REPORTS

15. J. R. Ross, K. H. Nam, J. C. DAuria, E. Pichersky, Arch.

Biochem. Biophys. 367, 9 (1999).
16. N. Dudareva et al., Plant Cell 12, 949 (2000).
17. N. Misawa et al., J. Bacteriol. 177, 6575 (1995).
18. We thank P. Hamman and A. Malek for DNA sequencing and M. Kumagai for helpful comments on the
manuscript. This work was supported by European
Community grant QLK3-CT-2000-00809.

Supporting Online Material

www.sciencemag.org/cgi/content/full/300/5628/2089/
DC1
Materials and Methods
Figs. S1 to S3
References
1 April 2003; accepted 2 June 2003

A Targeting Motif Involved in

Sodium Channel Clustering at the
Axonal Initial Segment
Juan Jose
Garrido, Pierre Giraud, Edmond Carlier, Fanny Fernandes,
Anissa Moussif, Marie-Pierre Fache, Dominique Debanne,
Be
ne
dicte Dargent*
The sorting of sodium channels to axons and the formation of clusters are of
primary importance for neuronal electrogenesis. Here, we showed that the
cytoplasmic loop connecting domains II and III of the Nav1 subunit contains a
determinant conferring compartmentalization in the axonal initial segment
of rat hippocampal neurons. Expression of a soluble Nav1.2 II-III linker
protein led to the disorganization of endogenous sodium channels. The motif
was sufcient to redirect a somatodendritic potassium channel to the axonal
initial segment, a process involving association with ankyrin G. Thus, this
motif may play a fundamental role in controlling electrical excitability
during development and plasticity.
Neuronal action potentials are generated at
the axonal initial segment (AIS), and their
saltatory conduction in myelinated axons oc-

curs via the nodes of Ranvier (1, 2). These

processes require a precise distribution of
voltage-gated sodium channels accumulated

References and Notes

1. E. Pichersky, D. R. Gang, Trends Plant Sci. 5, 439

(2000).
2. P. Collins, Foods Ingred. Process. Int. 13, 23 (1992).
3. G. J. Lauro, Cereal Foods World 36, 949 (1991).
4. C. F. Timberlake, B. S. Henry, Endeavour 10, 31
(1986).
5. L. Zechmeister, Cis-Trans Isomeric Carotenoids, Vitamins A, and Arylpolyenes (Springer-Verlag, Vienna,
1962).
6. G. Britton, in Carotenoids, G. Britton, S. Laaen-Jensen,
H. Pfander, Eds. (Birkhauser Verlag, Basel, 1998), vol.
3, pp. 13147.
7. A. Z. Mercadante, A. Steck, H. Pfander, Phytochemistry 46, 1379 (1997).
8. A. Z. Mercadante, A. Steck, H. Pfander, J. Agric. Food
Chem. 45, 1050 (1997).
9. S. H. Schwartz, B. C. Tan, D. A. Gage, J. A. D. Zeevaart,
D. R. McCarty, Science 276, 1872 (1997).
10. S. H. Schwartz, X. Qin, J. A. D. Zeevaart, J. Biol. Chem.
276, 25208 (2001).
11. F. Bouvier, C. Suire, J. Mutterer, B. Camara, Plant Cell
15, 47 (2003).
12. Materials and methods are available as supporting
material on Science Online.
13. B. Watillon et al., Plant Mol. Biol. 36, 909 (1998).
14. D. S. Skibbe et al., Plant Mol. Biol. 88, 751 (2002).

www.sciencemag.org SCIENCE VOL 300 27 JUNE 2003

Fig. 1. The cytoplasmic II-III region of Nav1.2 contains an AIS

localization signal. (A) Schematic
representation of the neuronal
sodium channel Nav1.2. Four homologous domains (I to IV) are
connected by large cytoplasmic
linkers (I-II, II-III, and III-IV); color
code as in (B). (B) Diagram of
CD4Ct and CD4 Nav1.2 II-III
constructs. The cytoplasmic II-III
linker of Nav1.2 (amino acids
983 to 1203; green) was fused to
the human CD4 receptor with a
deleted C terminus (CD4Ct). (C
and D) CD4Ct displays a nonpolarized surface distribution,
whereas the surface localization
of CD4 Nav1.2 II-III is restricted
to the AIS of transfected rat hippocampal neurons. (E and F) Like
endogenous sodium channels,
CD4 Nav1.2 II-III is resistant to
Triton X-100 extraction before
cell xation and immunostaining. Somatodendritic domains
and AIS were identied by staining for MAP2 (C) and ankyrin G
(D to F). Scale bar, 20 m.

