From the Department of Molecular and Cellular
Neurobiology, Vrije Universiteit, Amsterdam, 108HV Netherlands, the
§ Departments of Physiology and Biophysics, and Anatomy and
Cell Biology, University of Calgary, Calgary T2N 4N1, Canada, and the
Department of Biological Sciences,
University of Alberta, Edmonton T6G 1E9, Canada
Received for publication, October 29, 2002, and in revised form, November 25, 2002
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ABSTRACT |
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We report here that unlike what was suggested for
many vertebrate neurons, synaptic transmission in Lymnaea
stagnalis occurs independent of a physical interaction between
presynaptic calcium channels and a functional complement of SNARE
proteins. Instead, synaptic transmission in Lymnaea
requires the expression of a C-terminal splice variant of the
Lymnaea homolog to mammalian N- and P/Q-type calcium
channels. We show that the alternately spliced region physically
interacts with the scaffolding proteins Mint1 and CASK, and that
synaptic transmission is abolished following RNA interference knockdown
of CASK or after the injection of peptide sequences designed to disrupt
the calcium channel-Mint1 interactions. Our data suggest that Mint1 and
CASK may serve to localize the non-L-type channels at the active zone
and that synaptic transmission in invertebrate neurons utilizes a
mechanism for optimizing calcium entry, which occurs independently of a
physical association between calcium channels and SNARE proteins.
Calcium entry through voltage-gated calcium channels triggers the
release of synaptic vesicles in the presynaptic nerve terminal. It is
thought that the high calcium buffering capacity of neurons necessitates that the sensor for calcium-triggered exocytosis be
situated close to the source of calcium entry, i.e. near the inner mouth of presynaptic calcium channels (1-3). Accordingly, a
region within the cytoplasmic linker connecting domains II-III of
mammalian N- and P/Q-type calcium channels, termed the synaptic protein
interaction (synprint) site, was shown to bind tightly to a
number of different proteins of the presynaptic vesicle release complex, including syntaxin1A, SNAP-25, and synaptotagmin1 (4-6). In
mammalian neurons, the requirement for a physical complex between presynaptic calcium channels and the
SNARE1 complex is supported
by the observation that injection of peptides corresponding to the
synprint site blocks synaptic transmission, presumably
through a competitive decoupling of the SNARE protein-calcium channel
complex (2, 7-8).
The physiology of invertebrate synapses appears to correlate closely
with their mammalian counterparts. With few notable exceptions, most
vertebrate proteins involved in synaptic transmission have structurally
conserved orthologs in invertebrates (9). Surprisingly, however, none
of the invertebrate calcium channels identified from
Drosophila or Caenorhabditis elegans (10) appear
to carry a structural motif that would resemble synprint,
indicating that a correlate role of synprint does not exist
in invertebrates. However, this postulate has not yet been tested
directly because invertebrate models that are suitable for genetic
analysis of synaptic transmission in the central nervous system (such
as C. elegans and Drosophila) are not amenable to
direct physiological analysis at the level of single pre- and
postsynaptic neurons. Conversely, most invertebrate model systems that
are amenable for physiological analysis, until recently, lacked
molecular information that is required to interpret the physiological data.
To determine how transmitter release in invertebrates occurs in the
absence of calcium channels with a synprint motif, we have
taken advantage of identified Lymnaea neurons, which are directly accessible to both physiological and molecular analysis at the
level of single pre- and postsynaptic neurons. We have identified the
sequences of the major calcium channel subunits and key synaptic
elements, including SNARE proteins that are expressed in the
presynaptic neuron. We provide evidence that synaptic transmission is
dependent on a Lymnaea homolog of mammalian presynaptic
calcium channels that does not associate with the SNARE complex. Yet, despite the lack of a synprint region in the
Lymnaea channel isoform, the injection of mammalian
synprint peptide into Lymnaea synapses still
inhibits synaptic transmission. Thus, we challenge the current thinking
of an unequivocal role of synprint in the molecular assembly of synaptic proteins with calcium channels at the active synaptic zone.
Instead, we show that synaptic transmission in Lymnaea is critically dependent on the expression of an alternatively spliced C-terminal region of the presynaptic channel homolog that physically and functionally interacts with the scaffolding proteins Mint1 and
CASK. Our data suggest that these proteins might be involved in
anchoring presynaptic calcium channels at the active zones, thus
optimizing calcium entry for transmitter release.
Molecular Cloning and Identification of Lymnaea
Orthologs--
Parts of novel Lymnaea cDNAs were
identified by PCR cloning using degenerate primers (see Table I),
designed from regions with high sequence identity in aligned protein
orthologs described in the GenBankTM data base (NCBI,
Bethesda) using cDNA that was synthesized from brain ganglia RNA of
adult Lymnaea stagnalis.
cDNA clones were isolated by screening of
Conceptual translations were derived from the longest open reading
frame. When the protein translation initiation site was uncertain,
start sites were inferred from closely related orthologs described in
GenBankTM. Lymnaea orthologs and results of
comparison of sequence of putative Lymnaea proteins with
human orthologs are shown in Table I.
Sequence Alignments--
BLASTP retrieved orthologs
(GenBankTM) of Lymnaea proteins were aligned by
modified progressive pairwise, multiple alignment in PILEUP
(UNIX-based, GCG Wisconsin Package 2002, Accelrys, Madison, WI) and
were visually displayed (PLOTSIMILARITY, Accelrys).
Quantitative PCR of Lymnaea Genes in Identified
Neurons--
Primer pairs for qPCR detection of Lymnaea
gene transcripts were designed with Primer Express 1.0 software
(Applied Biosystems). Each candidate primer pair generated an amplicon
of the expected size of 85-120 bp, and PCR efficiencies were tested on
Lymnaea central nervous system cDNA using serial
dilutions of template. Primer dimers were identified by measurable
product in the absence of qPCR template. All qPCR primer pairs (see
Table I) averaged an amplification efficiency of ~1.9-2.0.
cDNA templates for the qPCR were prepared from single, identified
neurons with axons attached by gentle mechanical suction with pulling
pipette from each of six Lymnaea brain ganglia preparations. cDNA were synthesized as follows: 1) directly from freshly isolated neurons; 2) as unpaired pre- and postsynaptic neurons after 18 h
of primary culture on hemolymph-coated coverslips; or 3) as synaptically paired neurons cultured for 18 h (see primary cell culture technique described below for more details).
