice variants, giving rise to an even larger number of possible channel combinations and behaviors. Mutations of several channel proteins have been shown to be a causative factor in neurological disorders, making the calcium channel subunits target for therapeutic interventions (Burgess and Noebels, 1999).

The ₂ family consists of three genes. The first subunit identified was ₂-1 in rabbit skeletal muscle (Ellis et al., 1988). Five tissue-specific splice variants exist (Angelotti and Hofmann, 1996), but a functional significance of the splicing has not been established. Two new ₂ family members were subsequently identified in human and mouse, and were named ₂-2 and -3 (Klugbauer et al., 1999). The novel ₂ subunits are 56 and 30% homologous to ₂-1 at the amino acid level and share a number of structural motifs. The subunits have similar hydrophobicity profiles and all contain several potential N-glycosylation sites. Northern analysis has shown that ₂-1 is ubiquitously expressed, ₂-2 is found in several tissues including brain and heart, and ₂-3 is brain-specific (Klugbauer et al., 1999). An association of ₂-2 with tumors has been suggested, and the mouse homolog is a candidate for the ducky epileptic phenotype (Gao et al., 2000).

₂-1 consists of two proteinsa highly glycosylated ₂ that is believed to be extracellularly located, and a smaller  protein that anchors the ₂ to the cell membrane (Brickley et al., 1995; Wiser et al., 1996). These proteins are coded for by a single gene, the product of which is translated as a precursor polypeptide that is post-translationally cleaved (De Jongh et al., 1990). The ₂ and  associate by disulfide bridges that form between the numerous cysteine residues found in both proteins.

The effects of ₂-1on the biophysical properties of ₁ subunits depend on the expression system and subunits used. The subunit invariably increases the current density of calcium channels by increasing the amount of functional channel at the cell surface, and has been reported to allosterically alter the activation and inactivation of several ₁ subunits (Singer et al., 1991; Bangalore et al., 1996, Felix et al., 1997, Klugbauer et al., 1999). ₂-1 is known to enhance dihydropyridine binding to L-type channels and -conotoxin GVIA to N-type channels (Brust et al., 1993; Felix et al., 1997). ₂-2 and -3 significantly enhance and modulate the current through a number of HVA and LVA channels (Klugbauer et al., 1999; Gao et al.; 2000, Hobom et al., 2000). The action of these subunits depends on the ₁ subunit expressed and ₂-2 and -3 may preferentially interact with Ca_v2.3 (_1E), Ca_v2.1 (_1A), or even Ca_v3.1 (_1G).

Gabapentin (GBP) is an antiepileptic drug that has also found application in pain and anxiolytic disorders (Welty et al., 1993; Beydoun et al., 1995). GBP binds to rat brain (Hill et al., 1993) and skeletal muscle homogenates (Gee et al., 1996), with lower binding seen in heart, lung, and pancreas (Suman-Chauhan et al., 1993). GBP was subsequently found to bind specifically to ₂-1 of voltage activated calcium channels (Gee et al., 1996). Because voltage activated channels are involved in controlling the electrical excitability of neurons, it has been postulated that this drug reduces calcium current by modulating ₁ indirectly through its association with ₂-1 (Gee et al., 1996).

The purpose of this study was to characterize the gross structure and properties of the novel ₂-2 and -3 subunits. It was found that ₂-1, -2, and -3 are similarly processed and post-translationally modified. ₂-1 and -2 bound GBP with differing affinities, but the more distantly related ₂-3 did not.

	Materials and Methods

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Transfection of COS-7 Cells. Plasmids containing the ₂ subunits, or pcDNA3 (Invitrogen, Groningen, The Netherlands) as control, were transiently transfected in COS-7 cells, using FuGene 6 (Roche, Mannheim, Germany). ₂-1 and -3 had been previously cloned into the expression vector pcDNA3, and ₂-2 into pcDNA3.1/V5/His-TOPO (Klugbauer et al., 1999). The cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Karlsruhe, Germany) containing 10% fetal calf serum, 100 U of penicillin, and 100 µg/ml streptomycin at 37°C, 6% CO₂; the cells were seeded at 1.5 × 10⁶ cells per 100-mm culture plate. After 24 h, the cells were transfected in antibiotic-free medium with 6 µg of DNA using 18 µl of FuGene reagent per plate. The medium was replaced with complete medium after 16 h and harvested 60 to 72 h after the start of transfection.

