Pharmacological disruption of calcium channel trafficking by the alpha2delta ligand gabapentin.
ABSTRACT The mechanism of action of the antiepileptic and antinociceptive drugs of the gabapentinoid family has remained poorly understood. Gabapentin (GBP) binds to an exofacial epitope of the alpha(2)delta-1 and alpha(2)delta-2 auxiliary subunits of voltage-gated calcium channels, but acute inhibition of calcium currents by GBP is either very minor or absent. We formulated the hypothesis that GBP impairs the ability of alpha(2)delta subunits to enhance voltage-gated Ca(2+)channel plasma membrane density by means of an effect on trafficking. Our results conclusively demonstrate that GBP inhibits calcium currents, mimicking a lack of alpha(2)delta only when applied chronically, but not acutely, both in heterologous expression systems and in dorsal root-ganglion neurons. GBP acts primarily at an intracellular location, requiring uptake, because the effect of chronically applied GBP is blocked by an inhibitor of the system-L neutral amino acid transporters and enhanced by coexpression of a transporter. However, it is mediated by alpha(2)delta subunits, being prevented by mutations in either alpha(2)delta-1 or alpha(2)delta-2 that abolish GBP binding, and is not observed for alpha(2)delta-3, which does not bind GBP. Furthermore, the trafficking of alpha(2)delta-2 and Ca(V)2 channels is disrupted both by GBP and by the mutation in alpha(2)delta-2, which prevents GBP binding, and we find that GBP reduces cell-surface expression of alpha(2)delta-2 and Ca(V)2.1 subunits. Our evidence indicates that GBP may act chronically by displacing an endogenous ligand that is normally a positive modulator of alpha(2)delta subunit function, thereby impairing the trafficking function of the alpha(2)delta subunits to which it binds.
- SourceAvailable from: Annette C Dolphin[Show abstract] [Hide abstract]
ABSTRACT: The auxiliary α2δ-1 subunit of voltage-gated calcium channels is up-regulated in dorsal root ganglion neurons following peripheral somatosensory nerve damage, in several animal models of neuropathic pain. The α2δ-1 protein has a mainly presynaptic localization, where it is associated with the calcium channels involved in neurotransmitter release. Relevant to the present study, α2δ-1 has been shown to be the therapeutic target of the gabapentinoid drugs in their alleviation of neuropathic pain. These drugs are also used in the treatment of certain epilepsies. In this study we therefore examined whether the level or distribution of α2δ-1 was altered in the hippocampus following experimental induction of epileptic seizures in rats, using both the kainic acid model of human temporal lobe epilepsy, in which status epilepticus is induced, and the tetanus toxin model in which status epilepticus is not involved. The main finding of this study is that we did not identify somatic overexpression of α2δ-1 in hippocampal neurons in either of the epilepsy models, unlike the upregulation of α2δ-1 that occurs following peripheral nerve damage to both somatosensory and motor neurons. However, we did observe local reorganisation of α2δ-1 immunostaining in the hippocampus only in the kainic acid model, where it was associated with areas of neuronal cell loss, as indicated by absence of NeuN immunostaining, dendritic loss, as identified by areas where microtubule-associated protein-2 immunostaining was missing, and reactive gliosis, determined by regions of strong OX42 staining.Neuroscience 03/2014; · 3.12 Impact Factor
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ABSTRACT: Introduction: Chronic pain conditions are serious clinical concerns. Its genesis is closely associated with sensitization of nociceptive primary sensory neurons (nociceptors) and dorsal horn neurons by various pain mediators produced during inflammation and tissue injury. Growing evidence showed that increasing cell surface trafficking of pain-facilitating receptors is an important mechanism underlying the peripheral and central sensitization. Areas covered: We summarized the progress of this area over the past decade by showing that inflammation, tissue damage or pain mediators facilitate cell surface trafficking of pain-facilitating receptors such as transient receptor potential vanilloid-1, transient receptor potential ankyrin-1, voltage-gated sodium channel 1.8, P2X3 and EP4 in primary sensory neurons, GluR1 and GluR2 of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, NR1 