From the Max-Planck-Institut für Experimentelle
Medizin, 37075 Göttingen, Germany, the Departments of
§ Molecular Genetics and ** Biochemistry, Howard Hughes
Medical Institute, The University of Texas Southwestern Medical Center,
Dallas, Texas 75235, the ¶ Departments of Pharmacology,
Physiology, and Neuroscience, New York University, New York, New York
10016, and the
Department of Biology, University of California
at San Diego, La Jolla, California 92093-0366
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ABSTRACT |
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-Latrotoxin is a potent neurotoxin from black
widow spider venom that binds to presynaptic receptors and causes
massive neurotransmitter release. A surprising finding was the
biochemical description of two distinct cell surface proteins that bind
-latrotoxin with nanomolar affinities; Neurexin I
binds
-latrotoxin in a Ca2+-dependent
manner, and CIRL/latrophilin binds in a Ca2+-independent
manner. We have now generated and analyzed mice that lack neurexin I
to test its importance in
-latrotoxin action.
-Latrotoxin binding
to brain membranes from mutant mice was decreased by almost 50%
compared with wild type membranes; the decrease was almost entirely due
to a loss of Ca2+-dependent
-latrotoxin
binding sites. In cultured hippocampal neurons,
-latrotoxin was
still capable of activating neurotransmission in the absence of
neurexin I
. Direct measurements of [3H]glutamate
release from synaptosomes, however, showed a major decrease in the
amount of release triggered by
-latrotoxin in the presence of
Ca2+. Thus neurexin I
is not essential for
-latrotoxin action but contributes to
-latrotoxin action when
Ca2+ is present. Viewed as a whole, our results show that
mice contain two distinct types of
-latrotoxin receptors with
similar affinities and abundance but different properties and
functions. The action of
-latrotoxin may therefore be mediated by
independent parallel pathways, of which the CIRL/latrophilin pathway is
sufficient for neurotransmitter release, whereas the neurexin I
pathway contributes to the Ca2+-dependent
action of
-latrotoxin.
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INTRODUCTION |
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-Latrotoxin is a potent neurotoxin from black widow spider
venom that triggers neurotransmitter release (reviewed in Ref. 1).
-Latrotoxin is thought to elicit neurotransmitter release by binding
to presynaptic receptors. Only nanomolar concentrations of
-latrotoxin are required to trigger exocytosis, suggesting the
presence of high affinity receptors. Binding activates an unknown
signal transduction cascade that leads to exocytosis of virtually all
synaptic vesicles.
-Latrotoxin also induces neurotransmitter release
in the absence of Ca2+ in a manner that circumvents the
normal Ca2+-dependent stimulation pathway for
synaptic vesicle exocytosis (2). The mechanism of action of
-latrotoxin and the nature of the intracellular signaling cascades
involved are unknown.
Two membrane proteins that bind -latrotoxin with high affinity have
been isolated from brain. The first such protein identified was
neurexin I
, which was isolated by affinity chromatography of brain
proteins on immobilized
-latrotoxin (3, 4). cDNA cloning
revealed that neurexin I
is a member of a family of three genes
(4-6). Each of the neurexin genes contains two promoters that direct
synthesis of
- and
-neurexins. Neurexins are highly polymorphic
and neuron-specific in expression (7). Their structure and interactions
with putative endogenous ligands (neuroligins and neurexophilin)
suggest a receptor function (8-10). Experiments with recombinant
neurexin I
confirmed that it binds to
-latrotoxin with high
affinity but showed that this interaction is completely dependent on
Ca2+ (11). Despite the wealth of biochemical data on
neurexins and their interactions with endogenous ligands and
-latrotoxin, the in vivo roles of neurexins are
unknown.
The observations that -latrotoxin binds to neurexin I
only in the
presence of Ca2+ but triggers neurotransmitter release also
in the absence of Ca2+ suggested that there must be a
second
-latrotoxin receptor in addition to neurexin I
. Based on
this finding, a second high affinity receptor for
-latrotoxin called
CIRL or latrophilin was identified (12, 13). Surprisingly, CIRL has no
structural similarity to neurexins. The presence of two distinct high
affinity binding proteins for
-latrotoxin in brain raises the
possibility that
-latrotoxin may physiologically act via two
independent receptor pathways or that the in vitro binding
of
-latrotoxin by one of the two putative receptors may represent an
artifact. In the current study, we have addressed this question using
knockout mice. We have generated a mouse line in which expression of
neurexin I
was abolished and investigated the importance of neurexin
I
for brain function and
-latrotoxin action. Our data confirm
that neurexin I
constitutes a major
-latrotoxin receptor. We show that neurexin I
is not required for
-latrotoxin action in the absence of Ca2+ but is essential for full
-latrotoxin
action in the presence of Ca2+.