Membrane Preparations. Tissues from BALB/c mice were crushed under liquid nitrogen using a mortar and pestle. The powder was homogenized in buffer A (20 mM MOPS, pH 7.4, 300 mM sucrose, 2 mM EDTA, 1 mM iodoacetamide, 1 mM orthophenanthroline, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1 µg/ml antipain) by two 20-s bursts with a Polytron homogenizer (Kinematica AG, Switzerland) and 8 strokes in a power-driven Potter pestle (Braun Biotech, Germany). Cell debris was pelleted by centrifugation at 5000 g for 10 min at 4°C. Low-speed pellets were re-extracted as above and the supernatants combined. The supernatants were centrifuged at 70,000 g for 30 min at 4°C. The pellets were resuspended in buffer A and stored in aliquots at 80°C. Membranes from transfected COS-7 cells were prepared similarly, with the following modifications: cells were harvested by scraping into 1× phosphate-buffered saline, pelleted by low speed centrifugation, resuspended in 5 mM Tris-Cl, 5 mM EDTA, pH 7.4, containing protease inhibitors (as above) and incubated on ice for 15 min before homogenization. The pellets resulting from ultracentrifugation were resuspended in 50 mM MOPS, pH 7.4, plus protease inhibitors. The protein content was determined by the Bradford method using BSA as a standard.

Anti-Peptide Antibodies. Antibodies were raised in rabbits by Gramsch Laboratories (Schwabhausen, Germany) against peptides of the human ₂-2 (amino acids 98-115) and murine ₂-3 (amino acids 59-76) subunits (Klugbauer et al., 1999). These sequences were selected because they have low homology to each other and to ₂-1, are found after the putative signal peptide sequence, and have no potential N-glycosylation sites. The antibodies were affinity purified using a SulfoLink coupling gel (Pierce, Rockford, IL) with bound antigen. Antibodies were eluted with 4.5 M MgCl₂, and concentrated using Centricon YM-50 centrifugal filter devices (Millipore Corporation, Bedford, MA). The antibody was made to 1 mg/ml BSA in phosphate-buffered saline and stored at 20°C in aliquots.

Western Blotting. Proteins were separated by discontinuous SDS-PAGE in 5 or 7.5% resolving gels. The membrane preparations were denatured in Laemmli sample buffer (with or without 0.1 M dithiothreitol) by boiling for 3 min. Typically, 50 to 100 µg of tissue or 3 µg of COS-7 microsomal membrane preparation was loaded per lane. After electroblotting and blocking in 3% BSA, the nitrocellulose membranes were incubated for 2 h at room temperature with primary antibodies used at 1/750 for ₂-2 and 1/500 for ₂-3. A commercially available anti-₂-1 monoclonal antibody (ABR, Golden, CO) was used at a concentration of 1/500. Incubation with the secondary goat anti-rabbit- or goat anti-mouse-horseradish peroxidase antibodies (Dianova, Germany) proceeded for 1 h at room temperature at a dilution of 1/7000. Antibody binding was detected using the ECL system (Amersham Pharmacia Biotech, Freiburg, Germany).

Deglycosylation Assay. Membrane protein (70 µg) was denatured by boiling in 0.1 M -mercaptoethanol, 0.5% SDS for 5 min. The reaction was brought to 15 mM Tris-Cl, pH 8.0, 20 mM orthophenanthroline, and 1% Triton X-100. Two units of N-glycosidase F (Roche, Mannheim, Germany) were added and the reaction allowed to proceed for 5 h at 37°C. The protein was separated by SDS-PAGE gel and analyzed by Western blotting as described above.

Gabapentin Binding Assay. COS-7 membrane preparations (10 to 30 µg) were incubated in 200 µl volumes with various concentrations of [³H]gabapentin (143 Ci/mmol, custom synthesized by Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK) in 10 mM HEPES/KOH, pH 7.4, for 30 min at room temperature. The protein was precipitated using 8 ml of ice-cold precipitation buffer (22.5 mM HEPES, pH 7.4; 11.25% polyethylene glycol 6000; 11.25 mM CaCl₂) and filtered over GF/B filters soaked in the same buffer. Two further 8-ml volumes of precipitation buffer were used as washes. The activity of the filters was counted in a scintillation counter. Concentrations greater than 20 nM were achieved by adding nonradioactive gabapentin to the required concentration. The corrected binding was calculated using the equation, Total GBP bound = (specific dpm) × [1 + (concentration nonradioactive gabapentin/concentration [³H]gabapentin)].