and NR2 of N-methyl-d-aspartate receptors and acid-sensing ion channels 1 in dorsal horn neurons and P2X4 in spinal microglia. The anti-allodynic effects of gabapentin was mediated by blocking surface trafficking of α2δ1 and α2δ2 subunits of voltage-gated calcium channels in primary sensory and dorsal horn neurons. Expert opinion: Pain mediators stimulate forward surface trafficking of their own and/or other pain-facilitating receptors to amplify pain intensity and duration. Enhancing surface abundance of pain-facilitating receptors in nociceptors and dorsal horn neurons is an important mechanism underpinning chronic pain states. Targeting specific trafficking events of pain-facilitating receptors may open a novel therapeutic avenue to more efficiently treat chronic pain conditions.Expert Opinion on Therapeutic Targets 02/2014; · 4.90 Impact Factor
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ABSTRACT: To evaluate perioperative administration of gabapentin as an adjunct for analgesia in dogs undergoing amputation of a forelimb. Randomized, controlled trial. 30 client-owned dogs. Procedures-On the day before surgery, a baseline pain evaluation was performed in each dog by use of multiple pain assessment methods. Dogs then received gabapentin (10 mg/kg [4.5 mg/lb], PO, once, followed by 5 mg/kg [2.3 mg/lb], PO, q 12 h for 3 additional days) or a placebo. On the day of surgery, dogs were anesthetized and forelimb amputation was performed. Fentanyl was infused after surgery for 18 to 24 hours; use of other analgesics was allowed. In-hospital pain evaluations were repeated at intervals for 18 hours after surgery, and owners were asked to evaluate daily their dog's activity, appetite, and wound soreness for the first 3 days after discharge from the hospital. Results were analyzed by use of a repeated-measures ANOVA. Pain evaluation scores did not differ significantly between gabapentin and placebo groups in the hospital or at home after discharge. As an adjunct to other analgesics and anesthetics, gabapentin, at the dose and frequency used in this study, did not provide a significant benefit for the management of acute perioperative pain in dogs undergoing forelimb amputation. The small sample size and number of other confounding factors, such as aggressive use of other analgesics, limited the likelihood of detecting a benefit of gabapentin. Other gabapentin doses or dosing regimens warrant further study.Journal of the American Veterinary Medical Association 04/2010; 236(7):751-6. · 1.72 Impact Factor
Pharmacological disruption of calcium channel
trafficking by the ?2? ligand gabapentin
Jan Hendrich*, Alexandra Tran Van Minh*, Fay Heblich*, Manuela Nieto-Rostro*, Katrin Watschinger†, Jo ¨rg Striessnig†,
Jack Wratten*, Anthony Davies*, and Annette C. Dolphin*‡
*Laboratory for Cellular and Molecular Neuroscience, Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom;
and†Institut fu ¨r Pharmazie, Abteilung fu ¨r Pharmakologie und Toxikologie, Universita ¨t Innsbruck, A-6020 Innsbruck, Austria
Edited by Richard W. Tsien, Stanford University School of Medicine, Stanford, CA, and approved January 15, 2008 (received for review September 20, 2007)
The mechanism of action of the antiepileptic and antinociceptive
drugs of the gabapentinoid family has remained poorly under-
stood. Gabapentin (GBP) binds to an exofacial epitope of the ?2?-1
acute inhibition of calcium currents by GBP is either very minor or
absent. We formulated the hypothesis that GBP impairs the ability
of ?2? subunits to enhance voltage-gated Ca2?channel plasma
membrane density by means of an effect on trafficking. Our results
conclusively demonstrate that GBP inhibits calcium currents, mim-
icking a lack of ?2? only when applied chronically, but not acutely,
both in heterologous expression systems and in dorsal root-
ganglion neurons. GBP acts primarily at an intracellular location,
requiring uptake, because the effect of chronically applied GBP is
blocked by an inhibitor of the system-L neutral amino acid trans-
porters and enhanced by coexpression of a transporter. However,
it is mediated by ?2? subunits, being prevented by mutations in
either ?2?-1 or ?2?-2 that abolish GBP binding, and is not observed
for ?2?-3, which does not bind GBP. Furthermore, the trafficking of
?2?-2 and CaV2 channels is disrupted both by GBP and by the
mutation in ?2?-2, which prevents GBP binding, and we find that
GBP reduces cell-surface expression of ?2?-2 and CaV2.1 subunits.
