α2δ Ca2+Channel Subunit from Porcine Brain: Development of a Radioligand Binding Assay for α2δ Subunits Using [3H]Leucine

ANALYTICAL BIOCHEMISTRY ARTICLE NO.

255, 236 –243 (1998)

AB972447

Isolation of the [3H]Gabapentin-Binding Protein/a2d Ca21 Channel Subunit from Porcine Brain: Development of a Radioligand Binding Assay for a2d Subunits Using [3H]Leucine Jason P. Brown, Visaka U. K. Dissanayake, Alex R. Briggs, Mihajlo R. Milic, and Nicolas S. Gee1 Parke-Davis Neuroscience Research Centre, Cambridge University Forvie Site, Robinson Way, Cambridge, CB2 2QB, United Kingdom

Received March 26, 1997

The novel antiepileptic agent gabapentin (Neurontin) binds with high affinity to the a2d subunit of a voltage-dependent Ca21 channel. We report here a simple purification scheme for detergent-solubilized a2d subunits from porcine brain. This involves sequential chromatography on Q-Sepharose, Cu21-charged iminodiacetic acid–Sepharose, wheat germ lectin–agarose, and Mono Q. The purified protein was essentially homogeneous by SDS–polyacrylamide gel electrophoresis with a subunit Mr of 145,000. Using [3H]gabapentin as the radiolabeled tracer and (S)-3-isobutyl g-aminobutyric acid to define nonspecific binding, the overall purification factor was 2760-fold and the apparent yield 26.6%. We also developed and validated a novel binding assay for a2d Ca21 channel subunits using the ligand pair L-[3H]leucine/L-isoleucine. Even in binding assays of crude brain membrane fractions, [3H]leucine proved to be remarkably stable and specific for the a2d Ca21 channel subunit. [3H]Leucine offers several advantages over custom-labeled [3H]gabapentin: it has a higher specific activity, is relatively inexpensive, and is available from commercial sources. © 1998 Academic Press

Gabapentin (Neurontin) is an antiepileptic agent indicated for the treatment of seizures refractory to conventional drug therapies (1). More recently, gabapentin has also shown considerable promise for the treatment of anxiety (2, 3) and pain (3–5). Gabapentin

was designed as a lipophilic analogue of GABA2 and, as expected of a putative GABA-mimetic agent, displayed anticonvulsant activity in animal seizure models (6 – 8). Subsequent research however has provided little evidence of any direct interaction of gabapentin, at therapeutically relevant concentrations, with the principal molecular targets that influence GABAergic neurotransmission: GABA receptors (9), GABA transaminase, and the GABA uptake transporter (10, 11). A novel high-affinity binding site in the central nervous system for [3H]gabapentin was described in 1993 (9). The rank order of affinity of gabapentin analogues at this site mirrors the rank order of anticonvulsant activity for these compounds in animal models, suggesting that the [3H]gabapentin binding protein may modulate neuronal excitability (8). Recently, the binding protein was isolated from porcine brain and identified as the a2d subunit of a voltage-dependent Ca21 channel (VDCC) (12). VDCCs are heterooligomeric complexes composed of transmembrane a1 and a2d subunits, together with a hydrophilic b subunit; a transmembrane g subunit is also present in skeletal muscle (13). The a2d subunit, which is the product of a single gene, is proteolytically cleaved to yield separate a2 and d chains that remain linked via disulfide bridges (14). The a2d subunit is anchored to membranes through a hydrophobic domain in the 25-kDa d polypeptide (15, 16), though transmembrane domains in the 145-kDa a2 component have also been postulated (15, 17). While the physiological role of the a2d subunit is unclear, it has been shown in heterologous expression systems Abbreviations used: GABA, g-aminobutyric acid; ECL, enhanced chemiluminescence; VDCC, voltage-dependent Ca21 channel; IDA, iminodiacetic acid; WGA, wheat germ lectin–agarose. 2

1

To whom correspondence should be addressed. Fax: 01223 249106. E-mail: [email protected].