Downloaded from www.sciencemag.org on August 21, 2015

natural bixin isomer and traces of bixin dimethyl ester (Fig. 2E). Bixin aldehyde and
norbixin were not modified in incubation
with boiled enzymes or with protein extract
from E. coli harboring empty vector. Thus,
BoBADH and BonBMT represent bixin aldehyde dehydrogenase and norbixin methyltransferase, respectively, and support the reaction sequence proposed for bixin synthesis
(Fig. 1B).
Because the seeds of B. orellana are the
only natural source of industrially produced
bixin, we investigated whether bixin could be
produced in E. coli pACCRT-EB engineered
to accumulate lycopene (17). We transformed E. coliproducing lycopene with the
plasmid pUC19-LCD-BADH-nBMT coding
for B. orellana lycopene cleavage dioxygenase, bixin aldehyde dehydrogenase, and norbixin methyltransferase (12). HPLC analysis
of the lipid extract of transformed E. coli
harboring pUC19-LCD-BADH-nBMT revealed the accumulation of a new derivative
corresponding to bixin (Fig. 3A). No such
change was observed in E. coli cells transformed with an empty vector control (Fig.
3B). The average production level of bixin in
E. coli was 5 mg/g dry weight.
Bixin is one of the oldest pigments used
by humans and is increasingly in demand
because of the consumer ban on the chemically synthesized azo dye (2, 4). Bixin synthesis involves an unprecedented oxidative
remodeling of lycopene, a common intermediate which is the precursor of -carotene,
provitamin A. Given the feasibility of engineering bixin in a heterologous host such as
E. coli, we assume that coexpressing the three
cloned genes in sink organs such as tomato
fruit, which accumulates massive amounts of
the necessary precursor lycopene, should lead
to an alternative and competitive source for
natural bixin production.

2091

REPORTS
Fig. 2. Perturbation of
sodium channel organization by expression of
GFPNav 1.2 II-III. (A
and B) Effect of GFP
Nav1.2 II-III expression
on the peak amplitude
of Na current. Wholecell outward Na currents were recorded
from hippocampal neurons expressing either GFP or GFPNav1.2 II-III. Superimposed current traces evoked by depolarization from 90 mV to 40 mV are
shown. (C) GFP was visualized in soma and throughout dendrites and

at high density in these two specific membrane microdomains defined by the segregation of the ankyrin GIV spectrin complex
(3 6). At the molecular level, sodium channels from rat brain are composed of an
subunit, the pore-forming protein Nav1, and
the auxiliary subunits, 2 and 1 or 3 (2).
The fact that subunits interact with proteins
of the L1 family of adhesion molecules (7, 8),
with ankyrin G (9), and with tenascin R and
C (2) raises the possibility that subunits
contribute to sodium channel localization at
specific sites in the neuronal membrane.
However, tagged subunits expressed in hippocampal neurons are uniformly distributed
(10), indicating that information for sorting
and clustering of sodium channels is probably
carried by the pore-forming protein Nav1.
In the central nervous system, Nav1.2 sodium channels are distributed on unmyelinated axons, including the AIS (11, 12). To
define the molecular determinant accounting
for sodium channel compartmentalization at
the AIS, we assessed whether any of the large
intracellular regions of Nav1.2 contained sufficient information for sorting and specific
membrane organization. We analyzed the
surface distribution of chimeric proteins containing CD4 fused to one of the cytoplasmic
regions of Nav1.2 (Fig. 1A) (13) after transfection in rat hippocampal neurons. During in
vitro development, these cells have the intrinsic ability to form an AIS acting as a diffusion barrier (14, 15) and to accumulate sodium channels in the proximal region of the
axon (16), as observed in cultured spinal
motoneurons (17). A chimera containing the
C terminus of Nav1.2 (CD4 Nav1.2 Ct) is
targeted to axons (13). However, at the steady
state, the surface distribution of CD4 Nav1.2
Ct is located in more distal regions of the
axon than is ankyrin G (13). Thus, the C
terminus of Nav1.2 does not contain sufficient information for sodium channel localization at the AIS. We further analyzed the
Institut National de la Sante
et de la Recherche
Me
dicale Unite
464, Institut Jean Roche, Universite
de
la Me
diterrane
e, Faculte
de Me
decine Secteur-Nord,
Boulevard P. Dramard, 13916 Marseille Cedex 20,
France.
*To whom correspondence should be addressed. Email: dargent.b@jean-roche.univ-mrs.fr

2092

axons (arrowhead), whereas GFPNav1.2 II-III was concentrated in the AIS. Inset:
Clustering of endogenous channels was immunodetected in GFP-positive neurons but was absent in GFPNav1.2 II-IIIpositive neurons. Scale bar, 20 m.