The freshly isolated and cultured cells were transferred as a
collective into a prepared microcentrifuge tube in a minimal volume of
culture medium (<3 µl) for immediate total RNA isolation and first
strand synthesis, using a protocol as described by Van Kesteren
et al. (14). For qPCR, primer pairs at 2 pmol each were used
in a final PCR volume of 20 µl, using a master-mix of SYBR Green PCR
core reagents. The threshold line for determining the value of the
cycle of threshold, Ct, was set on 0.3 In Vitro Binding and Yeast Two-hybrid Analyses--
Bacterial
fusion proteins used in binding assays were constructed by inserting
PCR-amplified DNA in-frame into pTRCHis or pRSET (Invitrogen) or pGEX
(Amersham Biosciences). Fusion proteins for studying yeast two-hybrid
interactions were prepared in-frame with bait LexA DNA binding domain
of pHybLex and prey B42 activation domain of pYESTrp2. The following
fusion protein constructs for expression were prepared (name, protein
residue numbers, vector) as follows: rat Cav2.2 synprint:
718-963, pTRCHisC; rat stx1A: 1-268, pGEX-KG;
LCav2a N terminus: 1-67,
pYESTrp2; LCav2a I-II linker:
346-509, pYESTrp2, LCav2b
II-III linker: 714-888, pRSETA. LCav2a
III-IV linker: 1148-1205, pYESTrp2; LCav2a C terminus (CT) CT1: 1596-2141,
pRSETC, pGEX4T-1, pYESTrp2; LCav2a CT2:
1996-2141, pGEX4T-1, pYESTrp2; LCav2a
CT3: 1447-1722, pYESTrp2; Lstx1A: 1-270, pGEX-4T-1, pHybLex; LSNAP25: 1-220, pGEX-4T-1; LsytI:
1-418, pGEX-4T-1, Lmint: 916-1138, pHybLex;
LCASK: 576-861, pHybLex.
Detailed methods for the bacterial expression, purification, and
in vitro binding assay of the fusion proteins have been
described previously (11-12). All fusion protein constructs were
verified by sequencing and evaluated for measurable expressibility on a size-selected SDS-PAGE gel, using Coomassie Blue protein stain. Preparation of protein extracts of Lymnaea brain ganglia and
Western blot analysis were performed as described previously (11).
Lymnaea syntaxin1A was detected by commercial antibody
(ANR-002, Alomone Labs, Jerusalem, Israel). For analysis of two-hybrid
interactions, yeast bait and prey fusion vectors were transformed into
L40 Saccharomyces cerevisiae strain, and the binding
affinity of the interactions was measured by Primary Cell Culture--
Identified neurons of L. stagnalis were isolated and cultured in either a soma-soma
configuration or neurite-neurite configuration as described previously
(13). In knockdown experiments, identified neurons were initially
plated on hemolymph-pretreated glass coverslips (to prevent neuronal
adhesion) in conditioned medium bathed in 10 µM
antisense/mismatch DNA oligonucleotide probes (14) or 10 µg of dsRNA
(for RNA interference) for 3 days. The oligonucleotide or dsRNA-treated
neurons were then transferred and paired on
poly-L-lysine-pretreated glass coverslips in the presence
of CM. In some knockdown experiments, RNAi-treated neurons were tested
for non-synaptic release using a "sniffer cell" (15) Transmitter
release was detected from a dopamine-releasing cell, RPeD1, using one
of its postsynaptic neurons, VK, which displays a depolarizing response
to dopamine.
Antisense and RNAi Treatment--
15-mer double-stranded DNA
antisense calcium channel probes were designed across the start site of
LCav2: GAACGTGGCCATCCA (95-109), a
sequence shared in all Lymnaea isoforms identified. Mismatch
probes for LCav2 consisted of three base
changes to the antisense sequence
GAAGGTGCCCAACCA. Before treatment
to neurons, antisense/mismatch probes were reconstituted in
Lymnaea saline, passed through a 0.8-µm filter, boiled for
2 min, and cooled on ice.
For RNA interference technique, dsRNA probes were synthesized by the
transcription in both sense and antisense strands (MEGASCRIPT; Ambion,
Austin, TX) using T7 and T3 promoters in Bluescript II KS+
vector between base pairs 2237 and 2710 (II-IIII loop
LCav2a), 2237-2761 (II-III loop
LCav2b), 6083-6520 (C-terminal LCav2a), and 1893-2750
(LCASK). RNA probes were resuspended in Lymnaea
saline, boiled for 2 min, incubated at 65 °C for 15 min, and then
allowed to cool to room temperature, enabling single complementary RNA
strands to anneal properly. RNA samples were analyzed by agarose gel electrophoresis.
Electrophysiology--
Synaptic transmission was monitored by
simultaneous intracellular recordings of pre- and postsynaptic neurons
using hardware and acquisition/analysis software as described
previously (13). Calcium channel activities of VD4 neurons were
measured using established whole-cell recording technique (16).
Lymnaea Neurons Express Homologs of Mammalian Calcium Channels and
SNARE Proteins--
The Lymnaea soma-soma synapse is a
rapidly emerging model for investigating synaptic function. Until
recently, the full potential of this system had not been realized due
to lack of genomic information. We have thus used degenerate PCR
cloning and screening of Lymnaea cDNA libraries of whole
brain ganglia to identify orthologs of presynaptic vesicle release
proteins and of the pore-forming
LCav2 is a structural homolog of both
mammalian N- (Cav2.2) and P/Q-type (Cav2.1)
calcium channels considered responsible for transmitter release at
mammalian synapses (18-19), and of invertebrate gene relatives in
Drosophila (DmCa1A/cac) and C. elegans (unc-2) (20-22) (see Fig.
