(Received for publication, May 2, 1995; and in revised form, November 13, 1995)
From the
Gabapentin (1-(aminomethyl)cyclohexane acetic acid; Neurontin)
is a novel anticonvulsant drug, with a mechanism of action apparently
dissimilar to that of other antiepileptic agents. We report here the
isolation and characterization of a
[H]gabapentin-binding protein from pig cerebral
cortex membranes. The detergent-solubilized binding protein was
purified 1022-fold, in a six-step column-chromatographic procedure,
with a yield of 3.9%. The purified protein had an apparent subunit M
of 130,000, and was heavily glycosylated. The
partial N-terminal amino acid sequence of the M
130,000 polypeptide, EPFPSAVTIK, was identical to that reported
for the
subunit of the L-type Ca
channel from rabbit skeletal muscle (Hamilton, S. L., Hawkes, M.
J., Brush, K., Cook, R., Chang, R. J., and Smilowitz, H. M.(1989) Biochemistry 28, 7820-7828). High levels of
[
H]gabapentin binding sites were found in
membranes prepared from rat brain, heart and skeletal muscle. Binding
of [
H]gabapentin to COS-7 cells transfected with
cDNA was elevated >10-fold over controls,
consistent with the expression of
protein, as
measured by Western blotting. Finally, purified L-type
Ca
channel complexes were fractionated, under
dissociating conditions, on an ion-exchange column;
[
H]gabapentin binding activity closely followed
the elution of the
subunit.
[
H]Gabapentin is the first pharmacological agent
described that interacts with an
subunit of a
voltage-dependent Ca
channel.
Gabapentin (1-(aminomethyl)cyclohexane acetic acid; Neurontin)
is a novel antiepileptic drug that is orally active in various animal
models of epilepsy, including maximal electroshock in rats and
pentylenetetrazol- or audiogenically induced seizures in
mice(1, 2, 3) . Gabapentin has also been
shown to be effective in decreasing the frequency of seizures in
medically refractory patients with partial or generalized
epilepsy(3, 4) . Although originally synthesized as a
lipophilic -aminobutyric acid (GABA) (
)analogue,
capable of penetrating the blood-brain barrier, gabapentin does not
possess a high affinity for either GABA
or GABA
receptors, does not influence neural uptake of GABA and does not
inhibit the GABA-metabolizing enzyme, GABA transaminase (EC
2.6.1.19)(3, 5) . Moreover, gabapentin does not affect
voltage-dependent sodium channels (the site of action of several
antiepileptic drugs, including phenytoin, carbamazepine, and valproate)
and is inactive in assays for a wide range of other neurotransmitter
receptors, enzymes, and ion channels(5, 6) .
A
single high affinity (K = 38
± 2.8 nM) binding site for
[
H]gabapentin in rat brain has been
described(7) . Radioligand binding to brain membranes was
potently inhibited by a range of gabapentin analogues and by several
3-alkyl-substituted analogues of GABA, although GABA itself was only
weakly active. Other antiepileptic drugs including phenytoin, diazepam,
carbamazepine, valproate, and phenobarbitone were inactive. Gabapentin
(IC
= 80 nM) and (RS)-3-isobutyl-GABA (IC
= 80 nM)
were the most active compounds identified(7) . The (S+)-enantiomer of 3-isobutyl-GABA was significantly more
active than the (R-)-enantiomer both in displacing
[
H]gabapentin binding and in preventing maximal
electroshock seizures in mice(8) . These data strongly suggest
that the protein defined by [
H]gabapentin plays
an important role in controlling the excitability of neurons.
Despite extensive research the mechanism of action of gabapentin
remains unclear. In vivo behavioral studies have suggested the
possible involvement of the glycine co-agonist site of the NMDA
receptor complex in the anticonvulsant action of gabapentin;
intracerebroventricular administration of D-serine (a glycine
site agonist) reversed the protection afforded by gabapentin against
chemically induced seizures in mice(9) . However, radioligand
binding assays have not shown gabapentin to inhibit
strychnine-insensitive [H]glycine binding to
brain membranes, or to influence the binding of
[
H]MK-801 to the NMDA receptor
channel(7) . Other reports provide some evidence for an
interaction between gabapentin and an L-system amino acid transporter;
gabapentin crosses the intestinal membrane by a saturable process that
is competitively inhibited by leucine(10) . Moreover, the
binding of [
H]gabapentin to brain membranes is
potently inhibited by neutral L-amino acids and moderately
inhibited by the L-system substrate, BCH(11) . On the other
hand, membranes prepared from tissues known to exhibit L-system
transport activity (e.g. kidney) appear to lack
[
H]gabapentin binding sites(7) . Thus,
the relationship between the L-system transporter and the
[
H]gabapentin-binding protein remains unclear.