Fig. 3. Identication of the

AIS motif of neuronal sodium channels. (A) Schematic
representation of mutations
generated in Nav1.2 II-III to
dene the AIS motif. For
each mutant, the percentage of transfected CD4positive hippocampal neurons in which staining was
restricted to the AIS is indicated, taking as 100% the
total population of transfected neurons (i.e., CD4positive neurons). Data are
means SD from three to
seven different experiments;
n denotes the total number
of neurons analyzed for each
mutation. In parallel, the resistance of mutated proteins
to detergent extraction
(Triton X-100) was determined. (B) Alignment of the
AIS motif sequences in rat
neuronal voltage-dependent
sodium channels. Gray
shading indicates similarity
between amino acids; nonconserved residues are underlined. Amino acid abbreviations: A, Ala; D, Asp; E,
Glu; F, Phe; G, Gly; I, Ile; L,
Leu; M, Met; N, Asn; P, Pro; R,
Arg; S, Ser; T, Thr; V, Val. (C)
Surface expression of CD4Nav1.2 II-III mutants. (D) Nav1.6 clustering at the AIS of 4-week-old hippocampal
neurons. (E) Addition of the intracellular II-III region of Nav1.6 restricts surface distribution of CD4 to the AIS.
Scale bar, 20 m.

other cytoplasmic regions. The addition of

the II-III linker from the Nav1.2 subunit to the
human CD4 receptor deleted of its cytoplasmic tail (CD4Ct; Fig. 1B) resulted in a
surface distribution of the chimera markedly
restricted to the AIS (Fig. 1C), which was
identified by the absence of a somatodendritic marker, MAP2, and by staining for
ankyrin G (3) (Fig. 1D). In contrast, CD4Ct
was uniformly distributed at the cell surface
(Fig. 1C). Endogenous sodium channels and
CD4 Nav1.2 II-III were nonextractable by
detergent before cell fixation and immunostaining (Fig. 1, E and F), like the axonal cell
adhesion molecule L1 (14).
To confirm a role for the II-III linker in
organizing sodium channels at the AIS, we
examined whether its overexpression per-

turbed endogenous sodium channels. Na

current was measured by whole-cell patchclamp recording on hippocampal neurons expressing either green fluorescent protein
(GFP) or a chimera in which Nav1.2 II-III
was fused to the C terminus of GFP (GFP
Nav1.2 II-III). The peak amplitudes of outward Na currents recorded in GFPNav1.2
II-IIIpositive neurons were reduced by 50%
(n 29) relative to neurons expressing GFP
alone (n 30) (Fig. 2, A and B). No modification of inactivation kinetics or of the voltage dependence of activation or inactivation
was observed (fig. S1). The distribution of
GFPNav1.2 II-III differed remarkably from
that of GFP. The latter was uniformly distributed throughout dendrites, axons, and soma
(Fig. 2C). In contrast, GFPNav1.2 II-III was