1A). From an alignment of
various Cav2 homologs (Fig. 1, C and
D), a high degree of sequence conservation is seen in the
Single-cell Analysis of Transcript Expression in an Identified
Presynaptic Neuron--
To demonstrate the expression of the
identified calcium channel genes, we examined mRNA transcripts in
single identified pre- and postsynaptic neurons that are major players
in our established soma-soma and neurite-neurite synapse models (13,
24). To ensure unequivocal identification of the cell type, we first
acquired an expression profile of cell type-specific markers in three
identified neurons as follows: visceral dorsal 4 (VD4), left pedal
dorsal 1 (LPeD1), and right pedal dorsal 1 (RPeD1) which form a
critical component of a central respiratory rhythm-generating network. Cells were individually isolated from the intact ganglia and subjected to quantitative, real time PCR (qPCR) analysis. For
cholinergic/peptidergic presynaptic VD4 neurons, we detected
characteristically high LVAchT (vesicular acetylcholine
transporter) and LFMRFamide heptapeptide gene expression
levels (Fig. 2). In contrast, the
postsynaptic serotonergic neuron (LPeD1) and giant dopamine cell
(RPeD1) typically display the higher expression levels of
LVMAT, the vesicular transporter for monoamines (Fig. 2,
inset). We then performed real time qPCR in the presynaptic
VD4 neuron, using specific primers for the three major types of
voltage-gated calcium channels. As seen in Fig. 2, all types of calcium
channel
To minimize the possibility that an unidentified
LCav2 domain II-III linker splice
isoform with a synprint-like motif could exist in
Lymnaea neurons, we examined the relative abundance of the
LCav2 II-III linkers relative to the
domain I-II linker region of LCav2 which
is highly conserved (23) and, thus, a measure of the overall levels of
LCav2 transcript. As seen in Fig. 2, when
we used primers that specifically detected common sequences in both
LCav2a and
LCav2b but did not produce any gel bands
of longer sizes (not shown), we found that the expression of the short
II-III linker was strikingly similar to that of the I-II linker.
These data suggest that the identified short II-III linkers of
LCav2a and
LCav2b predominate in VD4, and if an
isoform of LCav2 with an identifiable
synprint-containing domain II-III loop were to exist, it
can only be of low and almost immeasurable abundance.
Gene Knockdown of Lymnaea Presynaptic Calcium Channels Perturbs
Synaptic Transmission--
To assess the role of
LCav2 channels in neurotransmitter
release, the presynaptic cell VD4 was pre-treated with antisense and
subsequently paired with its postsynaptic partner LPeD1. Specifically, antisense (15-mer) probes were designed at the start site of
LCav2 channels, and presynaptic VD4
neurons were pre-incubated at a concentration of 10 µM
for 3 days before synaptic pairing with postsynaptic LPeD1 neuron.
Synapses were subsequently tested with simultaneous intracellular
recordings. Unlike control conditions, or in cells injected with
mismatch antisense probes (Fig.
3A, n = 12),
excitatory synaptic transmission was perturbed between the soma-soma
paired cells (Fig. 3A, n = 9) in which VD4
cells were selectively treated with
antisense-LCav2 prior to pairing with
LPeD1. Under these experimental conditions, the postsynaptic LPeD1
cells, however, continued to exhibit an excitatory response to
exogenously applied ACh, suggesting that the antisense treatment did
not affect the postsynaptic cell (Fig. 3A). Pre-treatment of
VD4 with LCav1-antisense had only a minor
effect on synaptic transmission that appeared as a fatiguing of the
postsynaptic response over time (not shown).
To complement the antisense experiments, we utilized RNA interference
(RNAi) techniques to degrade catalytically mRNA in VD4 prior to its
pairing with LPeD1. Double-stranded RNA probes were designed against
the short II-III linkers found in both
LCav2a and
LCav2b. VD4 was incubated for 3 days
prior to soma-soma pairing in 8-10 µg of these RNAi probes. To test
for the effectiveness of the RNAi knockdown, total HVA barium currents
were measured in whole-cell patch configuration (Fig. 3B).
RNA interference of LCav2 resulted in a
dramatic depression of HVA currents, leaving only a residual current
with noticeably different inactivation kinetics, which may be due to
LCav1, the other HVA channel type
identified in VD4. Synaptic transmission was completely abolished with
selective RNAi knockdown of the gene containing the identified short
II-III linker (Fig. 3C, n = 7). As RNAi
knockdown was targeted specifically to this
LCav2 sequence, the absence of a postsynaptic response indicates that neurotransmission relies exclusively on a calcium channel homolog that lacks a
synprint motif in the II-III linker.
From the experiment shown in Fig. 3C, it was unclear whether
the lack of synaptic transmission was due to the inability of the VD4
neuron to release neurotransmitter or a consequence of a change in
synaptic architecture that prevented the postsynaptic cell from
detecting transmitter released from VD4. To discriminate among these
possibilities, the dopaminergic neuron RPeD1 was pre-treated with
LCav2 RNAi as described above.
Transmitter release was detected from RPeD1 as a function of
non-synaptic electrophysiological responses in a "sniffer" cell
(15). After RNAi treatment, RPeD1 somata were plated on
poly-L-lysine-coated dishes, and a freshly isolated soma of
one of its postsynaptic neurons (visceral K, VK) was manipulated
in close proximity to RPeD1 to detect induced release of transmitter.
RNAi treatment of RPeD1 rendered this cell incapable of transmitter
release (Fig. 3D, n = 13). In contrast, under control conditions, 100% of RPeD1 cells released dopamine that
was detected at some distance by the sniffer cell (Fig. 3D, n = 9). These data indicate that the loss in synaptic
transmission between VD4 and LPeD1 neurons was likely due to a direct
effect on neurotransmitter release.
Taken together, the above data indicate that, like in mammalian
neurons, Cav2 calcium channels are
required for transmitter release in Lymnaea neurons.
Lymnaea Synaptic Transmission Depends on SNARE Proteins but Not on
Their Interactions with the Lca22 Channels--
Previous
work (25) in other invertebrate systems suggests that synaptic
transmission in Lymnaea may also rely on the SNARE proteins
syntaxin1A and SNAP-25. As shown in Fig.
4A, injection of 1 µM Botulinum toxin (Bt) C1 or BtE into presynaptic VD4
neurons paired with LPeD1 abolished synaptic transmission between these neurons (Fig. 4A; n = 6 and 5, respectively), without affecting the response of the postsynaptic
neuron to exogenously applied ACh, the transmitter used at the
VD4-LPeD1 synapse (not shown). Hence, as expected a functional
complement of LSNAREs in the presynaptic cell is required
for transmitter release.
The absence of the synprint motif in
LCav2, however, predicts that
Lymnaea synaptic proteins such as syntaxin1A, SNAP-25, and
synaptotagmin1 should not be able to interact with the II-III linker.
