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The Journal of Neuroscience, February 15, 2002, 22(4):1266-1272
The Core Membrane Fusion Complex Governs the Probability of
Synaptic Vesicle Fusion But Not Transmitter Release Kinetics
Michael F. A.
Finley1, 2,
Sejal M.
Patel1,
Daniel V.
Madison2, and
Richard H.
Scheller1, 2
1 Howard Hughes Medical Institute and
2 Department of Molecular and Cellular Physiology, Stanford
University, Stanford, California 94305
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ABSTRACT |
Synaptic vesicle fusion is driven by the formation of a
four-helical bundle composed of soluble N-ethylmaleimide
sensitive factor (NSF) attachment protein receptors (SNAREs). Exactly
how the structural interactions that lead to the formation of this complex relate to neurotransmitter release is not well understood. To
address this question, we used a strategy to "rescue" synaptic transmission after proteolytic cleavage of the synaptosome-associated protein of 25 kDa (SNAP-25) by botulinum neurotoxin E (BoNtE). Transfection of CA3 hippocampal pyramidal cells with BoNtE-resistant SNAP-25 restored synaptic transmission. Additional mutations that alter
the interaction between SNAP-25 C-terminal coil and the other SNARE
coils dramatically reduce transmitter release probability but leave the
kinetics of synaptic responses unaltered. These data indicate that at
synapses, SNARE interactions are necessary for fusion but are not the
rate-limiting step of neurotransmission.
Key words:
soluble N-ethylmaleimide-sensitive factor
(NSF) attachment protein receptors (SNAREs); SNAP-25; botulinum
neurotoxin E (BoNtE); hippocampus; synaptic transmission; vesicle
fusion
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INTRODUCTION |
Release of neurotransmitter requires
the fusion of synaptic vesicles with the presynaptic plasma membrane.
Three proteins, collectively known as soluble
N-ethylmaleimide sensitive factor (NSF) attachment protein
receptors (SNAREs), are required for transmitter release: VAMP
(synaptobrevin), located on the vesicle, syntaxin, and SNAP-25, located
primarily on the presynaptic membrane (Südhof and Scheller, 2001 )
(see Fig. 1A). As a critical part of this process,
the SNAREs bind together in a parallel, four-helical bundle, with one
helix each contributed by VAMP and syntaxin and two contributed by
SNAP-25 (Poirier et al., 1998; Sutton et al., 1998), to form an
extremely stable, SDS-resistant complex that requires NSF-mediated
ATP-hydrolysis to be dissociated (Söllner et al., 1993). Each of
these helices contains a heptad repeat such that hydrophobic residues
from each helix are oriented toward the center of the bundle,
contributing to thermal stability (Sutton et al., 1998). In addition,
each helix contains a highly conserved inward-facing polar residue at
its center (either glutamine or arginine) that contributes to an ionic
layer in the middle of the four-helix bundle. It is hypothesized that
this ionic layer acts to align the four helices in register with one
another (Sutton et al., 1998). The energy released by the formation of
this complex may drive the fusion reaction that leads to transmitter
release (Hanson et al., 1997; Lin and Scheller, 1997).
Exactly how the structures of the SNARE molecules relate to their
function in synaptic vesicle fusion is under active investigation. Clostridial neurotoxins (Schiavo et al., 2000), highly specific proteases that cleave individual SNARE proteins, have been used extensively as a powerful tool to examine the function of toxin-cleaved SNARE proteins during neuromuscular or CNS synaptic transmission, as
well as for chromaffin cell secretion. In these studies, the investigation of molecular interactions was restricted to the cleavage
products produced by these proteases. The role of particular SNARE
protein amino acid residues has been investigated by overexpressing mutated forms of the protein in chromaffin cells and monitoring release
of norepinephrine from large, dense-core vesicles (Chen et al., 1999;
Criado et al., 1999; O'Sullivan et al., 1999; Wei et al., 2000).
However, it has been difficult to extend the study of precise molecular
interactions among SNAREs to CNS synapses, where neurotransmitter is
packaged in small, clear vesicles and transmitter release occurs on a
faster time scale. We sought to investigate the structure-function
relationship of SNAP-25 in functional mammalian CNS synapses by
combining the specificity of clostridial neurotoxin cleavage with the
ability to transfect organotypic hippocampal slices with mutated forms
of SNAP-25. For the first time, this approach has provided an
opportunity to directly examine the effects of SNAP-25 mutants on
transmission at a fast, excitatory CNS synapse.