27 JUNE 2003 VOL 300 SCIENCE www.sciencemag.org

REPORTS
Fig. 4. Involvement of
ankyrin G in targeting
membrane proteins
bearing the AIS motif.
(A) The AIS motif of
Nav1.2 is sufcient to
target the voltagedependent
channel
Kv2.1 to the AIS. Top:
Schematic representations of HA-Kv2.1 and
HAKv2.1-Nav1.2. A
segment of the C terminus of Kv2.1 encompassing the PRC motif
was substituted by a
segment of Nav1.2
containing the AIS motif. Bottom: In permeabilized neurons, HAKv2.1 was distributed
both in soma and
proximal
dendrites,
whereas HAKv2.1Nav1.2 was restricted
to the AIS (identied
by ankyrin G staining).
(B) Coimmunoprecipitation of 270-kD
ankyrin GGFP and
HAKv2.1-Nav1.2. HEK
cells were cotransfected either with
ankyrin GGFP and
HAKv2.1-Nav1.2 or with ankyrin GGFP and HA-Kv2.1, and then extracted with detergent. Extracts were
subjected to immunoprecipitation with antibody to HA. Immunoprecipitation inputs and pellets were analyzed
by Western blotting with antibody to GFP. (C) Colocalization of ankyrin GGFP and HAKv2.1-Nav1.2 in HEK
cells. (D) Overexpression of ankyrin GGFP induces a mislocalization of HAKv2.1-Nav1.2. Hippocampal
neurons were cotransfected with the indicated constructs and subjected to HA immunostaining 1 day later.
Colocalization of ankyrin GGFP and HAKv2.1-Nav1.2 is evident not only at the AIS (arrows) but also in the
soma. GFP expression did not affect segregation of HAKv2.1-Nav1.2 at the AIS, whereas somatodendritic
HA-Kv2.1 clusters did not colocalize with ankyrin GGFP. Scale bar, 20 m.

highly concentrated at the AIS but absent

from dendrites, although faint fluorescence
was detected in soma (Fig. 2C). This distribution was displayed by 71.5% of transfected
cells (n 76). We next quantified the number of AISs positive for sodium channel clustering. Nearly all hippocampal neurons expressing GFP alone (96%; n 69) displayed
staining of endogenous sodium channels at
the AIS (Fig. 2C, inset). In contrast, only
14% of cells (n 76) expressing GFP
Nav1.2 II-III exhibited detectable sodium
channel staining. These findings suggest that
the II-III linker is critical for sodium channel
organization at the AIS.
To identify the critical determinant at the
sequence level, we generated mutants of
CD4 Nav1.2 II-III containing either a truncated C terminus or internal deletions (Fig.
3A). Analysis of the steady-state distribution
of mutants defined a 27-residue motif between amino acids 1102 and 1028 that governed chimera compartmentalization at the
AIS (Fig. 3A). The AIS motif contained clusters of acidic residues similar to sorting motifs that regulate the trafficking of the endoprotease furin (18) and aquaporin 4 (19). The

critical role of the AIS motif was further

supported by an internal deletion (10981111; Fig. 3, A and C). Impaired compartmentalization of mutated CD4 Nav1.2 II-III
chimeras in the AIS was systematically associated with a loss of resistance to detergent
extraction (Fig. 3A). This suggests that the
sorting motif we have identified may also act
as a retention motif, as in the case of furin
(18) and the vesicular monoamine transporter
VAMT2 (20). The AIS motif identified in
Nav1.2 is highly conserved in Nav1.6, the
nodal sodium channel type (21), and in the
neuronal sodium channels Nav1.1 and Nav1.3
(22) (Fig. 3B). It is also relatively well conserved in Nav1.4 and Nav1.5, the skeletal and
cardiac muscle sodium channels (22). Nav1.6
was consistently visualized at the AIS of
cultured hippocampal neurons (Fig. 3D), as
reported in vivo in Purkinje neurons (5).
When the cytoplasmic II-III linker of Nav1.6
was fused to CD4, the resulting chimera was
highly enriched at the AIS (Fig. 3E) (80% of
cells displayed cell surface CD4 staining restricted to the AIS; n 429, seven independent experiments) and was resistant to detergent extraction. During the biogenesis of