Our evidence indicates that GBP may act chronically by displacing
an endogenous ligand that is normally a positive modulator of ?2?
subunit function, thereby impairing the trafficking function of the
?2? subunits to which it binds.
pore-forming ?1 subunit, associated with a membrane-
anchored, predominantly extracellular, ?2? subunit (for review
see ref. 1) and an intracellular ? subunit (for review see ref. 2).
Mammalian genes encoding four ?2? subunits have been iden-
tified (for reviews see refs. 2 and 3). The topology of the ?2?
protein was first determined for ?2?-1 and is thought to gener-
alize to all ?2? subunits (for reviews see refs. 1 and 4). They are
type I transmembrane proteins, the exofacial ?2subunit being
disulfide-bonded to a transmembrane ? subunit, formed by
posttranslational cleavage of the ?2? preprotein (5).
The mechanism of action of the antiepileptic and antinoci-
ceptive drugs of the gabapentinoid family has remained poorly
an analog of ?-amino-butyric acid (GABA), but is now believed
to have no effect on GABA receptors or transporters (for review
see ref. 6). The first key to understanding the mechanism of
action of GBP came from purification of the GBP-binding
protein from porcine brain (7), which was identified as the ?2?-1
auxiliary subunit of VGCCs. It is now known that GBP binds to
an exofacial epitope present in both the ?2?-1 and ?2?-2 subunits
(for reviews see refs. 1 and 8). However, although it was
originally reported that GBP application results in acute inhi-
bition of calcium currents (9, 10), in most studies, acute inhibi-
tion by GBP is either very minor or absent (for review see ref.
1). Furthermore, electrophysiological studies of synaptic trans-
mission are also equivocal, with some studies reporting inhibi-
tion by GBP (11), and other studies reporting no inhibition (12).
oltage-gated Ca2?channels (VGCCs) are heteromeric com-
plexes. The CaV1 and CaV2 subfamilies are made up of a
When neurotransmitter release is measured, there appears to be
little or no effect on depolarization-stimulated release of several
different neurotransmitters, but inhibition of release when this
has been enhanced by specific mediators (13–15). In general,
none of these studies points to the mechanism of action being a
simple inhibition of calcium currents.
We formulated the hypothesis that GBP impairs the ability of
?2? subunits to enhance VGCC plasma membrane density, via
an effect on trafficking. This would be in agreement with the
finding that in vivo responses to gabapentinoid drugs are fairly
slow in onset (16), and GBP does not inhibit acute pain (for
reviews see refs. 6 and 17).
Inhibition of CaV2.1 and CaV2.2 Currents by Chronic, but Not Acute,
GBP. We first compared the ability of GBP to inhibit calcium
currents, either acutely, or when included for 40 h in the culture
medium after transfection of tsA-201 cells with CaV2.1/?4 and
?2?-2. Chronic incubation with GBP (1 mM) reduced currents
formed with the WT ?2?-2 by 72.2 ? 4.2% at ?15 mV (Fig. 1A).
Furthermore, whereas the coexpression of ?2? subunits typically
shifted the voltage dependence of steady-state inactivation to
more negative potentials (Fig. 1B), this was depolarized by 9.3
mV in the continued presence of GBP (Fig. 1B). Thus, chronic
GBP mimicked a lack of influence of ?2? on the currents. In
CaV2.1/?4 was transfected into a cell line stably expressing ?2?-2,
where peak currents were 53.4 ? 14.0% smaller when cells were
cultured with 1 mM GBP [supporting information (SI) Fig. 5A].
Furthermore, the steady-state inactivation was again depolar-
ized by 11 mV by GBP (SI Fig. 5B), and the currents showed
significantly slower inactivation (SI Fig. 5 C and D). In contrast,
when GBP was applied either acutely for 10 min (Fig. 1C), or for
3–6 h before recording (n ? 8, data not shown), it had no effect
on IBain the same system.