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0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

ISOLATION AND ASSAY OF a2d Ca21 CHANNEL SUBUNITS

that the a2d subunit, in conjunction with a b subunit, enhances the functional expression of the pore-forming a1 subunit (16, 18, 19). We report here a rapid purification scheme for neuronal a2d Ca21 channel subunits. We also describe a simple radioligand binding assay for a2d subunits using commercially available reagents. MATERIALS AND METHODS

Materials Pig brains were obtained from the local abattoir and transported to the laboratory on ice. Chromatography media were from Pharmacia Biotech Ltd. (Milton Keynes, Bucks, UK). Mini-protean II precast gradient gels and reagents for silver staining were from Bio-Rad Laboratories (Hemel Hempstead, Herts, UK). GF/B filters were from Whatman International (Maidstone, Kent, UK). [3H]Leucine (141 Ci/mmol, code TRK 170) and [3H]gabapentin (50 Ci/mmol; custom synthesis) were from Amersham International (Amersham, Bucks, UK). Gabapentin and (S)-3-isobutyl GABA were obtained from Warner Lambert (Ann Arbor, MI). All other reagents were obtained from Sigma Chemical Co. (Poole, Dorset, UK) or from FSA Supplies (Loughborough, Leicester, UK). Methods Radioligand binding assays. [3H]Gabapentin binding assays were carried out essentially as described by Gee et al. (12). Polyethylenimine-soaked filters were used both for membranes and for detergent-solubilized proteins. Saturation data were transformed and analyzed using LIGAND version 3.0 (Elsevier-Biosoft). Preparation of detergent-solubilized brain membranes. Pig cerebral cortical membranes were prepared as described by Gee et al. (12) with minor modifications: (i) membranes were subjected to an additional wash with 25% (v/v) glycerol/12.5 mM Hepes/KOH, pH 7.4 (at 4°C), to reduce the concentration of chelators; (ii) a glass–Teflon homogenizer was used instead of a Waring blender. Solubilization of membranes using Tween 20 was performed as previously described (12). Q-Sepharose chromatography. Tween 20-solubilized proteins were loaded at 4 ml/min on to a QSepharose Fast Flow column (2.6 cm i.d. 3 37 cm) equilibrated in 0.08% Tween 20/10 mM Hepes/KOH, pH 7.4 (at 4°C). The column was washed with equilibration buffer until the A280 of the effluent reached the baseline value. Bound proteins were eluted at 1 ml/min with a linear gradient of NaCl (0 –750 mM) in a total volume of 1 liter of equilibration buffer. Fractions (10 ml) were collected and 25-ml aliquots of alternate frac-

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tions assayed for [3H]gabapentin binding activity. In some experiments [3H]leucine was used instead of [3H]gabapentin (see Results). Immobilized metal ion chromatography. Before each run, the iminodiacetic acid (IDA)–Sepharose column (2.5 cm i.d. 3 13.5 cm) was stripped of residual metal ions with 12 ml of 0.5 M EDTA/NaOH, pH 8.0 (at 21°C), washed with 200 ml of water, and charged with 24 ml of 0.3 M CuSO4. The column was then washed successively with 200 ml of water, 100 ml of buffer B [9:1 (v/v) buffer A:1 M imidazole], and 200 ml of buffer A [450 mM NaCl/0.08% Tween 20/10 mM Hepes/KOH, pH 7.4 (at 21°C)]. Pooled material from the Q-Sepharose step was applied at 2 ml/min after equilibration to 21°C and the eluent corresponding to the void volume of the column discarded. Fractions were then collected until the A280 value returned to baseline. This unretarded material was pooled (pool I1) and the column was then step-eluted with 200 ml of 10% buffer B (in buffer A) (pool I2), followed by 100 ml of buffer B (pool I3). Wheat germ lectin chromatography. The breakthrough material from the previous step (pool I1) was loaded at 10 ml/h onto a wheat germ lectin–agarose (WGA) column (1 cm i.d. 3 5 cm) equilibrated in 0.08% Tween 20/450 mM NaCl/10 mM Hepes/KOH, pH 7.4 (at 4°C). The column was washed with 6 ml of equilibration buffer and eluted with buffer containing 0.35 M N-acetyl-D-glucosamine at 1 ml/h. Active fractions (each 1 ml) were pooled and diluted fivefold with 0.08% Tween 20/10 mM Hepes/KOH, pH 7.4 (at 21°C), to reduce the ionic strength of the sample. Mono Q chromatography. The sample was passed through a 0.22-mm filter and applied to a Mono Q HR 5/5 FPLC column equilibrated in buffer A [0.08% Tween 20/10 mM Hepes/KOH, pH 7.4 (at 21°C)]. Bound proteins were eluted with a linear NaCl gradient (0 –750 mM) in a total volume of 100 ml of buffer A. Elution continued with 10 ml of buffer containing 750 mM NaCl, followed by 10 ml of buffer A. Fractions (each 1 ml) were collected and analyzed by both radioligand binding and SDS–polyacrylamide gel electrophoresis. HPLC analysis of [3H]leucine. [3H]Leucine was incubated with brain membranes for 50 min under standard assay conditions. The assay contents were centrifuged though a 0.22-mm nylon membrane at 10,000g for 3 min and the filtrate was applied to a reversedphase C18 HPLC column. The column was eluted at 1 ml/min with a gradient of 0 –100% acetonitrile in 0.1% trifluoroacetic acid in a total volume of 60 ml. SDS–polyacrylamide gel electrophoresis and Western blotting. SDS–polyacrylamide gel electrophoresis was performed using either 6% gels or 4 –20% gradient gels