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EXPERIMENTAL PROCEDURES |
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Genomic Cloning of Neurexin Genes--
A mouse genomic library
was screened for the 5 ends of the neurexin genes by high stringency
hybridization as described (14). Clones were isolated, mapped, and
sequenced using general molecular biology techniques (14, 15).
Sequences were analyzed on a personal computer using DNA-STAR
software.
Generation and Maintenance of Knockout Mice--
A knockout
vector was constructed from the genomic clone for neurexin I as
diagrammed in Fig. 1. Embryonic stem cells were electroporated with the
vector and selected with G418 (Life Technologies, Inc.) and FIAU
essentially as described (16). Resistant embryonic stem cell clones
were analyzed by polymerase chain reaction for homologous recombination
(primers used: 676 [GAGCGCGCGCGGCGGAGTTGTTGAC] and 918 [AGCCAATACTTCTGGGAAGACAGACT]) and confirmed by Southern blotting of
genomic DNA digested with SpeI with the probe indicated in
Fig. 1. Positive clones were injected into blastocysts, resulting in
the generation of a single mouse line that was bred to homozygosity and
genotyped by Southern blotting. To analyze the effect of the mutation
in the neurexin I
gene on mouse survival, mice heterozygous for the
neurexin I
mutation were mated with each other, and the number of
adult surviving offspring was determined by genotyping.
Antibodies and Immunoblot Analysis-- Antibodies against the cytoplasmic tails of neurexins were raised in rabbits using recombinant bacterially expressed proteins in which the N terminus of the cytoplasmic tail sequence was fused to a hexahistidine sequence for purification. Antibodies were affinity-purified on immobilized glutathione S-transferase fusion proteins of the same sequences, and their specificity was confirmed using recombinant neurexins. All other antibodies were described previously (17).
-Latrotoxin Binding Measurements--
-Latrotoxin binding
measurements were performed by a rapid centrifugation assay essentially
as described (18). Mouse brains were homogenized in 0.15 M
NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 7.8, and
crude membranes were prepared by centrifugation. The specific binding
of 0.5 nM 125I-
-latrotoxin to crude brain
membranes from mice was analyzed in triplicates in a 0.15-ml volume
with 0.2 mg of protein in the presence of 2 mM
Ca2+ or 2 mM EDTA. A 50-fold excess of
unlabeled
-latrotoxin was added to control for nonspecific binding.
Ca2+-independent binding was defined as
-latrotoxin
binding observed in the presence of EDTA, and
Ca2+-dependent binding was defined as the
difference between total
-latrotoxin binding measured with
Ca2+ and Ca2+-independent binding. Binding
assays for Scatchard analyses were performed similarly except that
-latrotoxin concentrations from 0.17 to 17.0 nM were
used.
Embryonic Cultures and Electrophysiology--
Cultures from
embryonic hippocampus from wild type or mutant mice were prepared as
described (19) and analyzed by electrophysiological recordings (20).
Spontaneous miniature excitatory postsynaptic currents were monitored
as a function of the application of 1 nM -latrotoxin in
the presence or the absence of Ca2+. To analyze the
currents when there was a high degree of overlap of miniature
excitatory currents (especially after
-latrotoxin application),
currents were integrated over 200-ms intervals. The charge transfer
obtained in this manner is proportional to the frequency of miniature
excitatory postsynaptic currents, the quantal release rate (21).
Measurements of [3H]Glutamate Release from
Synaptosomes--
Synaptosomes were prepared from the neocortex of
adult mice by a Percoll gradient centrifugation method modified from
Ref. 2. The crude mitochondrial fraction (P2) was
resuspended in 8.5% (v/v) Percoll suspended in 0.25 M
sucrose, 5 mM HEPES-NaOH, pH 7.4, and layered on top of an
12%/20% Percoll step gradient in the same buffer. After
centrifugation at 16,000 × g for 20 min, synaptosomes
were recovered from the 12%/20% Percoll interface. Percoll was
removed by the addition of 30 volumes of 0.32 M sucrose and
centrifugation. Pelleted synaptosomes were resuspended in 2 ml of
ice-cold gassed (95% O2/5% CO2)
Krebs-Henseleit-HEPES buffer (KHH buffer), pH 7.4 (composition in
mM: NaCl 118, KCl 3.5, CaCl2 1.25, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, HEPES-NaOH 5 at pH 7.4, glucose 11.5, 0.1 g/liter bovine serum albumin (Sigma catalog number A6793)).