A high concentration of GBP was used initially, because our
hypothesis required GBP either to be taken up by system-L
transporters and subsequently to bind intracellular ?2? subunits
and affect forward trafficking or, alternatively, to bind directly
to cell surface ?2? subunits and affect trafficking at the level of
endocytosis or recycling. For both of these sites, it will be
Author contributions: J.H. and A.T.V.M. contributed equally to this work; J.H., A.T.V.M.,
A.T.V.M., M.N.-R., K.W., J.S., and A.D. contributed new reagents/analytic tools; F.H. and
A.C.D. analyzed data; and A.C.D. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
‡To whom correspondence should be addressed at: Department of Pharmacology, Univer-
sity College London, Gower Street, London, WC1E 6BT, United Kingdom. E-mail:
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
March 4, 2008 ?
vol. 105 ?
competing with other large neutral amino acids (18, 19), and
both isoleucine and leucine are present at 800 ?M and valine at
400 ?M in the culture medium. However, plasma concentrations
of 70–120 ?M GBP are clinically relevant (20), and chronic
of IBa, by 45.0 ? 9.9% (Fig. 1 D and E). It also slowed the
inactivation of the currents, again a hallmark of calcium channel
currents not influenced by ?2? (SI Fig. 6 A and B). In the
prolonged presence of GBP, the CaV2.1/?4/?2?-2 current density
approached that observed in the absence of ?2? subunits
The effect of chronic GBP was also observed in the presence
of ?2?-1, because 1 mM GBP reduced peak CaV2.2/?1b/?2?-1
currents by 70.5 ? 7.3% (Fig. 1F), and depolarized the midpoint
for steady-state inactivation from ?55.5 ? 3.4 mV to ?39.7 ?
2.6 mV (n ? 4 for both, data not shown). These results also
provide evidence that GBP is not selective for a particular
subtype of CaV2 channel composition.
Prevention of the Effect of Chronic GBP by Mutation of ?2?-1 or ?2?-2.
Mutation of a single amino acid, R217A, in an RRR motif in
?2?-1 (21, 22), or the equivalent residue R282A in ?2?-2 (23), has
been found almost completely to abrogate GBP binding. To
demonstrate that the effect of chronic GBP was indeed due to
binding to ?2? subunits, we examined whether it would have any
effect on currents formed by CaV2.1/?4 cotransfected with
R282A-?2?-2. Chronic incubation with GBP (1 mM) did not
inhibit these currents (Fig. 2A) or result in any shift in the
steady-state inactivation (Fig. 2B). Furthermore, there was no
effect of GBP on currents formed with CaV2.2/?1b/R217A-?2?-1
(Fig. 2C), or CaV2.1/?4/?2?-3 (peak IBa was ?286.58 ? 32.3
pA/pF at ?15 mV for control, n ? 8; compared with ?258.7 ?
46.8 pA/pF for chronic GBP, n ? 9). These results agree with the
fact that none of these ?2? subunits bind GBP (data not shown).
Evidence for an Intracellular Site of Action of GBP. The in vivo
potency of GBP has been shown to depend both on binding to
?2? subunits and on substrate activity for system-L amino acid
transporters (24, 25), attributed to the requirement for the
zwitterionic drug to pass the blood–brain barrier (18). To
determine whether the effect of chronic GBP also required its
uptake at the single-cell level (as outlined in Fig. 2D), we
included the inhibitor 2-(?)-endoamino-bicycloheptene-2-
carboxylic acid (BCH) in the medium, either alone or together
with GBP for 40 h. BCH has a very low affinity for displacement
of GBP binding to ?2?-1 and ?2?-2 subunits (26), and it had no
effect on IBaamplitude when applied alone at 10 mM. The peak
current density at ?15 mV was ?330.2 ? 84.7 pA/pF (n ? 7) for
control and ?333.0 ? 73.2 pA/pF (n ? 8) in the presence of
BCH. However, chronic BCH prevented the effect of chronic
its steady-state inactivation (data not shown). This effect of BCH
is likely to result from its ability to block GBP uptake (18).