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FIG. 1. Flow chart of the purification scheme for the a2d subunit. The flow diagram gives an overview of the purification scheme for pig brain a2d subunits. Refer to Methods for specific information on each step.

with the Laemmli buffer system (20). Gels were stained using the Bio-Rad silver-staining kit according to the manufacturer’s instructions. Western blotting was performed as previously described using ECL detection (12). Protein determination. Protein concentration was determined by the method of Bradford (21) using bovine serum albumin as a standard. RESULTS

Isolation of Neuronal a2d Subunits A flow chart for the purification scheme is shown in Fig. 1. Pig cerebral cortical membranes were prepared as described by Gee et al. (12) with minor modifications (see Methods); the yield of membrane protein was 1.7fold higher using this modified protocol. Tween 20solubilized membrane proteins were fractionated on a Q-Sepharose ion-exchange column. A broad peak of [3H]gabapentin binding activity was observed (Fig. 2a). The peak fractions were pooled, warmed to 21°C, and applied to a Cu21-charged IDA–Sepharose column

(Fig. 2b). The column breakthrough material (pool I1) contained 81% of the applied [3H]gabapentin binding activity. Step-elution with buffers containing imidazole (10 and 100 mM) yielded additional pools, I2 and I3, containing 13 and 6% of the binding activity, respectively. Rechromatography of I2 on a high-resolution ion-exchange column gave a profile of [3H]gabapentin binding activity that was essentially identical to that for the purified protein (data not shown). Pool I3 could not be analyzed in this way as the binding activity was lost after freezing in the presence of high concentrations of imidazole. However, the presence of a2d subunits in all three fractions was confirmed by Western blotting using an a2-specific polyclonal antibody (Fig. 3). There is a hint that the a2d subunits in pool I1 migrate more slowly than those in pools I2 and I3. Only the major pool, I1, was subjected to further purification. The WGA column retained ;90% of the [3H]gabapentin binding activity and was eluted isocratically with 0.35 M N-acetyl-D-glucosamine (Fig. 2c). The stabilization of the A280 baseline at a level above that observed under equilibration conditions is explained by the higher intrinsic optical activity of the elution buffer. The peak of activity from the Mono Q column (Fig. 2d) was symmetrical and a single 145,000 Mr polypeptide whose intensity paralleled the level of [3H]gabapentin binding activity was observed on SDS– polyacrylamide gels (data not shown). The overall purification factor was 2760-fold over starting membranes and the apparent yield 26.6% (Table 1). A reduction in the apparent KD for [3H]gabapentin was observed during the purification procedure; thus, some of the yields are overestimated (see later). Figure 4 shows an SDS gel of pooled samples from each stage of the purification. The Mono Q pool contains a single band which corresponds to the a2 component of the purified subunit. In our hands (12) and as noted by others (17, 22) the d subunit stains either poorly or not at all on SDS–polyacrylamide gels. The yield of pure protein was 0.15 mg, which corresponds to 0.9 mg of a2d subunit per gram of pig cerebral cortex. Radioligand Binding Assay for a2d Subunits [3H]Gabapentin is available only as a custom-labeled product and requires occasional repurification by HPLC to remove uncharacterized degradation products. Certain L-amino acids, particularly those with uncharged aliphatic side chains, potently and competitively displace [3H]gabapentin binding from brain