	Results

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Analysis of ₂ Protein Structure. The specificity of the peptide antibodies was tested using membrane preparations of COS-7 cells transfected with the various ₂ proteins. The commercial ₂-1 antibody did not recognize ₂-2 and -3 preparations. Similarly, the peptide antibodies only recognized the ₂ against which they were raised (Fig. 1). The antibodies were also preincubated with an excess of peptide antigen before incubation with the protein blots to test for cross-reactivity. The bands corresponding to the ₂ proteins were not detected, indicating that the antibodies are specific (results not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   The anti-2-1, -2, and -3 antibodies are specific. The specificity of the antibodies was tested using membrane preparations of COS-7 cells transiently transfected with 2 constructs. Three micrograms of each membrane preparation was denatured in the presence of DTT and analyzed by Western blotting. The subunit transfected is indicated above the lanes. The antibodies applied, anti-2-1, 2-2, and 2-3, are shown below the blots.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   The 2 proteins are highly glycosylated and consist of two proteins. The sizes and structures of 2-1, -2 and -3 were assessed by Western blotting. Changes in the apparent molecular mass of 2-1, -2, and -3 were observed upon reduction (A). Brain membrane preparations were separated by SDS-PAGE with (+) and without () DTT, and analyzed by immunoblotting. Deglycosylation of denatured, reduced brain preparations using N-glycosidase F (NGF +) led to a shift in the protein mobility. For all three proteins this corresponded to a loss of approximately 30 kDa relative to untreated () controls (B). The antibodies used are indicated above the blots.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Western blot showing the tissue distribution of 2-1, 2, and -3 in mouse tissues. Membrane preparations of the indicated tissues were probed with 2-1, 2-2, and 2-3 antibodies. Forty micrograms of brain was used for 2-1 and -2 and 100 µg for the 2-3 blots. One hundred micrograms of the other tissues was loaded. Both 2-1 and -2 gels were electrophoresed under reducing conditions, whereas that for 2-3 was nonreducing.

Gabapentin Binding Assays. Binding of GBP to the ₂ subunits was assessed using COS-7 cells that overexpressed the proteins. Membrane preparations were incubated with [³H]gabapentin for 30 min. The protein was subsequently precipitated, captured on filters, and measured by scintillation counting. ₂-1 and ₂-2 were found to specifically bind GBP, whereas ₂-3 did not show any binding (Fig. 4). Cells transfected with pcDNA3 alone as a control did not interact with GBP. ₂-1 bound GBP with a relatively high affinity, and a dissociation constant (K_d) of 59 ± 6 nM (S.E.M.) was calculated by Scatchard analysis of data from three experiments (Fig. 5). ₂-2 was found to bind GBP with a K_d of 153 ±14 nM (S.E.M.) in six independent experiments. In both cases, GBP seemed to bind to a single binding site, as judged by the Scatchard plots. The contribution of ₂ carbohydrate side chains to GBP binding was also investigated. The binding of [³H]GBP to native mouse brain membranes following deglycosylation for 18 h with N-glycosidase F was not significantly altered relative to controls incubated without enzyme (not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Gabapentin binds to 2-1 and -2, but not to 2-3. The binding of [3H]gabapentin to membranes of COS-7 cells transiently transfected with 2-1, -2, and -3 was measured. Thirty micrograms of protein was incubated with 20 nM [3H]gabapentin. No binding was observed with membrane preparations of cells transfected with pcDNA3 alone. The data are from three separate experiments done in duplicate. The error bars show the standard error of the mean. The inset shows Western blots showing the expression and cleavage of the proteins in the preparations used.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Gabapentin binds to 2-1 and -2 with different affinities. Ten micrograms of protein was incubated with the indicated concentrations of gabapentin. A representative saturation curve for each subunit is shown, with the corresponding Scatchard plot. Kd values of 59 ± 6 nM (S.E.M.) were calculated for 2-1 (A) and 153 ± 14 nM (S.E.M.) for 2-2 (B). Three independent experiments for 2-1 and six for 2-2 were performed, with each assay done in duplicate. Specific binding was determined in the presence of 10 µM unlabeled gabapentin.