Indeed, as shown in Fig. 4, there was no measurable association of the
Lymnaea II-III linker with GST-Lstx1A,
GST-LSNAP25, or GST-Lsyt1 (Fig.
4B) in direct binding assays, whereas comparable amounts of
Lymnaea synaptic proteins readily bound to the positive control, rat His6 synprint (Fig. 4B).
We also did not detect binding of these proteins to the
LCav2 C terminus (Fig. 4C), N
terminus, domain I-II linker, and domain III-IV linker region in vitro, or in a yeast two-hybrid assay (not shown).
Moreover, neither the II-III linker nor the C-terminal region bound
syntaxin1A in protein extracts of Lymnaea whole brain
ganglia (Fig. 4C), indicating that syntaxin1A is not coupled
to these regions of the LCav2 channel via
adaptor proteins. In contrast, there was consistent,
dose-dependent binding of rat synprint to Lymnaea syntaxin1A from brain extracts (Fig.
4C).
Overall, these data indicate that Lymnaea orthologs of
syntaxin1A, SNAP-25, and synaptotagmin1, although potentially capable of interacting with rat synprint, do not associate with the
major cytoplasmic regions of the Lymnaea homolog of
presynaptic calcium channels and thus are unlikely to interact with
this channel altogether.
Synprint Peptide Injection into VD4 Perturbs Synaptic
Transmission--
Synprint peptides injected into mammalian
SCG neurons disrupt synaptic transmission, perhaps by competitively
inhibiting the interaction between syntaxin1A and/or SNAP-25 to N-and
P/Q-type channels (7). Because the Lymnaea channel ortholog
lacks synprint and consequently does not appear to associate
with these proteins, one would expect that mammalian
synprint peptide injected into Lymnaea VD4
neurons should not affect neurotransmission. Surprisingly, the
injection of 8-10 pM of rat synprint peptide
into VD4 neurons 1-4 h prior to recording interfered with synaptic
transmission between VD4-LPeD1 pairs (Fig.
5, n = 13). In 7 of 13 synprint-injected cells, a complete blockade of transmission
was observed. In the remaining synapses a marked
use-dependent perturbation of synaptic transmission was
noted, such that during a series of 10 action potentials, the
postsynaptic response became reduced to 32% of the amplitude of the
initial EPSP (Fig. 5, C and D). These
results suggest that the site of synprint peptide action was
initially inaccessible but became incrementally exposed to
synprint by repetitive presynaptic stimuli. The presence of
the synprint peptide per se did not affect
current densities or the biophysical properties of Lymnaea
calcium currents (not shown), indicating that the effects of these
peptides occurred independently of an action on calcium channels. To
ensure that the effects of the synprint peptide were specific, we generated a peptide of the domain II-III linker region of
the Lymnaea Cav2 calcium channel which, as shown
in Fig. 4B, is incapable of interacting with syntaxin 1, SNAP-25, or synaptotagmin. Injection of this peptide into VD4 neurons
did not significantly affect synaptic transmission between the paired
cells (Fig. 5, A, B, and D). This
observation is reminiscent of the inability of rat L-type calcium
channel II-III linker peptides to alter synaptic transmission in rat
SCG neurons (7), and supports the specificity of the
synprint peptide effects illustrated in Fig. 5.
Overall, our data suggest that synaptic transmission between
Lymnaea neurons exhibits many of the hallmarks of mammalian
neurotransmission but seems to occur without physical tethering of
presynaptic vesicle release complex to a synprint region in
presynaptic calcium channels. Mammalian synprint peptides
can nonetheless interfere with neurotransmitter release perhaps by
inactivating other synaptic elements that make use of the
synprint motif.
Alternative Splicing of the Non-L-type Channel Regulates Lymnaea
Neurotransmission--
As noted above, two splice variants of the
LCav2 channel with different C-terminal
tails are expressed in VD4 neurons (Fig. 2). To determine the
significance of this observation for synaptic transmission, we designed
a specific RNAi probe against the variant with the extended C terminus,
and we assessed its effect on synaptic transmission between VD4 and
LPeD1. The selective depletion of the longer C-terminal
LCav2 variant completely abolished
synaptic transmission in 6 of the 9 cells examined, and in the
remaining synapses, EPSP amplitude was reduced to 2.1 ± 0.2 mV
(see Fig. 6A). The loss in
synaptic transmission was accompanied by a significant reduction
(22.4 ± 3.01 pA/pF, n = 7; p = 0.001 versus control) in HVA current densities in
RNAi-treated cells (Fig. 6B). Although this reduction was
substantial, it was nonetheless significantly (p = 0.02) smaller than that seen in Fig. 3B which was designed
to eliminate all LCav2 expression. Indeed, the data shown in Figs. 3B and 6B are
consistent with the observation that the
LCav2a channel comprises a larger fraction of the total LCav2 calcium
mRNA transcript level (see Fig. 2).
The notion that an alternately spliced C-terminal sequence appears to
be essential for synaptic release raises the possibility that this
region might be involved in protein-protein interactions at the
synapse. To test this possibility, we injected a peptide (CT1, see also
Fig. 7A) directed against this
region into VD4 neurons 1 h prior to recording. As shown in Fig.
6, C and D, this resulted in a dramatic
inhibition of synaptic transmission, consistent with the notion that
the peptide may have competitively inhibited an interaction between the
non-L-type channel C-terminal region and an essential presynaptic
protein.