FIG. 2. Purification of the [3H]gabapentin binding protein/a2d subunit from pig brain. (a) Q-Sepharose chromatography of detergentsolubilized membranes (100,000g supernatant). Fractions 44 –72 were pooled; (b) Cu21-IDA chromatography. Three pools, I1, I2, and I3, were taken corresponding to the breakthrough material, fractions 10 –15 and fractions 25–27, respectively; (c) wheat germ lectin chromatography. Fractions 1–23 were pooled; (d) Mono Q chromatography. Fractions 32–50 were pooled.

ISOLATION AND ASSAY OF a2d Ca21 CHANNEL SUBUNITS

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FIG. 3. Western blot of brain membranes and Cu21-IDA pools I1, I2, and I3. Samples were electrophoresed on a 6% SDS–polyacrylamide gel before transfer to nitrocellulose. Track 1, Brain membranes (2.5 mg); track 2, pool I1 (0.56 mg); track 3, pool I2 (0.85 mg); and track 4, pool I3 (6.87 mg). The blot was probed with an anti-a2 rabbit polyclonal and anti-rabbit HRP conjugate followed by ECL detection.

membranes (23). We therefore evaluated [3H]leucine as a possible alternative radiolabel for the a2d subunit. First, the metabolic stability of [3H]leucine was assessed by reversed-phase HPLC (Fig. 5). Purity values following incubation of the radiolabel with or without membranes for 50 min were 94 and 96%, respectively. Second, saturation analyses of brain membranes and purified a2d subunits were performed with [3H]gabapentin and L-[3H]leucine using (S)-3-isobutyl GABA and L-isoleucine, respectively, to define nonspecific binding. Table 2 shows that for each sample both radioligands gave similar KD values. However, Bmax values for L[3H]leucine binding to membranes and to purified a2d subunits were 1.66- and 1.43-fold higher, respectively, than those obtained with [3H]gabapentin. To examine the specificity of [3H]leucine for the a2d subunit in more detail we compared the binding activity profiles for Tween 20-solubilized brain membrane proteins after fractionation on a Q-Sepharose column. As can be seen in Fig. 6, the profiles are very similar, although the [3H]leucine trace is offset on the vertical axis. The only notable difference is a minor peak in the

FIG. 4. Analysis of fractions from the a2d purification scheme. Samples were resolved on a 4 –20% gradient gel under reducing conditions. M, marker proteins. Apparent subunit Mr values (31023) are indicated. Track 1, brain membranes (10 mg); track 2, Tween 20-solubilized proteins (10 mg); track 3, Q-Sepharose pool (10 mg); track 4, Cu21-IDA column pool I1 (1 mg); track 5, wheat germ lectin– agarose pool (1 mg); track 6, Mono Q pool (1 mg); and track 7, sample buffer control.

[3H]leucine profile centered on fraction 40. This [3H]leucine-specific binding protein which represents ;3% of the total activity has not been further characterized. In a separate purification, the elution profiles obtained with [3H]gabapentin and [3H]leucine for the Cu21-IDA column were essentially identical, and column yields and fold purification data for other steps of the a2d purification scheme were in close agreement (data not shown). DISCUSSION

The purification of the [3H]gabapentin binding protein/a2d subunit from porcine brain was achieved in a four-step column chromatographic procedure which takes approximately 6 days to complete. The purity of the preparation was confirmed by SDS–polyacrylamide gel electrophoresis using a 4 –20% gradient gel to cover a broad range of molecular weights. The final purification factor using [3H]gabapentin as the radiolabeled tracer was 2760-fold and the yield 26.6%. These values

TABLE 1

Purification of the a2d Subunit/[3H]Gabapentin Binding Protein Sample

Total binding activity (pmol)

Total protein (mg)

Specific activity (pmol/mg)

Yield (%)

Purification (fold)

Apparent KD (nM)

Membranes Solubilized membranes Q-Sepharose Cu21-IDA Wheat germ lectin Mono Q

779 500 485 374 331 207

1548 792 124 27.6 0.40 0.15

0.50 0.63 3.91 13.6 828 1380

100 64.2 62.3 48.0 42 26.6

1 1.26 7.82 27.2 1656 2760

92 6 13 (n 5 3) 66a 17a 12a 11a 9.4 6 0.29 (n 5 3)

a

n 5 1.