Synaptosomes were loaded with 3H-labeled glutamate by a
5-min incubation in KHH buffer containing 0.17 µM
[3H]glutamate (24 mCi/mmol; NEN Life Science Products) in
a 95% O2/5% CO2 atmosphere at 34 °C.
Afterward synaptosomes (100 µl) were captured on a glass fiber filter
(GF/B) in a superfusion chamber, overlaid with 50 µl of a 50% slurry
of Sephadex G-25 in KHH buffer, and superfused (flow rate, 0.8 ml/min)
at 34 °C using two stimulation protocols: 1) Synaptosomes were
superfused with KHH buffer for at least 12 min for equilibration, and
then the perfusate was collected for 2 min to establish the base-line release rate of [3H]glutamate, and 0.5 nM
-latrotoxin was added to the superfusion buffer for 1 min followed
by continued superfusion with KHH buffer without
-latrotoxin. The
perfusate was collected throughout the procedure. 2) Synaptosomes were
superfused with Ca2+-free KHH buffer (KHH buffer in which
0.1 mM EGTA was present instead of 1.25 mM
CaCl2) gassed with 95% O2/5% CO2
for at least 12 min for equilibration. Then the perfusate was collected
for 2 min to establish the base-line release rate of
[3H]glutamate, after which 0.5 M sucrose was
added to the superfusion buffer for 30 s to stimulate release of
glutamate. Thereafter the synaptosomes were superfused for an
additional 2.5 min with Ca2+-free KHH buffer to
re-equilibrate them, and finally a 1-min pulse stimulation of
-latrotoxin in the same buffer was applied followed by continued
superfusion without
-latrotoxin. The perfusate was continuously
collected. [3H]Glutamate levels in the perfusate were
determined by scintillation counting. The fractional release rate was
calculated by dividing the amount of radioactivity released at any
given time point by the total amount of radioactivity remaining with
the synaptosomes at that point. Two mice from each genotype were
analyzed in independent experiments.
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RESULTS |
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Structure of the 5 End of the Neurexin I
Gene--
Upon
screening a mouse genomic library, we isolated a clone containing a
single large exon from the 5
end of the neurexin I
mRNA (Fig.
1). Sequence analysis revealed that the
exon encoded the N terminus of neurexin I
, including the signal
peptide, the first LNS domain (LNS domains are repeat sequences found
in laminins, neurexins, and sex hormone-binding globulins; Ref. 26),
and the first epidermal growth factor-like sequence. The exon ended at
the 5
boundary of the first site of alternative splicing in neurexin
I
corresponding to residue 253 (Fig. 1). In addition to the 5
end
of the coding region, the exon also contained the entire
5
-untranslated region that we previously sequenced in rat and bovine
cDNA clones (0.89 kilobase pairs; Refs. 5, 7), suggesting that the
exon present in this genomic clone constitutes the first exon of the
gene and that the 5
-flanking sequences present in the genomic clone
represent the neurexin I
promoter.
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Generation of Knockout Mice for Neurexin I--
Using the
neurexin I
genomic clone, we constructed a targeting vector in which
the entire first exon and several kilobases of 5
-flanking sequence
(presumably containing the neurexin I
promoter) were replaced by a
neomycin gene cassette as a positive selectable marker (Fig. 1). The
short arm of the targeting vector was constructed from the sequence of
the first intron and followed by two copies of a Herpes simplex virus
thymidine kinase gene cassette for negative selection. The long arm of
the vector was obtained from further 5
-flanking sequences (Fig. 1). As
a result, the entire first exon and part of the promoter of the
neurexin I
gene were deleted in the targeting vector.
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Mice Lacking Neurexin I Are Viable and Fertile--
Mice
carrying the deletion of the first exon of the neurexin I
gene were
bred to homozygosity and analyzed. Homozygous mutant mice were
indistinguishable in appearance from wild type mice. They were fertile
and survived for more than a year. The only abnormality that we
observed was that female knockout mice were less able than control mice
to attend to litters, either their own mutant litter or wild type
substituted control litter. As a consequence, when mouse pups were
cared for by neurexin I
-deficient females, more pups died
independent of the genotype. These data indicate that the neurexin I
mutation does not cause a major impairment in mouse survival or brain
functions but may have subtle behavioral effects. Exact definition of
these potential behavioral changes will require extensive behavioral
analyses.