To obtain further evidence that GBP uptake is required for its
effect, we used Xenopus oocytes, which express only a low level
of endogenous system-L transporter activity (27). We found that
chronic incubation with 200 ?M GBP only significantly inhibited
CaV2.2/?1b/?2?-2 currents (by 57%, Fig. 2F) when a system-L
transporter protein LAT4 (27) was coexpressed. Expression of
LAT4 had no significant effect on the peak IBaat ?5 mV, which
was ?0.49 ? 0.12 ?A (n ? 34) and ?0.41 ? 0.06 (n ? 41) ?A
in the absence and presence of LAT4, respectively, from four
We also found that chronic incubation of oocytes for 40 h in
the presence of L-leucine (400 ?M) significantly enhanced IBa
only when LAT4 was coexpressed (Fig. 2G). Furthermore,
L-leucine did not enhance the small currents obtained in the
absence of ?2?, despite the presence of LAT4 (Fig. 2G). This is
applied chronically but not acutely. (A) (Left) Current density–voltage (IV)
relationships for CaV2.1/?4/?2?-2 currents in the absence or presence of
chronic GBP (1 mM, red circles, n ? 11) for ?40 h (or H2O as control, black
squares, n ? 13) from immediately after transfection of tsA-201 cells until
recordings were performed. The reduction in peak IBaat ?15 mV was statis-
tically significant (P ? 0.0013, Student’s two-tailed t test). (Right) Examples of
mV in 5-mV increments, under control conditions and in the presence of GBP.
Calibration bars refer to both sets of traces. (B) Steady-state inactivation data
Data are fit to a single Boltzmann equation. The V50,inactwas 37.6 ? 2.1 mV in
the presence of ?2?-2, ?30.8 ? 4.4 mV in the absence of ?2? (dashed line), and
?28.3 ? 2.7 mV in the continued presence of GBP (P ? 0.0103 compared with
for 10 min had no effect on CaV2.1/?4/?2?-2 currents. Examples of currents
resulting from step potentials from ?90 mV to ?10 mV, under control con-
ditions and after application of GBP for 10 min, to a cell whose initial current
peak current (red trace) after application of GBP was 99.0 ? 7.1% of its initial
value, before GBP application (black trace). (D) IV relationships for CaV2.1/?4/
?2?-2 currents from cells cultured in the absence or presence of 100 ?M GBP
applied chronically as described in A. GBP (red circles, n ? 10) and control
(black squares, n ? 14). The reduction in peak IBaat ?15 mV was statistically
significant (P ? 0.0215, Student’s two-tailed t test). (E) The percentage of
inhibition of peak current density by GBP (100 ?M, hatched red bar, n ? 10;
and 1 mM, solid red bar, n ? 11) was determined for each experiment and
The statistical significance of the reduction was determined relative to the
data in the presence of ?2?-2 and absence of GBP for each individual set of
experiments;*, P ? 0.0013;**, P ? 0.021;***, P ? 0.00004, Student’s
two-tailed t test. (F) (Left) IV relationships for CaV2.2/?1b/?2?-1 currents in the
equivalent amount of H2O as control (black squares, n ? 8). (Right) Examples
of currents resulting from step potentials from ?90 mV to between ?15 and
presence of GBP (red traces). Calibration bars refer to both sets of traces.
GBP is effective to inhibit IBaafter heterologous expression, when
Hendrich et al.
March 4, 2008 ?
vol. 105 ?
no. 9 ?
compatible with the view that an endogenous low-molecular-
weight ligand, such as L-leucine itself, may normally occupy the
GBP-binding sites on ?2?-1 and ?2?-2 and be a positive modu-
lator required for the full functionality of ?2? subunits (1, 23).
The existence of an endogenous ligand was previously suggested
by the observation that the apparent affinity for GBP is in-
creased by 5- to 10-fold upon dialysis or partial purification of
?2? subunits (19, 23).
In agreement with this hypothesis, for both R217A-?2?-1 and
R282A-?2?-2, the peak currents were consistently smaller, com-
pared with the WT ?2? subunits, by ?62.5% and 34.5% respec-
tively (see also refs. 23 and 28). However, they were significantly
greater than in the absence of any ?2? subunit, where IBaat ?15
mV was ?33.9 ? 5.8 pA/pF for CaV2.2/?1b (n ? 9, P ? 0.05) and
?65.1 ? 8.8 pA/pF for CaV2.1/?4 (n ? 13, from ref. 24). This is
compatible with the hypothesis that the RRA mutant ?2?
subunits are defective either in forward trafficking or in main-
taining mature VGCC complexes at the plasma membrane.