nodes of Ranvier, Nav1.2 is replaced by

Nav1.6 (23, 24). It is thus conceivable that the
AIS motif is involved in sodium channel
clustering at the nodes of Ranvier and possibly in the unexpected localization of neuronal
sodium channels in the heart (25).
We next investigated whether the AIS
motif of Nav1.2 was sufficient to relocalize a
potassium channel in hippocampal neurons.
We constructed a hemagglutinin (HA)
tagged Kv2.1-Nav1.2 chimera (Fig. 4A) in
which the region encompassing the proximal
restriction clustering (PRC) (26) motif was
replaced by amino acids 1082 to 1203 of
Nav1.2 II-III. HA-Kv2.1 displayed a somatodendritic distribution with donut-shaped
clusters (Fig. 4A) (26). In contrast, HA
Kv2.1-Nav1.2 was restricted to the AIS (Fig.
4A) (97% of transfected cells; n 194). We
confirmed that HAKv2.1-Nav1.2 was inserted at the cell surface and was resistant to
Triton X-100 extraction (figs. S2 and S3).
Genetic studies have shown that the suppression of ankyrin G gene expression impaired
sodium channel localization at the AIS (3, 5).
Thus, we next addressed whether the AIS
motif associated with ankyrin G by coexpressing 270-kD ankyrin GGFP (27) either
with HAKv2.1-Nav1.2 or HA-Kv2.1 in HEK
cells. Ankyrin GGFP coimmunoprecipitated
with HAKv2.1-Nav1.2, but not with HAKv2.1 (Fig. 4B). The two partners colocalized
in the plasma membrane and also intracellularly (Fig. 4C). These data provide evidence
for an association between 270-kD ankyrin
GGFP and the AIS motif, a finding that was
not observed with an in vitro binding assay
(28). In hippocampal neurons, overexpressed
ankyrin GGFP was localized at the plasma
membrane of soma, in addition to its normal
distribution at the AIS (Fig. 4D) (27). In
addition to colocalization in the AIS, coexpressed HAKv2.1-Nav1.2 was associated
with mislocalized somatic ankyrin G and
somatic clusters were clearly visible (Fig.
4D). This was observed in 80% of the cells
coexpressing ankyrin GGFP and HA
Kv2.1-Nav1.2 (n 391, two independent
experiments). In contrast, HAKv2.1Nav1.2 remained confined in the AIS in
76% of cells coexpressing GFP alone (n
406). In cells coexpressing HA-Kv2.1 and
ankyrin GGFP, somatic clusters of
ankyrin G did not mirror HA-Kv2.1 aggregates (Fig. 4D). Thus, overexpression of
ankyrin G resulted in a mislocalization of
HAKv2.1-Nav1.2 in soma.
Our results offer insights into the intrinsic
signals for sodium channel targeting and
clustering at discrete sites in the neuronal
plasma membrane. We propose that segregation is specified by a 27amino acid motif
within linker II-III of the pore-forming Nav1
proteins. This signal is sufficient to relocalize
the somatodendritic cation channel Kv2.1, as

www.sciencemag.org SCIENCE VOL 300 27 JUNE 2003

2093

REPORTS
well as the cytosolic protein GFP, to the AIS.
However, additional motifs probably play a
role in establishing the differential distribution of certain types of sodium channel in
vivo. We also obtained evidence for the association of ankyrin G with the motif we have
identified. From a mechanistic point of view,
ankyrin G could be primarily involved in
anchoring sodium channels at the plasma
membrane. However, we cannot exclude the
possibility that the two partners could be
preassembled early in biogenesis and cotransported during sorting, as observed in the case
of presynaptic proteins (29, 30).
Note added in proof: During revision of
this report, Lemaillet et al. (31) identified a
conserved ankyrin-binding motif located
within the AIS motif.
References and Notes

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Facilitation of Spinal NMDA

Receptor Currents by Spillover of
Synaptically Released Glycine
Seifollah Ahmadi,1* Uta Muth-Selbach,2* Andreas Lauterbach,1*
Peter Lipfert,2 Winfried L. Neuhuber,3 Hanns Ulrich Zeilhofer1
In the mammalian CNS, N-methyl-D-aspartate (NMDA) receptors serve prominent roles in many physiological and pathophysiological processes including
pain transmission. For full activation, NMDA receptors require the binding of
glycine. It is not known whether the brain uses changes in extracellular glycine
to modulate synaptic NMDA responses. Here, we show that synaptically released glycine facilitates NMDA receptor currents in the supercial dorsal horn,
an area critically involved in pain processing. During high presynaptic activity,
glycine released from inhibitory interneurons escapes the synaptic cleft and
reaches nearby NMDA receptors by so-called spillover. In vivo, this process may
contribute to the development of inammatory hyperalgesia.
Glycine serves a dual role in central neurotransmission. It is not only the primary inhibitory
neurotransmitter in the spinal cord and brain
stem, but also an obligatory coagonist at excitatory glutamate receptors of the NMDA type
(13). Although glycine concentrations in the
cerebrospinal fluid are in the micromolar range
(46), which would fully activate this site (7),
glycine transporters located on glial cells can
1
Institut fur Experimentelle und Klinische Pharmakologie und Toxikologie, Universitat ErlangenNurnberg, Fahrstrasse 17, D-91054 Erlangen, Germany. 2Klinik fur Anasthesiologie, Moorenstrasse 5,
Universitatsklinikum Dusseldorf, D-40225 Dusseldorf,
Germany. 3Institut fur Anatomie, Krankenhausstrasse
9, Universitat Erlangen-Nurnberg, D-91054 Erlangen,
Germany.