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MATERIALS AND METHODS |
Molecular biology. Full-length mouse SNAP-25b
containing the toxin resistant mutation D179K (GAC to AAG) was
expressed using a pcDNA3 (Invitrogen, Carlsbad, CA) mammalian
expression vector lacking the neomycin-resistance gene
[("pcDNA3m") courtesy of Dr. T. F. J. Martin, University
of Wisconsin, Madison, WI]. Additional mutations were made to this
construct using PCR-based mutagenesis (QuickChange, Stratagene, La
Jolla, CA) and confirmed by sequencing. Epitope-tagged versions of
SNAP-25b were made by subcloning SNAP-25b from pcDNA3m into a
pcDNA3.1(+) (Invitrogen) vector with a multiple cloning site modified
to contain the myc sequence N-terminal to SNAP-25. For recombinant
protein expression, the full-length SNAP-25b constructs were subcloned
from pcDNA3m into pGEX and expressed as glutathione
S-transferase fusion proteins. Recombinant botulinum neurotoxin E (BoNtE) light chain (gift of the late Dr. Heiner Niemann,
Medizinische Hochschule, Hannover, Germany) was used as described
previously (Chen et al., 1999) to test cleavage of wild-type and mutant
versions of SNAP-25 in vitro. Wild-type SNAP-25 was
partially cleaved (>50%) by 10 nM and completely cleaved
by 25 nM BoNtE light chain, whereas no cleavage of
BoNtE-resistant SNAP-25 (D179K) was evident even at 400 nM (data not shown). pEGFP-C1 (Clontech,
Palo Alto, CA) was used for expression of green fluorescent protein (GFP).
Biolistic transfection. Organotypic hippocampal slices were
prepared as described previously (Pavlidis and Madison, 1999). Slices
were transfected using the Helios Gene Gun System (Bio-Rad, Hercules,
CA). Parameters were optimized on the basis of previous studies (Wong
et al., 1998) and advice from other users and Bio-Rad. Gold beads (1.6 µm diameter) were suspended in 100 µl of 50 mM spermidine. GFP and SNAP-25 expression
constructs were added (~1 µg DNA/mg beads) and coprecipitated with
1 M CaCl2 onto gold beads. DNA-bound beads were rinsed three times with 100% ethanol and resuspended in 100% ethanol (1.4 ml/10 mg beads) containing 0.02 mg/ml
polyvinylpyrrolidone. The lumen of Tefzel tubing (Bio-Rad) was coated
with the DNA-bound gold beads and subsequently cut into 0.5 inch
lengths to give "cartridges." The cartridges were stored at 4°C
with desiccant until time of transfection. Organotypic slices were
transfected within 1 hr of preparation. Cartridges were loaded into the
Helios Gene Gun, and the DNA-coated gold particles were propelled into
the tissue with a short burst of helium pressurized to 100-130 psi. A
subset of cells in which a gold particle had penetrated the nucleus
expressed the DNA (see Fig. 1). We typically observed one to three
GFP-positive pyramidal cells per slice (range, 0-10) in addition to a
variable number of glial cells and interneurons. Expression of GFP was
observed within 24 hr after transfection and lasted for up to 2 weeks.
Immunofluorescence. Slices were fixed in 4%
paraformaldehyde, rinsed three times in PBS, cryoprotected in 30%
sucrose (in PBS), and embedded in Tissue Tek (Sakura, Torrence, CA).
Cryostat sections (15 µm) of transfected slices were incubated with
an antibody against the myc antigen (mouse anti-myc primary 9E10; Santa
Cruz Biotechnology, Santa Cruz, CA) at 1:1000 for 4-16 hr at 4°C and
Texas Red anti-mouse secondary (Jackson ImmunoResearch, West Grove, PA)
at 1:200 for 1 hr at room temperature. GFP was imaged directly. Slices
were mounted in Vectashield, coverslipped, and visualized on a Zeiss
Axiophot microscope.
Electrophysiology. Paired, whole-cell patch-clamp recordings
in organotypic hippocampal slices were performed as described previously (Pavlidis and Madison, 1999), with minor adjustments. Recordings were made from 6- to 10-d-old cultured slices. Slices were
treated with BoNtE holotoxin (~40 µg/ml; Wako) via application of
toxin to the culture medium 24-72 hr before the time of recording. Pyramidal cells expressing GFP were targeted as the putative
presynaptic cells for whole-cell patch-clamp recording in slices
treated with BoNtE. To patch onto pyramidal cells expressing GFP, these
cells were first identified in the microscope under fluorescent
illumination from a mercury lamp (Zeiss) filtered through an Endow GFP
filter set with long-pass emitter (Chroma Technologies, Brattleboro, VT). The gold particle located in the nucleus of the cell [which was
visible under both fluorescent and differential interference contrast
imaging (DIC)] was subsequently used to relocate the GFP-positive cell
after switching to DIC for patch clamping. The presynaptic cell
electrode manipulator was attached to the recording stage so that the
slice could be moved beneath the objective to locate potential
postsynaptic cells. Using this arrangement, multiple postsynaptic cells
(typically two to three) were tested for each presynaptic cell.