Chronic GBP Reduces Plasma Membrane Expression of Calcium Chan-
nels. To probe further whether GBP affected VGCC trafficking,
we then examined the subcellular distribution of VGCC sub-
units, using CaV2.1 with an exofacial double hemagglutinin
(HA) tag (CaV2.1–2HA, Fig. 3 A–C). Under control conditions,
CaV2.1 and ?2?-2 were colocalized, both intracellularly (Fig. 3A)
and also at the plasma membrane, as seen most clearly in
nonpermeabilized cells (Fig. 3B). The plasma membrane expres-
sion in nonpermeabilized cells was quantified from low-
magnification images including those in Fig. 3C, in which
cell-surface expression is seen across the entire cell because of
the depth of the optical section (4.5 ?m for these images) and the
flattened nature of the cells. Chronic incubation with 100 ?M
and 1 mM GBP significantly reduced the expression of both
CaV2.1 and ?2?-2 at the plasma membrane (Fig. 3C) as well as
increasing the nonuniform intracellular clustering of both sub-
units (Fig. 3A). A very similar result was observed for GFP–
CaV2.2, which is expressed throughout transfected Cos-7 cells,
typically showing a fairly uniform distribution [SI Fig. 7 (29)].
Using permeabilized cells, we found that WT ?2?-2 colocalizes
with GFP-CaV2.2, both intracellularly and at the plasma mem-
brane (SI Fig. 7Ai). Chronic GBP application resulted in regions
of intracellular clustering in most cells, both for ?2?-2 and for
GFP-CaV2.2 (SI Fig. 7A ii and iii, quantified in SI Fig. 7B).
To quantify further the effect of GBP on cell-surface expres-
sion of ?2?-2, we used cell-surface biotinylation and found that
chronic incubation with GBP (100 ?M-1 mM) reduced the
proportion of ?2?-2 expressed at the cell surface (Fig. 3D). The
biotinylation procedure did not induce cell permeabilization,
and this was not affected by chronic incubation with GBP,
because no biotinylation of the intracellular protein Akt was
observed (Fig. 3D).
In contrast, chronic GBP did not reduce the level of R282A-
?2?-2 expressed at the cell surface (SI Fig. 8A). Furthermore, in
the presence of R282A-?2?-2, there was a less uniform distri-
bution of both the mutant ?2?-2 and GFP-CaV2.2 than that seen
cDNA (nonconducting Kir2.1-AAA (30) (Left) or with mLAT4 (27) (Right).
Oocytes were incubated without (black bars) or with 200 ?M GBP (red bars)
from 1 h after the time of injection until recording between 40 and 48 h later.
To combine data from several experiments, the mean peak control IBaat ?5
mV was normalized and the effect of GBP determined relative to control. The
numbers of determinations are shown on the bars. Statistical significance:**,
and Bonferroni’s post hoc test). (G) As in E, with the combinations of trans-
fected subunits indicated below the bars. Oocytes were incubated without
(black bars) or with (white bars) 400 ?M L-leucine. The statistical significance
is P ? 0.011 (*) for the effect of L-leucine in the mLAT4 condition and only in
the presence of ?2?-2.
by neutral amino acid transporter. (A) (Left) IV relationships for CaV2.1/?4/
R282-?2?-2 currents in the absence or presence of chronic GBP (1 mM, red
circles, n ? 12) for ?40 h or the equivalent amount of H2O as control (black
squares, n ? 12), from immediately after transfection until recordings were
performed. (Right) Examples of currents resulting from step potentials from
?90 mV to between ?15 and ?15 mV in 5-mV increments under control
as described in A. Chronic GBP (1 mM, red circles, n ? 5) and control (black
squares, n ? 7). (C) (Upper) IV relationships for CaV2.2/?1b/R217A-?2?-1 cur-
rents in the absence or presence of chronic GBP. The cDNAs were transfected
into tsA-201 cells that were then incubated with GBP (1 mM, red circles, n ?
Examples of currents resulting from step potentials from ?90 mV to between
?15 and ?15 mV in 5-mV increments under control conditions and in the
presence of 1 mM GBP. Calibration bars refer to both sets of traces. (D)
Diagram illustrating some of the various sites at which GBP (red circles) or the
may exert its effect on intracellular ?2? subunits during maturation and
trafficking of the VGCC complex to the plasma membrane and/or bind to cell
surface ?2? subunits and affect recycling from the plasma membrane. BCH is
a competitive inhibitor of system-L transport. (E) IV relationships for CaV2.1/
chronic treatment with GBP (1 mM), in the absence (red filled circles, n ? 7) or
presence (red open circles, n ? 10) of the inhibitor of system-L transport, BCH
(10 mM) applied chronically from 1 h before GBP. The reduction in peak IBaby
GBP alone at ?10 mV was statistically significant compared with control (P ?