Synaptic Transmission Depends on CASK and Mint1--
The
alternately spliced C-terminal region in
LCav2a contains a proline-rich region
with multiple PXXP consensus sites for putative association
with SH3 domains, as well as a characteristic (D/E)XWC motif
at the C-terminal, 3' end (see Figs. 1D and 7A). Both the (D/E)XWC and an upstream proline-rich region are
highly conserved in mammalian N- and P/Q-type calcium channels, where they have been shown to bind specifically to the first of two PDZ
domains of Mint1 and SH3 domains of CASK, respectively (26-27). To
determine whether LCav2 C terminus could
interact with Mint1 and CASK in vitro, we employed a yeast
two-hybrid assay, using the LCav2a C
terminus as bait and Lymnaea Mint1 and CASK as prey. We
detected a robust interaction (Fig. 7A) with CASK binding to
a fragment containing a proline-rich sequence (CT3), whereas Mint1
bound downstream specifically to the C-terminal tail containing DDWC
motif (CT2). Mint1 binding was virtually abolished when we introduced a
single amino acid mutation W2140K into the CT2 peptide sequence,
converting Lymnaea LCav2a DDWC sequence
to DDKC, the terminal amino acids reminiscent of rat
To test further the role of Mint1 and CASK in synaptic transmission, we
generated separate peptides corresponding to the CASK and Mint1 binding
regions, and we injected them individually into VD4 neurons 1 h
prior to recording. Whereas injection of the CASK interacting CT3
peptide did not affect synaptic transmission in 6 of 7 synaptic pairs
examined (Fig. 7C), the Mint1 interacting CT2 peptide
dramatically inhibited synaptic transmission such that 4 of 10 synapses
exhibited no detectable transmission, and in the remaining cells EPSP
amplitudes were dramatically reduced (Fig. 7C). Injection of
a DDKC mutant CT2 peptide (which is incapable of binding Mint1, see
Fig. 7A) resulted in normal EPSPs in 7 of 8 experiments,
suggesting that the effects of the Mint1-interacting peptide were
indeed specific for the Mint1-calcium channel interaction. Our data are
thus consistent with a mechanism in which Lymnaea synaptic
transmission is dependent on a Mint1-calcium channel interaction, as
well as on the presence of CASK.
To determine whether the expression of the calcium channel splice
variant and synaptic/scaffolding proteins are dynamically regulated
during synaptogenesis, we carried out qPCR analysis of pre- and
postsynaptic VD4 and LPeD1 neurons that were either separately cultured
or synaptically paired overnight prior to mRNA extraction but were
otherwise cultured under identical conditions. As shown in Fig.
7D, the expression of the
LCav2 channel splice variants were not
altered during synapse formation, consistent with our previous finding
that the development of calcium hotspots during synapse formation
appears to involve a redistribution of existing channels (16).
Furthermore, there were no obvious gene expression changes observed in
SNARE proteins or synaptotagmin1 (see also inset to Fig.
7D). Interestingly, whereas gene expression of Mint1 was
almost undetectable in unpaired neurons (see inset), robust
Mint1 expression was detected after pairing, indicating that Mint1 may
serve as key signaling element during synapse formation. We were unable
to perform a similar analysis for CASK, because CASK expression levels
were only barely over the detection limit (requiring >36 PCR cycles),
thus preventing us from reliably determining changes in CASK
expression. Nonetheless, given the profound effects of CASK RNAi
knockdown, together with the observations that both Mint1 and CASK bind
to specific calcium channel splice variant whose expression is
essential for synaptic transmission, suggests that these proteins could
regulate synaptic activity via their interaction with calcium channels.
Absence of Coupling of SNARE Complexes with Presynaptic Calcium
Channels--
The Lymnaea soma-soma synapse is an emerging
model that allows convenient access to identified single pre- and
postsynaptic neurons (13, 16). These synapses are morphologically and
electrophysiologically similar to those in vivo and require
new gene transcription and de novo protein synthesis to form
(13). Moreover, as in mammalian synapses, target cell-specific calcium
hotspots develop at the active zones during synapse formation, which
appear to result from a redistribution of existing calcium channels
rather than the synthesis of new channels (16). Detailed analyses of
the molecular players involved in Lymnaea synapse
formation/function have so far been hampered by a lack of information
about the Lymnaea genome. We have thus cloned the entire
complement of voltage-gated calcium channels, plus a number of key
synaptic and scaffolding proteins from Lymnaea to provide a
novel perspective regarding the relationship between SNARE proteins,
calcium channels, and scaffolding proteins during transmitter release
at the level of individual pre- and postsynaptic neurons.
Although the SNARE complexes are important for transmitter release from
Lymnaea neurons, this role appears to be independent of
their coupling with calcium channels. Our results therefore contrast
with those obtained in vertebrates where a direct association between
SNARE complexes and calcium channels was deemed necessary for
transmitter release (2, 5). Indeed, the synaptic protein interaction
site found in mammalian N-type and P/Q-type calcium channels (4-6) is
conspicuously absent in invertebrate Cav2 orthologs such as
in C. elegans, Drosophila (10), and
Lymnaea. It has remained unclear as to whether synaptic
transmission in invertebrates occurs independently of a calcium channel
SNARE protein interaction, or whether invertebrate calcium channels
might perhaps harbor a synaptic protein interaction site that is
structurally unrelated to that found in mammals (9, 28-31). Because
syntaxin1A, SNAP-25, and synaptotagmin1 mainly associate through
exposed, cytoplasmic surfaces, any putative synaptic binding domain on
the calcium channel would most likely be confined to one of the
intracellular loops. Yet, in pull-down assays and/or in yeast
two-hybrid assays, we were unable to detect a physical interaction of
these proteins with any of the major intracellular regions of the
LCav2 channel, indicating that they do
not directly associate with LCav2. The
injection of mammalian synprint peptides (but not
LCav2 II-III linker peptides)
nonetheless inhibited synaptic activity without affecting calcium
channel function, suggesting that these peptides are capable of
non-specifically interfering with synaptic transmission downstream of
calcium channels. Alternatively, synprint may act as an
interaction domain in other as yet unidentified proteins more distantly
associated with transmitter release. Our observation of a
synprint peptide effect in a synapse where calcium channels
are apparently uncoupled from SNAREs raises the possibility that
similar nonselective effects could have occurred when this peptide was
injected into mammalian SCG neurons to uncouple the SNARE complex from
voltage-gated calcium channels (2, 7). If so, this would weaken the
interpretation that a direct physical coupling between calcium channels
and the SNARE complex must be a prerequisite for transmitter release at
synapses to ensure proximity of calcium channels to the release sites.
The synprint region may perhaps serve to optimize the
efficiency of synaptic transmission or to serve as a site for
regulating calcium channel activity (2, 12, 19, 32-33). Thus, rather
than being universally required, the presence of a synaptic protein
interaction site in calcium channels may be an evolutionary
specialization in the vertebrates.