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-Latrotoxin Binding Is Impaired in the Neurexin I
Knockouts--
We prepared brain membranes from wild type mice,
neurexin I
knockout mice, and two different control knockout lines
and studied binding of 125I-labeled
-latrotoxin to these
membranes in the presence and the absence of Ca2+ (Fig.
4). In the knockouts, a major decrease of
Ca2+-dependent binding of
-latrotoxin was
observed. In contrast, no major changes in Ca2+-independent
binding were detected. To gain insight into the relative affinities of
Ca2+-dependent and Ca2+-independent
-latrotoxin binding sites, we performed Scatchard plot analyses of
-latrotoxin binding to brain membranes from wild type and knockout
mice. These analyses revealed that
Ca2+-dependent and Ca2+-independent
binding exhibit similar affinities (Fig.
5).
Ca2+-dependent binding accounted for a major
part of the total binding capacity but was largely absent in the
knockout mice, suggesting that neurexin I
constitutes the major
Ca2+-dependent high affinity binding site for
-latrotoxin in mice. In addition, a smaller decrease in the
Ca2+-independent binding of
-latrotoxin was occasionally
observed in the knockout mice (Fig. 5). The
Ca2+-dependent binding site has the same
affinity and a similar abundance as the high affinity
Ca2+-independent binding site that is probably provided by
the CIRL/latrophilin protein (12, 13).
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-Latrotoxin Still Activates Neurotransmission in Neurons Lacking
Neurexin I
--
To test if
-latrotoxin still stimulated
neurotransmitter release in neurexin I
-deficient neurons, we
cultured hippocampal neurons from knockout and wild type mouse embryos
and studied miniature excitatory postsynaptic currents as a function of
-latrotoxin. 1 nM
-latrotoxin, a concentration
considerably higher than the KD values of the
Ca2+-dependent and Ca2+-independent
binding sites (Fig. 5), was applied in the presence or the absence of
Ca2+. An increase in neurotransmitter release was observed
in both types of mice, with or without Ca2+ (data not
shown). These data demonstrate that neurexin I
-deficient mice still
respond to relatively high concentrations of
-latrotoxin, suggesting
that neurexin I
is not required for
-latrotoxin stimulated neurotransmitter release. However, it is difficult to quantitate the
number of synapses whose release events give rise to the signal in
electrophysiological experiments under the particular conditions used
here. As a consequence, the electrophysiological results give no
insight into the relative magnitude of the effect of
-latrotoxin in
the two types of mice.
Quantitation of [3H]Glutamate Release from
Synaptosomes Triggered by -Latrotoxin--
To directly measure
[3H]glutamate release from nerve terminals, we purified
synaptosomes from the neocortex of wild type and knockout mice and
loaded them with [3H]glutamate. We suspended the
[3H]glutamate-loaded synaptosomes in a superfusion
chamber, measured base-line release of [3H]glutamate
during superfusion with either Ca2+-containing or
Ca2+-deficient buffer, and then stimulated release with
-latrotoxin at low concentration (0.5 nM) or with
sucrose (0.5 M). When we superfused wild type synaptosomes
with
-latrotoxin in the presence of Ca2+, we observed a
large prolonged increase in [3H]glutamate release (Fig.
6A).
[3H]Glutamate release was activated by
-latrotoxin
within 30 s and lasted for at least 10 min, the length of the
experiment. In neurexin I
-deficient synaptosomes, however, the
extent of [3H]glutamate release stimulated by
-latrotoxin was significantly decreased (Fig. 6A). These
data could either mean that neurexin I
contributes to
-latrotoxin
action in the presence of Ca2+ and is required for full
action of
-latrotoxin or that there is a general decrease in the
amount of [3H]glutamate that can be released from
neurexin I
-deficient synaptosomes.
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DISCUSSION |
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Neurexins are a family of highly polymorphic cell surface proteins
with a receptor like structure. There are three - and three
-neurexins, each of which is alternatively spliced. Neurexins interact with at least two endogenous ligands:
-neurexins bind to
neurexophilin (10), and
-neurexins bind to neuroligins (8, 9). In
addition, neurexin I
but not other neurexins bind
-latrotoxin with high affinity (3, 4, 11). The structures and known ligands of
neurexins indicate the possibility of dual functions for neurexins as
cell adhesion molecules and signal transduction receptors (24, 25).
However, the exact in vivo roles of neurexins are
unclear.