0.0464, Student’s two-tailed t test). (F) IBawas measured in Xenopus oocytes
after injection of CaV2.2/?1b/?2?-2 cDNAs, together with either a control
www.pnas.org?cgi?doi?10.1073?pnas.0708930105Hendrich et al.
with WT ?2?-2, and less uniform colocalization of the R282A-
?2? subunit and GFP-CaV2.2 (SI Fig. 8 B and C). GBP had no
additional effect on the subunit distribution found with R282A-
?2?-2 (SI Fig. 8 B and C).
Chronic GBP Inhibits Native Calcium Currents in DRG Neurons. To
determine the relevance of our findings to native VGCCs in a
neuronal system relevant to the therapeutic use of GBP, we
examined whether GBP had a similar chronic effect on IBa
density in cultured adult dorsal root ganglion (DRG) neurons.
We found a marked reduction in peak high voltage-activated
(HVA) calcium currents (by 50.7 ? 11.2% at 0 mV, n ? 19, P ?
0.02), when DRGs were incubated with 1 mM GBP for 3 days
(Fig. 4A). Contamination with T-type current was avoided by
measuring peak current from a holding potential of ?40 mV,
although a similar result was obtained when measuring IBaat the
end of a 50-ms step from ?80 mV (Fig. 4A). In contrast, acute
application of GBP for 10 min had no effect on adult DRG IBa
Subsequent to the finding that GBP binds to certain VGCC ?2?
subunits (19), the evidence is now very strong that gabapentinoid
Cells were then fixed and permeabilized before immunocytochemical localiza-
tion of CaV2.1 (HA Ab, Left) and ?2?-2 [?2-2 (102–117) Ab, Center], by using a
in green and ?2?-2 in red, with regions of colocalization in orange–yellow).
bar: 30 ?m.) No signal was observed in nontransfected cells or in the absence of
permeabilized. The transfected cell(s) in each image are identified by an arrow.
Similar results were obtained in three independent experiments. (Scale bar: 30
?m.) (C) (Left) Merged images of nonpermeabilized cells taken with a magnifi-
The optical section for these images is 4.5 ?m, and, as the cells are flattened,
staining is seen over most of the cell surface. (Scale bar: 60 ?m.) (Right) Quanti-
and ?2?-2 (gray bars) from magnification ?20 images under control conditions
test. (D) The effect of GBP (100 ?M and 1 mM) was determined on cell-surface
(Left) Whole-cell lysate (WCL). (Right) Streptavidin pull-down of biotinylated
estimated from the ratio of the biotinylated ?2?-2 to the amount of ?2?-2 in the
WCL. Data are from five experiments; 4.93 ? 1.92% of total ?2?-2 was at the
(n ? 5, P ? 0.05, repeated-measures ANOVA and Bonferroni’s post hoc test).
Effect of chronic GBP on the plasma membrane localization of CaV2.1
relationships for HVA IBarecorded from rat DRGs cultured for 3 days in the
absence (black squares, n ? 22) or presence of GBP (1 mM, red circles, n ? 19).
(Upper Right) Example HVA currents from a holding potential (VH) of ?40 mV
to between ?30 and ?10 mV in 10-mV steps. Upper traces (black) control;
lower traces (red) chronic GBP (1 mM). IBawas measured at 20 ms, as indicated
by the dotted line. IBaat 0 mV was significantly inhibited by chronic GBP (P ?
0.02, Student’s two-tailed t test). (Lower) As above, except VHwas ?80 mV,
the dotted line. IBawas obtained in the absence (black squares, n ? 12), or
had no effect on native DRG HVA currents. (Left) Bar chart showing lack of
bar) to cells whose initial current amplitude had stabilized (n ? 6 for each).
(Right) Examples of currents resulting from a step potential from VH?40 mV
to ?10 mV under control conditions (black trace) and from the same cell after
application of GBP (red trace) for 10 min.
Effect of GBP on native calcium currents in DRGs. (A) (Upper Left) IV
Hendrich et al.