ISOLATION AND ASSAY OF a2d Ca21 CHANNEL SUBUNITS

241

FIG. 6. Comparison of elution profiles obtained with [3H]gabapentin and [3H]leucine after fractionation of Tween 20-solubilized brain membrane proteins on Q-Sepharose. Fractions were assayed with either 20 nM [3H]gabapentin or 20 nM [3H]leucine.

FIG. 5. Analysis of [3H]leucine by HPLC. (a) Control sample and (b) sample following incubation with brain membranes for 50 min as described under Methods. The retention times (min) of [3H]leucine were 7.8 (a) and 8.1 (b).

are overestimated by a factor of ;2 owing to the presence of endogenous inhibitory substances in the starting membranes (24). This is reflected in the high yield at the Q-Sepharose step— during which endogenous ligands are presumably washed out—and the reduction in apparent KD values as the scheme progresses.

The KD of 9.4 nM for purified a2d subunits probably approaches the true value. The procedure yielded 0.9 mg of purified a2d subunits per gram (wet weight) of pig cerebral cortex. The original six-step procedure (12), which led to the identification of the [3H]gabapentin binding protein as the a2d subunit, yielded 0.23 mg of purified protein per gram of tissue and took approximately 2 weeks to complete. The Cu21-IDA column effectively replaces the lentil lectin, gel filtration, and hydroxyapatite columns of the original procedure (12) and thus considerably simplifies the new scheme. The presence of three peaks of binding activity on the Cu21-IDA column suggests different molecular forms of the a2d subunit or different polypeptides each capable of binding [3H]gabapentin. The latter is unlikely as brain membranes contain only a single class of binding sites (9, 12, 23). Splice variants of the a2d subunit are known (15, 25, 26), but only a single type is found in brain (27, 28). Heterogeneity of the subunit could arise from subtle variations in the pattern of glycosylation or other posttranslational modifications. Western blots using an a2-specific polyclonal antibody confirmed that all three Cu21-IDA pools (I1, I2, and I3) contained a2d subunits. However, there is a hint in Fig. 3 that the a2d subunits in pool I1 migrate more slowly than those in pools I2 and I3. On the other hand, brain membranes (from which I1 is

TABLE 2

Saturation Analysis of Membranes and Purified a2d Subunit Using [3H]Gabapentin and L-[3H]Leucine KD (nM 6 SE)

Membranes Mono Q pool

Bmax (pmol/mg 6 SE)

Gabapentin

Leucine

Gabapentin

Leucine

Bmax ratio (leucine/gabapentin)

107.5 6 15.3 13.9 6 3.2

58.9 6 6.5 20.6 6 1.0

4.68 6 0.17 6963 6 232

7.80 6 0.55 9963 6 899

1.66 1.43

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BROWN ET AL.