*These authors contributed equally to this work.

To whom correspondence should be addressed. Email: zeilhofer@pharmakologie.uni-erlangen.de

2094

lower extracellular glycine in the vicinity of

synaptic NMDA receptors to subsaturating
concentrations (810). A physiological role of
glycine in the modulation of NMDA receptor
mediated synaptic transmission therefore appears possible. We have previously shown that
in the spinal cord dorsal horn the neuropeptide
nocistatin (NST) (11) specifically inhibits the
synaptic release of glycine [and -aminobutyric
acid (GABA)] without affecting the release of
L-glutamate (12). We have now used this peptide to show that spinal NMDA receptors can be
facilitated by spillover of synaptically released
glycine both in spinal cord slices and in vivo.
Glutamatergic and glycinergic input was activated simultaneously by electrical field stimulation. Excitatory and inhibitory postsynaptic
currents (EPSCs and IPSCs) were isolated
pharmacologically and recorded from superficial dorsal horn neurons in transverse rat

22.
23.
24.
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29.
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31.
32.

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S. K. Maier et al., Proc. Natl. Acad. Sci. U.S.A. 100,
3507 (2003).
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Trimmer, Neuron 25, 385 (2000).
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R. G. Zhai et al., Neuron 29, 131 (2001).
G. Lemaillet, B. Walker, S. Lambert, J. Biol. Chem.,
published online 25 April 2003 (10.1074/
jbc.M303327200).
We thank V. Bennett, A. Goldin, O. Pongs, and J.
Trimmer for cDNA and antibodies; L. Fronzarolli, F.
Jullien, and S. Hutter for excellent technical assistance; and M. Seagar, B. Winckler, and F. Castets for
critical reading of the manuscript. Supported by
grants from INSERM, Association Francaise contre les
Myopathies, and Fondation Me
dicale pour la Recherche ( J.J.G.).

Supporting Online Material

www.sciencemag.org/cgi/content/full/300/5628/2091/
DC1
Figs. S1 to S3
1 April 2003; accepted 12 May 2003

spinal cord slices (13). At a saturating concentration of 10 M (12), NST reversibly

reduced the amplitude of glycinergic IPSCs
(gly-IPCSs) by 36.5 7.2% (n 15, P
0.01, paired t test). By contrast, -amino-3hydroxy-5-methylisoxazole-4-propionic acid
(AMPA) receptormediated EPSCs (AMPAEPSCs) remained completely unchanged
[96.7 4.3% of control amplitudes, n 11,
P 0.33, see also (12)], whereas NMDAEPSCs were reversibly reduced by 29.2
3.2% (P 0.001) in 15 out of 17 neurons
(Fig. 1A).
Inhibition of NMDA-EPSCs was completely prevented when the slices were continuously
superfused with glycine (100 M) to permanently saturate NMDA receptors ( NMDAEPSC: 0.16 5.9%, n 6, P 0.67, ANOVA
followed by Scheffe s post hoc test). A significant contribution of GABA acting on ionotropic GABAA or G proteincoupled GABAB
receptors could be excluded, because all
experiments were performed in the continuous presence of the GABAA receptor
blocker bicuculline (10 M) and additional
application of the GABAB receptor antagonist CGP55845 (100 M) did not prevent
inhibition ( NMDA-EPSC: 37.2 10.2%,
n 8, P 0.01) (Fig. 1B).
A reduction in glycine release could interfere with NMDA receptormediated synaptic
transmission by at least two mechanisms.
First, glycine might act through presynaptic
strychnine-sensitive glycine receptors to facilitate the release of glutamate, an action that
has been demonstrated in the brainstem (14).
A significant contribution of this process to
the inhibition of NMDA-EPSCs described
here appears unlikely, because NMDAEPSCs were recorded in the presence of
strychnine (300 nM). Second, glycine might

27 JUNE 2003 VOL 300 SCIENCE www.sciencemag.org

A Targeting Motif Involved in Sodium Channel Clustering at the

Axonal Initial Segment
Juan Jos Garrido et al.
Science 300, 2091 (2003);
DOI: 10.1126/science.1085167

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