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drugs have their therapeutic effect via this route, because an
R217A ?2?-1 knockin mouse develops neuropathic pain in
response to nerve injury, but this is unresponsive to either GBP
or its analog pregabalin (28). We now show that GBP is an
inhibitor of VGCC trafficking, rather than a direct inhibitor of
calcium currents, and thus exerts its inhibitory effects primarily
on intracellular ?2? subunits. We have shown that ?2? subunits
have their main effect on VGCC trafficking (30), and that the
von Willebrand factor-A (VWA) domain is key to this process.
It is notable that GBP binds to the two ?2? subunits that have
VWA domains with perfect metal-ion adhesion site (MIDAS)
motifs (31). It has been postulated that all such VWA domains
undergo a conformational change on binding the protein ligand
of this domain (30, 31), and one might speculate that gabap-
entinoid drugs could interfere with this function of the VWA
domain. The reduction in cell-surface expression of ?2?-2 and
CaV2.1 in the presence of GBP and the reduced colocalization
of R282A-?2?-2 with CaV2.2 provides supporting evidence for
The findings presented here could account for the lack of
effect, or small responses, reported to acute GBP in different
experimental systems, both on calcium currents and on synaptic
transmission (for review see ref. 1). Previous studies examining
more prolonged exposure to GBP have also shown either no
inhibition or a small inhibition of current amplitude (32, 33). Our
finding that the effect of GBP requires its uptake into individual
cells explains the requirement for a relatively high concentration
of GBP, because it will need to outcompete large neutral amino
acids for uptake at this site. It is clear that the trafficking of
VGCCs and their insertion and removal from the plasma
membrane proceeds dynamically (see, for example, ref. 34), and
these rates are likely to depend on the physiological state of the
neurons, and the amount of ?2? subunits present, which both
enhance forward trafficking and decrease the turnover of
VGCCs (30, 35). Insertion of VGCCs into the plasma membrane
of presynaptic terminals or axons may occur rapidly under
certain conditions, for example, after the induction of neuro-
pathic pain, when ?2?-1 subunit levels are elevated. In this
regard, it has been reported that GBP slowly inhibited calcium
currents over a period of minutes in DRGs from ?2?-1-
overexpressing mice (mimicking the neuropathic pain state), but
not WT mice (36).
In conclusion, the action of GBP elucidated here, to inhibit
VGCC trafficking and plasma membrane expression, represents
a previously uncharacterized mechanism of drug action, and
points the way forward for development of screens for drug
Tissue Culture and Heterologous Expression of cDNAs. Details are provided in
Electrophysiology. Calcium channel currents were recorded essentially as de-
scribed (37, 38) and detailed in SI Methods.
Immunoblotting. Immunoblot analysis was performed essentially as described
(30). SDS/PAGE-resolved samples were transferred to PVDF membranes and
probed with relevant primary Abs as described (30) and with the appropriate
horseradish peroxidase-conjugated secondary Abs, followed by enhanced
(39) and detailed in SI Methods.
Biotinylation Assay At 72 h after transfection with the cDNAs described, cells
were rinsed three times with PBS and then incubated with PBS containing 1
mg/ml Sulfo-NHS-SS-Biotin (Pierce) for 30 min at 4°C. The biotin solution was
room temperature to quench the reaction. The cells were gently rinsed three
times with PBS and then lysed. Half of the cell lysate was loaded onto a 3–8%
Tris-Acetate gel to determine total protein expression. Equal amounts of
biotinylated proteins were precipitated by adding 50 ?l of streptavidin–
agarose beads (Pierce) and incubated overnight at 4°C. The streptavidin–
agarose beads were washed three times and incubated with 100 mM DTT in
2? Laemmli sample buffer for 1 h at 37°C. Eluted proteins were then resolved
by SDS/PAGE. Immunoblotting for the cytosolic protein Akt was used as a
control to determine that intracellular proteins were not biotinylated as a
result of cellular damage.
Statistical Analysis Data are given as means ? SEM, and the statistical tests
t test, as stated.
ACKNOWLEDGMENTS. We thank Dr. S. Bodoy (University of Barclona, Barce-
lona) for mouse LAT4 cDNA and Kanchan Chaggar (University College Lon-
don) for tissue culture. This work was supported by Medical Research Council
Grant G0700368, the Wellcome Trust, Austrian Science Fund Grant P17109,
and the British Council.
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