As a first step to probe the functions of neurexins and their role in
-latrotoxin action, we have now produced mice that lack neurexin
I
. Our data show that these mice are remarkably normal, suggesting
that neurexin I
is not an essential gene and not required for basic
nervous system functions. This finding suggests that neurexin I
is
functionally redundant or that it performs a more subtle function that
is not immediately apparent in the analysis performed here. The
presence of multiple neurexins with overlapping expression patterns (7)
would agree well with functional redundancy. This indicates that
neurexins may substitute for each other functionally. On the other hand
we found that the maternal behavior of the neurexin I
knockout mice
appears to be abnormal, suggesting that subtle defects may exist in the
single neurexin I
knockout. Future experiments using knockouts of
multiple neurexins and a detailed behavioral analysis will be required to resolve these questions.
-Latrotoxin is an excitatory neurotoxin that is a component of black
widow spider venom and produces massive neurotransmitter release after
binding to presynaptic nerve terminals (reviewed in Ref. 1). Although
-latrotoxin binding to neurexin I
requires Ca2+ (11),
-latrotoxin induces neurotransmitter release even in the absence of
Ca2+, indicating that a second receptor for
-latrotoxin
may mediate its toxicity in the nerve terminal. Such a receptor was
recently identified in CIRL/latrophilin, a membrane protein that
resembles G-protein-linked receptors and binds
-latrotoxin with high
affinity in the absence of Ca2+ (12, 13).
It is puzzling that neurons should express two distinct -latrotoxin
receptors with different binding properties
(Ca2+-dependent versus
Ca2+-independent) but similar affinities. Although the two
types of
-latrotoxin receptors have been characterized thoroughly
biochemically, it is unknown if they function as
-latrotoxin
receptors in vivo. To address this question, we analyzed
-latrotoxin action in the neurexin I
-deficient mice that we had
generated. Our data demonstrate that in the neurexin I
-deficient
mice, most of the Ca2+-dependent
-latrotoxin
binding activity is lost, whereas Ca2+-independent binding
is unchanged. The
-latrotoxin affinities of neurexin I
and CIRL
are very similar, as is the relative abundance of the
Ca2+-dependent binding site due to neurexin
I
and the Ca2+-independent binding site presumably due
to CIRL. Thus neurexin I
accounts for the bulk of the
Ca2+-dependent high affinity binding of
-latrotoxin. We found, however, that
-latrotoxin is still capable
of triggering neurotransmitter release in cultured hippocampal neurons
or synaptosomes from neurexin I
-deficient mice. Therefore neurexin
I
is not absolutely required for the excitotoxic action of
-latrotoxin. Nevertheless, we observed that in the presence of
Ca2+, glutamate release triggered by
-latrotoxin from
synaptosomes is decreased in the knockout mice compared with wild type
mice. If release is stimulated by
-latrotoxin or by sucrose in the absence of Ca2+, no change is observed, indicating that the
general release apparatus is intact. This finding also agree well with
the electrophysiological data and the mild phenotype of the knockout
mice. Viewed together, our data demonstrate that neurexin I
is not
essential for the ability of
-latrotoxin to trigger neurotransmitter
release but contributes to
-latrotoxin action in the presence of
Ca2+.
What could the action of neurexin I be compared with that of CIRL?
CIRL is the presumptive Ca2+-independent
-latrotoxin
receptor that probably mediates the
-latrotoxin-dependent activation of neurotransmitter
release observed in the neurexin I
knockout mice. Our finding that
in the presence of Ca2+, neurexin I
is required for a
full response to
-latrotoxin at low concentrations suggests two
possibilities. Either neurexin I
and CIRL represent independent
pathways for
-latrotoxin action that work in parallel, or neurexin
I
assists CIRL in the presence of Ca2+ in triggering
neurotransmitter release. In either case, neurexin I
represents a
target for
-latrotoxin. Futhermore, it is possible that
-latrotoxin may have as yet unidentified effects other than triggering neurotransmitter release, which could be mediated by neurexin I
. Taken together, with the identification of two
structurally different, presumably cooperative receptors the action of
-latrotoxin is much more complex than previously envisioned.
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ACKNOWLEDGEMENTS |
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We thank Dr. Y. A. Ushkaryov for the
initial isolation of genomic neurexin I clones; Dr. Y. Hata for the
generation of antibodies to His-tagged neurexin tails expressed in
bacteria; and M. S. Brown, and J. L. Goldstein for invaluable
discussions.
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FOOTNOTES |
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* This study was supported by a grant from the Souderforschungsbereich and by National Institutes of Health Grants MH50824 (to T. C. S.) and NS35098 and NS34937 (to A. G. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should addressed. Tel.: 214-648-5022;
Fax: 214-648-6426; E-mail: TSudho{at}MEDNET.SWMED.EDU.
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REFERENCES |
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