	Discussion

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This study was undertaken to clarify the gross protein structure and characteristics of the novel ₂-2 and -3 subunits. It was of particular interest to determine whether the characteristic traits of ₂-1, namely its composition of two proteins derived from a single precursor protein, and its high level of glycosylation, are conserved. Because GBP, a drug increasingly used for neurological disorders, binds to ₂-1, the novel subunits were also tested for drug binding.

Using antibodies directed against the putative ₂ of the ₂-2 and -3 subunits, it could be assessed whether precursor proteins are cleaved and form complexes via disulfide bridges. Both ₂-2 and -3 in native tissues were found to be composed of an ₂ and , which could be separated by reduction. The difference in mass of the nonreduced complex relative to the reduced protein roughly corresponds to that of  alone and was approximately 50 kDa for each of the ₂ proteins. The cleavage site of ₂-1 is between A⁹³⁴ and A⁹³⁵, when the signal sequence is not taken as part of the protein (De Jongh et al., 1990; Jay et al., 1991). The alanine at position 934 in ₂-1 is conserved in ₂-2 and -3, although the rest of the sequence in the region diverges. Although the ₂-1 cleavage site cannot be assumed to be the same as for the novel proteins, the approximate sizes of the  proteins and the conservation of the alanine suggest that this could be the case. If the alanine is assumed to be the first residue of  for both ₂-2 and -3, then the mass calculated for  of ₂-2 based on its amino acid content is 17.2 kDa, and for ₂-3, 15.1 kDa. The mass estimated indirectly is larger than that calculated using the amino acid sequence. This difference is likely to be due to the difficulty associated with inferring sizes based on shifts in SDS-PAGE migration of reduced and nonreduced proteins, as well as post-translational modifications such as N-glycosylation (both  proteins contain potential N-glycosylation sites). A similar pattern of modification has been observed for the  of ₂-1, which has a calculated mass of 19 kDa, and a migration of 24 and 27 kDa using antibodies against the  (De Jongh et al., 1990).

All three ₂ proteins were found to be highly glycosylated, with 30 kDa of the mass of the proteins consisting of oligosaccharides. Because glycosylation is essential for current stimulation by ₂-1 of Ca_v2.1 (_1A) (Gurnett et al., 1996), this is likely to be the case for the novel subunits as well.

The distribution of ₂ protein in mouse tissue was compared with that of the mRNA expression pattern (Klugbauer et al., 1999). ₂-3 was detected only in brain, which corresponds with the results of the Northern analysis. ₂-2 mRNA was originally described to be ubiquitously expressed, with the highest levels in brain, heart, pancreas, and skeletal muscle (Klugbauer et al., 1999). Lower levels of the mRNA were seen in other tissues after longer exposures. In this study, ₂-2 protein was detected at very low levels in skeletal muscle, although the Northern analysis had produced a signal comparable with brain. Gao et al. (2000) reported high levels of ₂-2 mRNA in human lung and cloned the subunit from a lung library. Although ₂-2 protein was observed at low levels in mouse lung in this study, it is possible that certain lung cell types express high levels. Whereas the Gao group showed overexpression of ₂-2 protein in various tumor cell lines, no data was presented on the endogenous levels in healthy lung tissue.

The specific binding of gabapentin to ₂-1 was the first described interaction between a regulatory subunit of voltage activated calcium channels and a pharmaceutical agent. The K_d of porcine brain ₂-1 was reported as 9.4 nM (Brown et al., 1998) but as 37.5 nM for porcine ₂-1 expressed in COS-7 cells (Brown and Gee, 1998) and 16 nM for rabbit ₂-1 in COS-7 cells (Gee et al., 1996). In this study, K_d values of 59 nM for ₂-1 and 153 nM for ₂-2 were determined. In a preliminary report by Su et al. (2000), ₂-2 is described as having two binding sites (K_d values of 147 and 25 nM). The reason for variations in K_d values is not clear, but may be attributable to species differences and assay methods. Because GBP is used for a variety of neurological disorders, it is interesting to note that it binds to two auxiliary calcium channel subunits that have been found to exert differing modulatory effects on ₁ pore subunits in heterologous expression systems.