Possible Role of LCav2a Channels in Presynaptic
Transmitter Release--
Despite the absence of a synprint
motif, calcium hotspots appear to form at the active zones during
synapse formation (16). Interestingly, this appears to involve a
redistribution of existing calcium channels, rather than the new
synthesis of channel protein (16), consistent with our observation that
transcript levels of individual calcium channel splice variants did not
change during synapse formation. Given that synaptic transmission in
Lymnaea appears to be critically dependent on the
LCav2a channel (i.e. the one
with the long C terminus that is capable of interacting with Mint1 and
CASK), we propose that interactions between the C terminus and these
proteins may be involved in channel clustering at the active zones and
perhaps in synaptic transmission per se. This would be
consistent with the fact that the Mint1 and CASK interaction domains
are highly conserved in synaptic calcium channels, and with recent
evidence suggesting that synaptic targeting of N-type calcium channels
in rat hippocampal neurons is dependent on the C-terminal region of
these channels (27).
Although Mint1 and CASK can both interact with the C-terminal region of
the channel and have both been implicated in protein targeting
functions, they do not appear to have identical cellular functions,
with CASK having a role in developmental processes and Mint1 having a
closer association with synaptic vesicle exocytosis (34-36). The
observations that the expression of Mint1 appeared to occur only
following cell-cell contact (see Fig. 7D) and that Lymnaea synaptic transmission was blocked by injection of
Mint1 interacting DDWC-CT2 peptides 1 h prior to recording suggest
that the Mint calcium channel interaction may mediate a more acute role
in synaptic transmission. In contrast, the CASK interaction may be of
greater importance in the earlier stages of synapse formation/channel
clustering. This would fit with our observation that injection of the
CASK interacting CT3 peptide 1 h prior to recording did not affect
synaptic transmission. We also note that the RNAi knockdown of CASK
that resulted in the loss of synaptic transmission was initiated prior
to synapse formation, again perhaps consistent with an early role of
CASK. However, although our data provide evidence for an important role
of CASK and the LCav2 C-terminal region,
we must acknowledge two potential caveats. First, because we did not
examine any putative effects of the CT3 peptide during earlier stages
of synapse formation, our data do not permit us to determine whether a
physical interaction between CASK and the C terminus of the
LCav2 channel is indeed of physiological
significance. Second, although a single point mutation in the CT2
peptide prevented both Mint1 binding and, in parallel, abolished the
physiological effects of this peptide, we cannot rule out the
possibility that another protein recognizing the Mint1-binding site on
the LCav2 calcium channel could be
antagonized by the CT2 peptide. Our experimental data at this stage do
not allow us to firmly implicate Mint1 in synaptic transmission. Further experiments will therefore be needed to prove a role of Mint1
in synaptic transmission, and to define the temporal sequence of the
involvement of CASK (and possibly Mint1) in synapse formation and
synaptic activity.
Taken together, it appears that an interaction of presynaptic calcium
channels with scaffolding proteins appears to be more fundamental for
synaptic transmission than direct coupling to syntaxin, SNAP-25 and
synaptotagmin. This may have implications for understanding the roles
of other types of presynaptic calcium channels that lack the
synprint motif, such as certain splice variants of the human
N-type channel (37) and apparently all types of non-vertebrate calcium
channels identified to date. Such differences likely contribute to the
observed synaptic diversity across cell types and species (38).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ZAP cDNA libraries
of the Lymnaea central nervous system. Full-length cDNA
clones were generated, using primer walking and 5'- and 3'-rapid
amplification of cDNA ends. PCR fragments were amplified with DNA
polymerases, Herculase, or TurboPfu (Stratagene), and final
sequences were assembled from at least three independent PCRs. All
sequencing was completed in both sense and antisense directions using
ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Inc., Foster City, CA).
Rn
(base-line subtracted values) with curves reaching maximally 6-8
Rn and base lines of ~0.02
Rn. Data were
considered only if the
CT values of control and
experimental samples were >8 cycles. The overall cDNA expression
levels per sample were normalized to the expression of controls
L-aldolase and to L-
-tubulin which
both show consistent expression for each cell type, and corresponded proportionally to the different cell diameters.
-galactosidase activity
using o-nitrophenyl-
-D-galactoside substrate,
qualitatively by filter lift according to manufacturer's instructions
(Hybrid Hunter, Invitrogen) and quantitatively in liquid extracts.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 subunits of the
structural superfamily of voltage-dependent calcium channels. Lymnaea calcium channel orthologs include
representatives of the major high voltage-activated (HVA) calcium
channel classes, L-type (LCav1) and
non-L-type (LCav2), and (LVA) T-type
(LCav3) channels, as well as a calcium
channel
subunit (LCavb). In addition,
we identified a representative of the functionally unidentified class,
dubbed U-type (17). These subunits (four
1 and one
)
are reminiscent of the representation in Drosophila and
C. elegans genomes (10) and likely provide a complete
complement of such genes in the Lymnaea nervous system.
subunit interaction site in the domain I-II linker (AID sequence) (23) and in the major transmembrane domains, but no apparent sequence identity is evident in the domain II-III linker, the
locus of the synaptic protein interaction (synprint) site in
vertebrate channels (Fig. 1, C and D). Indeed,
the II-III linker region in LCav2
appears considerably shorter than that of vertebrate Cav2
channels, and repeated attempts to identify an
LCav2 channel with a longer,
synprint-containing II-III linker in Lymnaea
cDNA were unsuccessful. In trying to do so, however, we isolated an
additional LCav2 isoform
(LCav2b) that varied by 17 amino acids in
the II-III linker (see Fig. 1). The apparent lack of the
synprint motif in Lymnaea and other invertebrates contrasts with the high sequence homology among presynaptic vesicle release proteins such as syntaxin1A, SNAP-25, and synaptotagmin1 (see
Table I). Taken together, these
observations indicate that although SNARE proteins are highly conserved
throughout evolution, the appearance of a synprint motif may
be an evolutionary specialization in vertebrates.
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Fig. 1.
Comparison of vertebrate and
invertebrate orthologs of Cav2 calcium channels.
A, phylogram of Cav2 1 subunits.
A single maximum parsimony tree obtained using the heuristic algorithm
in PAUP*4.0b8. Numbers on the branches are bootstrap support
values. B, transmembrane topology of the calcium channel
1 subunit, indicating the absence of the synaptic
protein interaction site in invertebrate calcium channels, and the
location of proline-rich and DDWC motifs in the C terminus.