To identify the molecular target for gabapentin, we have purified
and characterized a [H]gabapentin-binding protein
from pig cerebral cortex membranes. Partial N-terminal sequencing
identifies the protein as an
subunit of a
voltage-dependent Ca
channel. This identification is
supported by several lines of evidence, including heterologous
expression of rabbit skeletal muscle
cDNA in
COS-7 and HEK cells; and radioligand binding and immunoblotting
studies, after column fractionation of purified L-type Ca
channel complexes under dissociating conditions.
Figure 1:
Four of the six column-chromatographic
steps of the purification scheme. A, Q-Sepharose; B,
Sephacryl S-400; C, hydroxyapatite; D, Mono Q HR 5/5.
, binding activity; - - -, A
.
Figure 2:
SDS-polyacrylamide gel electrophoresis of
purified [H]gabapentin-binding protein eluted
from Mono Q. M, marker proteins. Apparent subunit M
values (
10
) of the
markers are indicated.
The retention of the
[H]gabapentin-binding protein by lectin columns
suggested that the protein was probably glycosylated. Incubation of the
reduced SDS-denatured protein with high concentrations of
glycopeptidase F led to a decrease in the apparent subunit M
from 130,000 to 105,000 (Fig. 3).
Purified [
H]gabapentin-binding protein that had
been subjected to heat denaturation without SDS and 2-mercaptoethanol
was resistant to the enzyme. Thus the
[
H]gabapentin protein is heavily glycosylated,
with N-linked oligosaccharide chains that are accessible to
glycopeptidase F only under fully denaturing conditions.
Figure 3:
SDS-polyacrylamide gel electrophoresis of
[H]gabapentin-binding protein showing the effect
of glycopeptidase F treatment. Prior to deglycosylation, samples of
[
H]gabapentin-binding protein (lanes
1-6) or buffer (lanes 7 and 8) were boiled
in either the absence (lanes 1-3 and 8) or the
presence (lanes 4-7) of SDS/2-mercaptoethanol. Lanes
1 and 4, no enzyme; lanes 2 and 5, 0.1
unit of enzyme; lanes 3 and 6-8, 0.5 unit of
enzyme.
Figure 4:
Inhibition of
[H]gabapentin binding to the purified protein.
Various amino acids and related compounds were tested for their ability
to inhibit radioligand binding. Each point in the dose-response curves
represents the mean from three separate experiments.
, (S+)-3-isobutyl GABA (IC
= 40
nM);
, (R-)-3-isobutyl GABA (IC
= 370 nM);
, L-leucine (IC
= 80 nM);
, D-leucine (IC
= 10,000 nM).
Figure 5:
Specific binding of
[H]gabapentin (A) and
[
H]nitrendipine (B) to membranes
prepared from various rat tissues. The results represent mean values
± S.E. of three separate experiments performed in
duplicate.
Figure 6:
A, binding of
[H]gabapentin to HEK (a), 2L (b), and COS-7 (c and d) cell membranes. The
results represent the mean values ± S.E. of four separate
experiments performed in duplicate (HEK and 2L cells) or two
experiments with replicates of six (COS-7 cells). The 2L line stably
expresses
and
subunits and is a derivative
of HEK 293. COS cells were transfected either with pcDNA3 alone (c) or with a pcDNA-3/
construct (d). The concentration of [
H]gabapentin
in the COS-7 experiments was 60 nM. B, immunoblotting
of COS-7 cell membranes using an affinity-purified anti-
antibody. Membranes were electrophoresed either under reducing (lanes 1-3) or non-reducing (lanes 4-6)
conditions. Lanes 1 and 4, sample c (5 µg of
protein); lanes 2 and 5, sample d (5 µg of
protein); lanes 3 and 6, 200 ng of purified pig brain
[
H]gabapentin-binding protein.
frag. marks the position of a
proteolytic degradation product of the pure protein. C,
Scatchard analysis of binding data obtained with COS-7 cell membranes
(sample d). The data shown are from one representative
experiment.