The effect of GBP on the physiological activity of calcium channels is not clearly understood. In patch-clamp studies with hippocampal granule cells, no effect of GBP was reported (Schumacher et al., 1998). However, in other studies, modest to dramatic changes in calcium current were noted. A reduction in the calcium current in isolated neurons (Stefani et al., 1998) and in rat neocortical slices (Fink et al., 2000) upon application of GBP has been described. Calabresi et al. (2000) found GBP to reduce most excitatory properties of striatal spiny neurons, which could account for the anticonvulsant effect of the drug. Dooley et al. (2000) have also shown that GBP and a related compound, pregabalin, reduce the release of norepinephrine when stimulated by potassium and electrical pulses. The calcium channels affected are not known, but candidates are L-type (Stefani et al., 1998) and P/Q (Fink et al., 2000; Meder and Dooley, 2000). No consistent effect of GBP on Ca_v1.2 (_1C), Ca_v2.1 (_1A), and Ca_v3.2 (_1G) currents in the heterologous human embryonic kidney 293 expression system were observed in this study. The complexity of the interaction between GBP and ₂ is further illustrated by a temperature dependent influence of ruthenium red, MgCl₂, and spermine on GBP binding (Taylor and Bonhaus, 2000). Initial studies on GBP binding using rat tissue homogenates showed strong binding in skeletal muscle and brain, where ₂-1 is most highly expressed (Gee et al., 1996). A much lower binding of the drug was seen in liver and kidney, which express considerable levels of the protein, as judged by Western blotting. A possible explanation for these conflicting results is that the binding of GBP to ₂ is modulated by other subunits (e.g., the ₁ pore protein). It is possible that the effects of GBP depend on the composition and environment of the channel. The lack of clinical side effects of the drug on skeletal muscle and other ₂-1 expressing tissues supports this view (Beydoun et al., 1995). GBP has also been shown to be an agonist of certain -aminobutyric acid_B receptors, and this has also been postulated to be involved in the clinical action of the drug (Ng et al., 2001).

Gabapentin binding is dependent on the presence of both  and  subunits, which do not have to be translated as a single precursor protein (Wang et al., 1999). Their interaction is important, however, because neither  nor  bind the drug when expressed alone (Wang et al., 1999). Cleavage of the precursor protein is also not required for binding (Brown and Gee, 1998). Mutation analysis of porcine ₂-1 by Brown and Gee (1998) led to the identification of a region (960-994) in -1, containing a zinc-like finger, that is important for gabapentin binding. Another study identified residues 206 to 222, 516 to 537, and 383 to 603 as essential for binding (Wang et al., 1999). Because ₂-2 has in this study been shown to bind gabapentin, it is instructive to compare the sequences identified as putative binding sites in previous reports (Fig. 6). Mutations of charged amino acids in ₂-1 was performed by Wang et al. (1999), and Arg²¹⁷ found to be the most important. This residue is found in ₂-2, but not in ₂-3, supporting its role in the binding of GBP. It is, however, not clear whether these sites are essential for the association of ₂ with , or whether they form a gabapentin-binding pocket.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Alignment of 2 sequences important for GBP binding. 2-1 is compared with 2-2 and 2-3 in the regions believed to be involved in GBP binding (Brown et al., 1998; Wang et al., 1999). Residues conserved in 2-1, 2-2 and 2-3 are italicized. Residues only found in 2-1 and -2 are underlined. A217 is shown in bold. The alignment of the sequences was done using Clustal V.

Gabapentin analogs have been developed that bind to ₂-1 with a higher affinity than gabapentin and are effective in an animal model of epilepsy (Bryans et al., 1998). Because ₂ subunits may associate preferentially with ₁ subunits in the brain, these drugs could have differing therapeutic actions if more than one of ₂ family binds these drugs.

In summary, we present results indicating that ₂-2 and -3 consist of two proteins, derived from a single transcript, that are associated by disulfide bonds. Both novel subunits are highly glycosylated. ₂-2, but not ₂-3, is capable of binding the antiepileptic drug GBP, which has a higher affinity for ₂-1 than for ₂-2. Further investigation into the molecular action of GBP and its analogs on voltage gated calcium channels should enable the fine-tuning of treatment of neurological disorders.