C, region size (mean and S.E.) of the cytoplasmic domain
I-II and II-III linkers in Cav1 and Cav2
representatives from invertebrates (jellyfish, flatworm, nematode, two
insects, snail, and squid) and vertebrates (tunicate, fish, marine ray,
frog, chicken, rabbit, hamster, mouse, rat, and human) and a human
Cav2.2 splice variant (
1B-
2) lacking the
synprint site (37). Note that the extended,
synprint-containing II-III linker region seems exclusive to
vertebrate Cav2 channels. D, similarity between
multialigned amino acid sequences of full-length, Cav2
calcium channel
1 subunits from invertebrates (C. elegans, Drosophila, and Lymnaea) and human
homologs (
1A,
1B, and
1E).
Highlighted with boxes are highly conserved regions
corresponding to the major transmembrane domains (D1 through D4), and
three cytoplasmic regions associated with putatively ascribed binding
partners:
subunits (AID), calcium, and calmodulin (EF-hand/IQ
motifs) and Mint1 ("(D/E)_WC" motif). The synprint
region contained in the II-III linker is not conserved among putative
full-length Cav2 isoforms.
Molecular cloning, identification, and primer pair sets for Lymnaea
genes
1 subunits, as well as the
subunit were
present in measurable abundance in VD4, including the two C-terminal
splice variants of both the LCav1 and
LCav2 channels (Fig. 2). In addition,
expression of a number of key synaptic proteins such as VAMP,
syntaxin1A, SNAP-25, synaptotagmin1, and nsec1/munc18-1 was also
detected (not shown, but see Fig. 7D). Hence,
Lymnaea VD4 neurons robustly express many of the proteins
thought to be involved in mammalian synaptic transmission.
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Fig. 2.
Quantification of calcium channel mRNAs
in VD4 neurons. Gene expression measurements using real time qPCR
of cDNA synthesized from mRNA of identified neurons freshly
isolated from six Lymnaea central nerve ring ganglia
(mean ± S.E., n = 6). Data are represented as the
cycle threshold of detection (CT), normalized to the
expression of control marker gene,
L-aldolase. Main panel,
expression profile of calcium channel 1 subunits
(LCav1 to Cav4) and
subunit in VD4. For both LCav1 to
LCav2, both the full-length, a
isoform (C-term +) and C-terminally truncated b
isoforms (C-term
) are detected. Inset,
expression profile of cell type-specific marker genes in identified
VD4, LPeD1, and RPeD1 neurons. For presynaptic VD4 neurons, the
cholinergic/peptidergic neuron phenotype in each sample is confirmed
(i.e. high LVAchT and LFMRFamide
heptapeptide gene expression). The postsynaptic serotonergic neuron
(LPeD1) and giant dopamine cell (RPeD1) shows the
expected serotonin/dopamine phenotype (i.e. high levels of
LVMAT).
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Fig. 3.
Transmitter release requires the
LCav2 channel. A,
isolated VD4 and LPeD1 neurons were treated with 10 µM,
15-mer antisense, or mismatch oligonucleotide probes directed against
the start site of LCav2. In pairs treated
with antisense probes (n = 9), trains of action
potentials induced in VD4 (arrow) failed to evoke a
postsynaptic response in LPeD1. In pairs treated with mismatch probes
(n = 12), however, action potentials in VD4
(arrow) elicit 1:1 EPSPs in the postsynaptic LPeD1 neuron.
LCav2 knockdown does not affect the
postsynaptic cell, which is still responsive to exogenously applied
ACh. B, RNAi knockdown of the
LCav2 gene in VD4 reduces the HVA barium
current densities, revealing altered kinetics of the remaining
currents. Sample recordings display representative currents for both
control (n = 6) and RNAi (n = 5)-treated cells. C, knockdown of the
LCav2 gene via RNA interference. When the
pre- and postsynaptic neurons were treated with 10 µg of RNAi
directed against the II-III linker of
LCav2, synaptic transmission was
perturbed (n = 7). Induced trains of action potentials
in VD4 (arrow) fail to elicit a postsynaptic response in
LPeD1. In contrast, under control conditions, induced action potentials
in VD4 (arrow) elicit 1:1 EPSPs in LPeD1. D,
knockdown of the LCav2 gene with RNAi
perturbs nonsynaptic release of transmitter. Transmitter release from
the dopamine releasing cell RPeD1 (n = 13) was detected
using a sniffer cell, VK, that depolarizes in response to
dopamine. Unlike control cells (n = 9), in RNAi
treated cells, trains of action potentials in RPeD1 (arrow)
fail to elicit a response in the sniffer cell VK.
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Fig. 4.
Lymnaea synaptic transmission
depends on SNARE proteins, but not on SNARE protein-calcium channel
interactions. A, effect of Botulinum toxins (Bt)
on synaptic transmission in Lymnaea. Unlike in control pairs
(n = 6), in VD4 neurons injected with BtC1
(n = 6) or BtE (n = 5), a train of
action potentials (arrow) in VD4 failed to elicit a
postsynaptic response in LPeD1. B, in vitro
binding of His6-LCav2a
II-III linker (left panel) or the positive control
His6-rat Cav2.2 synprint
(right panel) to incremental amounts of 20, 40, or 80 µl
of a 50% slurry of Lymnaea syntaxin1A, SNAP-25, and
synaptotagmin1. Western blots were probed with an Anti-Xpress antibody.
C, in vitro experiments involving the binding of
230 µg of Lymnaea brain ganglia extracts to immobilized
His6- LCav2b II-III linker
(80 µl), His6-LCav2a C
terminus (80 µl), and His6-rat Cav2.2
synprint (20, 40 and 80 µl) under identical experimental
conditions.
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Fig. 5.
Synprint peptides prevent
Lymnaea synaptic transmission. A and
B, sample recordings (A) and bar graph
(B) (mean EPSP amplitude ± S.D.) representing control
pairs or pairs in which the presynaptic VD4 neuron was injected with
rat Cav2.2 synprint, or the Lymnaea
Cav2 II-III linker. In ~50% (n = 13) of
the synprint injected pairs, synaptic transmission is
completely abolished (not shown). In the remaining synprint
injected pairs, the average EPSP amplitude is 2.26 ± 0.40 mV. In
control and LCav2 II-III linker injected
pairs, synaptic transmission was normal. Single action potentials
induced in VD4 (arrow) elicited 1:1 EPSPs with average
amplitude of 6.6 ± 0.96 mV (control, n = 6) and
6.6 ± 0.72 mV (LCav2 II-III
linker, n = 8). C and D, the
effect of synprint displays marked use dependence. In these
experiments, all neuron pairs were subjected to a series of 10 action
potentials with 30 s between each action potential. Unlike in
control cells, or in cells injected with
LCav2 II-III linker (not shown), in
synprint injected pairs (top panel), the EPSP
induced by the 10th action potential is on average 32% smaller than
that induced by the 1st action potential. The graph in D
demonstrates the use-dependent blockade of synaptic
transmission in synprint injected neurons (closed
circles). The remaining pairs injected with synprint do
not display synaptic transmission at any time during our recordings
(closed triangles). In control (open circles) and
LCav2 II-III linker injected (open
triangles) pairs, the amplitude of EPSPs remains relatively
constant.