Figure 7:
A, silver-stained SDS-polyacrylamide gel
of skeletal muscle L-type Ca channels purified on
Mono Q. The column was eluted with a linear NaCl gradient (0-750
mM), and 1-ml fractions were collected (total gradient volume
of 75 ml). Lanes 1-9 correspond to fraction numbers 24,
28, 32, 36, 40, 44, 48, 52, and 56, respectively; a 20-µl aliquot
of each fraction was analyzed; lane 10, molecular weight
markers. B, immunoblotting of Mono Q column fractions.
Nitrocellulose blots were prepared from two gels, each identical to the
one described above. Blots were probed either with a monoclonal
antibody against the skeletal muscle
subunit or with
a polyclonal antibody raised against the pig brain
subunit. Only the relevant portion of each blot is shown. C, binding of [
H]gabapentin to Mono Q
fractions. Assays were performed in triplicate (20 µl of
sample/assay) with 20 nM [
H]gabapentin.
Figure 8:
Binding assays and immunoblotting of
pooled wheat germ eluent Mono Q peak fractions. Nitrocellulose blots
were prepared from three identical SDS-polyacrylamide gels. Lane
1, wheat germ eluent (5 µl); lane 2, Mono Q peak 1
(40 µl); lane 3, Mono Q peak 2 (40 µl). Each blot was
probed with a different primary antibody and an appropriate horseradish
peroxidase-conjugated second antibody. Peroxidase activity was detected
using 3-amino-9-ethylcarbazole ( and
subunits) or luminol (
subunit).
[
H]Gabapentin binding assays on the same samples
were performed in triplicate.
The purification of the detergent-solubilized
[H]gabapentin-binding protein from pig brain was
achieved by sequential chromatography on Q-Sepharose, lentil lectin,
Sephacryl S-400, hydroxyapatite, wheat germ lectin, and Mono Q. The
protein in the Mono Q eluent was analyzed on a 4-20% gradient
SDS-polyacrylamide gel, which covers a broad range of molecular
weights. The amount of radioligand binding paralleled the intensity of
the M
130,000 band, although other faint
co-eluting bands were observed. The possibility that
[
H]gabapentin might bind to one of these
quantitatively minor components was excluded in other experiments (see
below). The final specific activity, based on the peak Mono Q
fractions, was 1584 pmol/mg. This value is approximately 5-fold less
than that expected, given a starting specific activity value of 1.55
pmol/mg for brain membranes. However, the low concentration of protein
in the Mono Q eluent, and the presence of Tween 20, which interfered
with the protein assay, precluded an accurate determination of specific
activity. The susceptibility of the protein to inactivation, which was
particularly evident in phosphate buffers, may also have been a
contributory factor. In all, we have prepared five batches of
[
H]gabapentin-binding protein using this, or a
very similar, procedure. Preparations of higher purity, though with
reduced microgram yields, could be obtained either by reducing the
amount of starting material, or by judicious selection of fractions to
be pooled. However, the purification scheme described here offers a
reasonable compromise between yield and purity.
The partial
N-terminal sequencing of the M 130,000 polypeptide
was a crucial step in the identification of the
[
H]gabapentin-binding protein as an
subunit of a Ca
channel. The
sequence homology with the
subunit predicted a
more widespread tissue distribution for the
[
H]gabapentin-binding protein than has previously
been acknowledged(7) . Indeed, radioligand binding assays
revealed high concentrations of [
H]gabapentin
binding sites not only in brain, but also in skeletal muscle and heart.