derived) do not show this effect. Three other gels (data not shown) also proved inconclusive and it is unclear whether the population of a2d subunits is heterogeneous. An alternative explanation for our findings is that pool I1 corresponds to dissociated a2d subunits, which are not retained by the column, and pools I2 and I3 to complexes of the a2d subunit with other VDCC components, at least one of which binds to the Cu21-IDA matrix. The stability of VDCC heterooligomers is dependent on the type of detergent used for solubilization (12, 17). In previous studies with digitonin-solubilized rabbit skeletal muscle VDCCs two peaks of [3H]gabapentin binding activity were observed on Mono Q; these were shown by immunoblotting to correspond to free a2d subunits and a1/a2d/b complexes (12). Thus, detergents such as Triton (17) and Tween 20 (12) which destabilize VDCCs and thus increase the proportion of free a2d subunits may give superior results if Cu21IDA columns are employed. WGA columns have frequently been used in the purification of the skeletal muscle L-type Ca21 channel complexes (31–34); the use of Cu21-IDA and WGA columns in series represents a powerful combination for the isolation of neuronal a2d subunits. To our knowledge this combination has not previously been employed in the study of VDCC subunits. The original identification of the a2d subunit as the molecular target for gabapentin relied on the purification and sequencing of a [3H]gabapentin binding protein from crude brain extracts. The binding protein was operationally defined by the difference in [3H]gabapentin binding observed in the presence and absence of a related competitive ligand, (S)-3-isobutyl GABA. As neither the precursor of gabapentin for radiolabeling nor (S)-3-isobutyl GABA is available commercially, we evaluated the ligand pair L-[3H]leucine/ L-isoleucine as an alternative means for measuring a2d subunits. Experiments with this combination of radioligand and unlabeled displacer were prompted by the observation that neutral L-amino acids have a high affinity for the [3H]gabapentin binding site (23). Initial concerns over possible metabolism and lack of specificity of an L-amino acid, such as [3H]leucine, proved unfounded; even in the presence of crude brain membranes, [3H]leucine was not metabolized over a period longer than the normal assay time. Saturation analysis of ligand binding to brain membranes and purified a2d gave KD values for [3H]leucine and [3H]gabapentin which were broadly consistent with the IC50 values reported for the unlabeled compounds at the [3H]gabapentin binding site (23, 24). The ratio of the Bmax values (leucine:gabapentin) for membranes is 1.66:1. However, this does not suggest a significantly lower specificity of [3H]leucine because a similar ratio of Bmax

values was obtained using purified a2d subunits. Theory suggests that we should obtain identical Bmax values for ligands recognizing the same purified protein; there are two possible explanations for this discrepancy. First, [3H]leucine may have an additional binding site on the a2d subunit. This is unlikely because Scatchard plots were linear and thus consistent with a single population of sites. Second, the quoted specific activity value for each radioligand may deviate from the true value. Imprecision in specific activity values could affect quantitative work, but this applies equally to any radioligand binding assay where the manufacturer’s analysis is generally accepted. The high specificity of [3H]leucine for the a2d subunit is underlined by the near-identical Q-Sepharose elution profiles obtained with [3H]gabapentin and [3H]leucine. A small peak preceding the main a2d peak is present for [3H]leucine but as it is baseline separated and quantitatively minor it does not obscure the fractions to be pooled. While we have focused on the ligand pair [3H]leucine/isoleucine, other neutral L-amino acids with a high affinity for the [3H]gabapentin binding site are known (23). In principle, these ligands could be used to develop assays analogous to the one described here. For example, L-methionine, which is available at very high specific activity values, might afford a highsensitivity assay for tissues and cells with low levels of a2d subunits. Most studies on VDCCs have focused on the L-type channel from rabbit skeletal muscle. Two factors have made this a particularly attractive target: (i) the relatively high density of L-type channel sites and (ii) the commercial availability of a1 subunitspecific radioligands (e.g., dihydropyridines) with which to monitor purification schemes. In a tissue such as brain multiple types of a1 subunits are known, each with different ligand specificities (13). In some cases VDCCs are defined only in terms of their electrophysiological properties and specific radiolabels are not available. [3H]Gabapentin has a number of advantages for monitoring the purification of neuronal a2d subunits and VDCCs: it does not appear to discriminate among splice variants (i.e., the brain, muscle, and heart forms) of the a2d subunit (12), nor does it appear to discriminate between species (9, 23). Thus, [3H]gabapentin offers a means of detecting all VDCCs under conditions that promote subunit association. These comments apply equally to L-[3H]leucine which can be used as a direct replacement for [3H]gabapentin in assays of neuronal a2d subunits. Apart from the fact that it is commercially available [3H]leucine has two other significant advantages over [3H]gabapentin: it is available at a higher specific activity, which leads to better signal-to-noise ratios, and it can be purchased in

ISOLATION AND ASSAY OF a2d Ca21 CHANNEL SUBUNITS

small quantities, thus obviating the need for ligand repurification. In summary, we have described a new purification method for a2d subunits from pig brain. We have also developed and validated a novel assay for the a2d subunit in which [3H]gabapentin and (S)-3-isobutyl GABA are replaced by L-[3H]leucine and isoleucine, respectively. The availability of a simple, inexpensive assay using commercially available reagents should facilitate the isolation and molecular characterization of a2d subunits and VDCC complexes from a variety of sources.

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