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Fig. 6.
Alternative splicing of
LCav2 is essential for
synaptic transmission. A, effect of selective RNAi knockdown
of the long form C-terminal splice variant of the
LCav2 Ca2+ channel on EPSPs.
The experimental conditions were as outlined in Fig. 3, B
and C, but the RNAi probe was directed against the
alternately spliced C-terminal region. Note that in 6 of 9 synapses
examined, synaptic transmission was undetectable, whereas in the
remaining pairs, EPSP amplitude decreased to about 30% of the control
value. B, current densities in control cells, and in cells
depleted of the LCav2 C-terminal splice
variant. The control data are the same as those shown in Fig.
3B. C and D, effect of injection of a
peptide (CT1) corresponding to the alternately spliced
C-terminal region in LCav2a on synaptic
transmission in form of sample traces (C) or a bar graph
(D). Note that this peptide drastically depresses EPSPs in
paired VD4/LPeD1 neurons.
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Fig. 7.
Mint1 and CASK regulate Lymnaea
neurotransmission. A, yeast two-hybrid assay
illustrating the binding of Mint1 and CASK to the alternatively spliced
C-terminal region of LCav2. Both Mint1
and CASK bind to this region but to separate domains with Mint1
interacting with the extreme C-terminal tail (CT2 region), and CASK
binding to an upstream proline-rich region (CT3 region). Site-directed
mutagenesis of the tail of the CT2 peptide from DDWC to DDKC abolishes
the Mint interaction. Data are expressed in form of -galactosidase
activity determined from liquid extracts. The top of the
figure depicts a graphic representation of the
LCav2 channel C-terminal region with the
location of CT peptides illustrated above. B, sample traces
showing that RNAi depletion of VD4 neurons of CASK greatly reduced
neurotransmission. In 4 of 7 cells, no EPSPs could be detected, whereas
in the remaining cells they were greatly reduced. C, effect
of injection of CASK interacting CT3 peptides and of wild type and DDKC
mutant LCav2 CT2 peptides into VD4
neurons on synaptic transmission. Note that the wild type CT2 peptide
disrupted neurotransmitter release, whereas the mutant CT2 peptide and
the CT3 peptide did not. D, qPCR analysis of gene expression
of cultured VD4 and LPeD1 neurons, comparing those cultured overnight
as VD4/LPeD1 synaptic pairs or unpaired. Whereas the expression of
synaptic proteins such as syntaxin1A (Lstx1A) and
synaptotagmin1 (Lsyt1) or calcium channels is
not regulated during synapse formation, the expression of
LMint1 is dramatically increased in paired cells.
Inset, gene expression levels of
Lsyt1 and LMint1 in unpaired neurons
and synaptic pairs. Data are represented as the cycle threshold of
detection (CT), normalized to the expression of
L-aldolase. Note the virtual absence of
LMint1 in unpaired neurons. Also note that these experiments
differ from those shown in Fig. 2, where only freshly isolated neurons
of a single identified type were used for analysis under each
condition.
1E
channels (Fig. 7A) (26). Hence, in analogy to what has been
reported for rat N-type calcium channels, Mint1 and CASK can bind to
the LCav2 carboxyl tail, raising the
possibility that these interactions might be critically involved in
neurotransmission. To test this possibility, we first treated VD4
neurons with RNAi to CASK. This resulted in block of synaptic activity
in 4 of 7 experiments and dramatically reduced EPSP amplitude in the
remaining cells (Fig. 7B), indicating that CASK is essential
for synaptic function. Moreover, this is consistent with the
possibility that an interaction between CASK and the
LCav2 C terminus is required to ensure
proper neurotransmitter release. There was no detectable change in
calcium current density or the biophysical characteristics of the
channels after CASK RNAi treatment or in the presence of the C-terminal
peptide (not shown), indicating that the observed effects were not due
to a change in calcium channel function. We were, however, unable to
detect consistent effects of Mint1 RNAi knockdown on synaptic
transmission (n = 25). This could in principle suggest
that Mint1 might not play a key role in synaptic transmission (but see
below). Alternatively, it is possible that the knockdown may have been
inefficient/incomplete or perhaps that compensation from another Mint
isoform could have occurred.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
---|
* This work was supported in part by operating grants from the Canadian Institutes of Health Research (to N. I. S. and G. W. Z.).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.
¶ Recipient of a postdoctoral fellowship award from the Human Frontiers in Science Program.
Supported by from Alberta Heritage Foundation for Medical
Research Studentships.
** Recipient of a Canadian Institutes of Health Research postdoctoral fellowship award.
§§ Recipient of a Senior Scholarship from Alberta Heritage Foundation for Medical Research and is a Canadian Institutes of Health Research Investigator. To whom correspondence should be addressed: Dept. of Physiology and Biophysics, University of Calgary, 3330 Hospital Dr., N. W., Calgary T2N 4N1, Canada. Tel.: 403-220-8687; Fax: 403-210-8106; E-mail: Zamponi@ucalgary.ca.
¶¶ Recipient of a Scientist Award from the Alberta Heritage Foundation for Medical Research and is a Canadian Institutes of Health Research Investigator.
Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M211076200
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ABBREVIATIONS |
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The abbreviations used are: SNARE, soluble NSF attachment protein receptors (where NSF is N-ethylmaleimide-sensitive factor); dsRNA, double-stranded RNA; ACh, acetylcholine; qPCR, quantitative, real time PCR; RNAi, RNA interference; HVA, high voltage-activated; LVA, low voltage-activated; VD4, visceral dorsal 4; Bt, Botulinum toxin; EPSP, excitatory postsynaptic potential.
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