The distribution of [
H]nitrendipine sites was
broadly similar to that for [
H]gabapentin,
although detailed differences in the relative levels of the binding
sites were apparent. However as [
H]gabapentin
probably labels both L- and non-L-type voltage-dependent Ca
channels, these differences are not unexpected. To further
confirm the identity of the [
H]gabapentin-binding
protein, we performed radioligand binding assays on cell lines
expressing the
subunit from rabbit skeletal
muscle. High levels of specific [
H]gabapentin
binding in the 2L cell line, which expresses both
and
calcium channel subunits, were
observed, whereas the parental HEK 293 line was almost devoid of
activity. COS-7 cells transfected with
cDNA
alone, expressed >10-fold higher levels of
[
H]gabapentin binding sites than control cells.
These data were consistent with expression of the
protein, as measured by Western blotting using a
polyclonal antibody raised against the
polypeptide.
The expressed
protein bound
[
H]gabapentin with high affinity (K
= 16 nM; n =
2), broadly similar to that determined for rat muscle membranes. These
studies argue strongly that [
H]gabapentin binds
to the
subunit in our purified preparation and
not to a minor contaminant.
Most studies on the structure and
functional domains of Ca channels have focused on the
L-type channel from rabbit skeletal muscle. This channel is a
heteromultimeric complex composed of an
subunit,
which forms the Ca
conducting pore, and three
accessory subunits:
,
, and
. Although
it is now well established that the
subunit is the
target for dihydropyridines, early preparations of the
``dihydropyridine receptor'' apparently contained only an
subunit(25) . It is now known that these
samples were ``contaminated'' with
subunits
that were not visible on SDS gels. In view of the heterologous
expression studies described above, the possibility of
[
H]gabapentin's binding to
subunits in our preparation of purified pig brain
was unlikely. However,
and
subunit cDNAs have been shown to enhance the cell surface
expression of co-transfected
subunits(21, 23, 26) . We considered
the possibility that transfection of
cDNA might
enhance the expression of host-derived
subunits. To address this, we isolated L-type Ca
channel complexes from rabbit skeletal muscle membranes.
Digitonin was employed as the solubilizing agent, as it is reported to
preserve the oligomeric structure of Ca
channels(20) . Purification was achieved using a
combination of lectin chromatography and ion-exchange chromatography,
as used by others(20, 25, 27) . To allow
fractionation of individual subunits, we planned to disrupt the
Ca
channel complexes by exchanging detergents.
However, we found that this step was not necessary; partial
dissociation occurred during chromatography on Mono Q, even in
digitonin-containing buffers. Two peaks of
[
H]gabapentin binding activity, which closely
followed the elution of the
polypeptide, were
observed. The
and
subunits were found only in
the second peak, presumably as
and
complexes. The earlier
elution, from ion-exchange columns, of dissociated
subunits is in agreement with another
study(20) . The profile of the
subunit was not assessed
by immunoblotting, although a faint band of the expected size (35 kDa)
was seen in the peak
fraction (Fig. 7A, lane 7). Data from heterologous
expression studies and purification experiments show conclusively that
the single high affinity [
H]gabapentin binding
site found in brain and muscle membranes is the
subunit. Moreover, it is clear that the binding of
[
H]gabapentin does not require the presence of
the
and
subunits. However, these subunits may
modulate the binding of [
H]gabapentin to the
subunit in hetero-oligomeric complexes. A slight
enhancement of binding to the
subunit, in the
presence of the
and
subunits, is suggested from
the data in Fig. 7(compare lanes 5 and 7) and Fig. 8(
immunoblot; compare lane 2 with lanes 1 and 3). It is interesting to note
that the
subunit itself modulates the binding of
-conotoxin to the
subunit of the N-type
Ca
channel (23) .
The biochemical
properties of the purified pig brain
[H]gabapentin-binding protein are strikingly
similar to those of the
subunit from rabbit
skeletal muscle; the muscle
subunit exhibits a
characteristic mobility shift on SDS-polyacrylamide gels, in the
presence and absence of reducing agents(24, 27) ;
moreover, the removal of N-linked carbohydrate from the
subunit, by exhaustive digestion with glycopeptidase
F, results in an apparent molecular mass of 105 kDa(28) . The
subunit is thought to be anchored to the
membrane by a hydrophobic segment located in the 25-kDa
peptide(24) , although transmembrane segments in the larger
140-kDa
component have also been postulated (29) . Although we have yet to confirm the identity of the
25-kDa component in our purified preparation by N-terminal sequencing,
the poor staining of the band on SDS-polyacrylamide gels is certainly a
characteristic feature of the
polypeptide(28, 30) .
The alteration in the
hydrodynamic properties of the pig brain
[H]gabapentin-binding protein, which occurs
during purification, is almost certainly explained by the dissociation
of the
and
subunits. Takahashi et al. (28) found that the skeletal muscle
subunit was associated with an
heterotrimeric complex in solutions containing either 0.5% digitonin or
0.1% CHAPS. However, in 0.5% Triton X-100, the
subunit was found to dissociate from the tripartite complex. Our
hydrodynamic data suggest that the
[
H]gabapentin-binding protein is associated with
other proteins immediately following solubilization with 0.4% Tween 20,
and that dissociation begins on the Q-Sepharose column. The presence of
both free and complexed
subunits may explain
trailing of the [
H]gabapentin binding activity
profile for this column.
The
physiological role of the subunit is not well
understood at present. Co-expression of
with the
and
subunits is known to be required for
efficient assembly and functional expression of Ca
channel complexes(21, 23, 26) . Since
the
subunit appears to be common to all
voltage-dependent Ca
channels(26, 35) , it is conceivable that
gabapentin modulates the activity of more than one type of neuronal
Ca
channel. In mouse spinal cord neurons gabapentin
blocked responses to BAY K 8644 (36) , but in other studies
gabapentin did not significantly affect L-, N-, or T-type
voltage-dependent Ca
channels (37) . However,
given the structural diversity of Ca
channels, as
revealed by molecular cloning studies(26, 35) , data
from a few electrophysiological studies should be interpreted with
caution. At least six genes encoding Ca
channel
subunits have been identified. Classes C, D, and S
are sensitive to dihydropyridines (L-type), class B is sensitive to
-conotoxin GVIA (N-type), and class A is sensitive to
-agatoxin IVA (P-type). Class E is resistant to the agents listed
above (B- and T-type channels). At least four genes encode
subunits, one gene encodes the
subunit, and
multiple splice variants of
,
and
have been described(26, 35) .
The potential combinational heterogeneity of Ca
channels at the structural level is enormous. It is possible that
gabapentin exerts functional effects only with particular
combinations of subunits. Moreover, these effects may be observed only
under conditions that mimic closely the excessive repetitive discharges
that characterize clinical epilepsy. Further studies on the cellular
electrophysiological actions of gabapentin, in a variety of systems,
are required before the action of the drug at Ca
channels can be fully understood.
Finally, the data reported
here show that the protein labeled by
[H]gabapentin in brain membranes is not the
L-system transporter. However, the high affinity interaction of certain
L-system substrates (e.g.L-leucine and L-methionine; (11) ) with the
subunit is intriguing. To our knowledge the effects of neutral amino
acids on the functional activity of voltage-dependent Ca
channels have not been investigated. However, endogenous ligands
such as these presumably compete with gabapentin in vivo for
the binding site on the
subunit. This perhaps
explains why the therapeutic concentration of gabapentin (2) is
well above the K
of the drug at the
[
H]gabapentin binding site. We cannot say from
present data whether the gabapentin binding site is located on the
or the
component, or whether it is
extracellularly or intracellularly disposed. However, the heavy
glycosylation of
(24, 28) and
weak labeling by hydrophobic photoaffinity probes (28) suggest
that the bulk of the
subunit is found at the
extracellular surface. Further studies on the topology of the
subunit and the precise location of the
[
H]gabapentin binding site are required.
In
summary, we have purified and characterized a high affinity
[H]gabapentin-binding protein from pig brain
membranes. N-terminal sequencing has identified the protein as an
subunit of a voltage-dependent Ca
channel. This conclusion is supported by tissue distribution
studies, by hydrodynamic data, by heterologous expression of cloned
cDNA in COS-7 and HEK cells, and by radioligand
binding and immunoblotting studies on fractionated Ca
channel subunits. [
H]Gabapentin is the
first ligand described that interacts with the
subunit. We suggest that modulation of voltage-dependent neuronal
Ca
channels may be important to the antiepileptic
action of gabapentin.