Postsynaptic cells were held at  85 mV (corrected for junction
potential) unless indicated otherwise. Whole-cell patch-clamp
recordings were established onto one or more potential postsynaptic
partners in the vicinity of the presynaptic cell (100-200 µm away).
The presynaptic cell was stimulated to fire action potentials (APs) by
passing depolarizing current through the whole-cell electrode at
0.1-0.2 Hz for 3-5 min while monitoring the postsynaptic current. If
a reliable response that was time locked to the AP was observed,
the cell was considered synaptically connected (see Fig.
2A, cell 2, B, 1).
We confirmed that the response was excitatory by depolarizing the cell
to 55 mV, a holding potential at which inhibitory currents become
outward using our internal solutions (our unpublished observations).
Nontransfected negative control cells were often tested with
paired-pulse (see Fig. 2B, 3) or
high-frequency (33-60 Hz; see Fig. 3B, 3,
C, 3) bursts of action potentials, or both, to
try to draw out potential responses. A minimum of six GFP-negative
pairs (two different presynaptic cells, each with three postsynaptic
partners) in the same slice as the rescue were tested in this way to
rule out the possibility that an apparent rescue might simply be
failure of the toxin to block transmission. Approximately 90% of
negative control cells showed no responses to AP stimulation. The
remaining 10% showed failure rates of >99% (96 pairs analyzed
corresponding to 15 DK rescues). Averages are reported ±SE. All
statistics were performed using Analyze-It software (Leeds, United
Kingdom) for Microsoft Excel.
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RESULTS |
In this study, we have used four mutants of SNAP-25 designed to
alter different functional aspects of this SNARE (Chen et al., 1999).
In the first of these mutants (DK), the point mutation D179K was
designed to render SNAP-25 resistant to BoNtE cleavage. This mutation
is located adjacent to the BoNtE cleavage site (between R180 and I181),
and the mutant is at least 40-fold more resistant to BoNtE cleavage
than the native SNAP-25 (see Materials and Methods). The other three
mutations incorporate the D179K mutation to make each of them toxin
resistant. The second mutant, D179K/M167A/I171A (MAIA), represents an
alteration in the hydrophobicity of the SNAP-25 C-terminal coiled-coil
domain by substituting smaller hydrophobic residues (the methyl group
of alanine) for the bulky methionine and isoleucine residues found at
the 2 and 1 layers of the four-helical bundle. A third
mutant, D179K/Q174A (QA), replaces the ionic residue at the central
layer of the coiled-coil. Finally, the D179K/K201STOP (STOP) mutant
lacks the six C-terminal amino acids, a region of the protein that
potentially links SNAP-25 to a calcium-sensing mechanism(s) (Capogna et
al., 1997; Trudeau et al., 1998). In addition, this region of the
protein contributes to the C-terminal hydrophobic layer closest to the
vesicle membrane (Poirier et al., 1998; Sutton et al., 1998).
To address the effects of these SNAP-25 mutants on excitatory,
glutamatergic synaptic transmission, we chose the organotypic hippocampal slice preparation because it provides the opportunity to
make electrophysiological recordings from individual synaptically connected pairs of neurons that have been transfected with mutant SNAREs. Synaptic transmission between pairs of CA3 pyramidal neurons in
organotypic hippocampal slices has been shown previously to be similar,
if not identical, to transmission in noncultured tissue (Debanne et
al., 1996; Pavlidis and Madison, 1999). These connections between two
individual CA3 pyramidal neurons likely consist of an average of ~10
synapses each (Pavlidis and Madison, 1999). In addition, this
preparation is amenable to transfection (Nakayama et al., 2000).
BoNtE-resistant SNAP-25 (DK) was introduced into organotypic
hippocampal slices along with GFP using biolistic-mediated gene
transfer. This method typically resulted in one to three GFP-positive
pyramidal neurons appearing in each slice (range, 0-10) (Fig.
1B). Because GFP and
SNAP-25 mutants were introduced via the same gold particles but on
separate constructs, we confirmed that expression of DK occurred in
GFP-positive pyramidal cells by cotransfecting SNAP-25 mutants with an
N-terminal myc tag. Immunofluorescent labeling revealed extensive
overlap between the expression of GFP and DK, even in fine axon-like
processes and varicosities (Fig. 1C). Further mutations in
SNAP-25, such as those in MAIA and STOP, did not alter the level of
expression or the localization of SNAP-25 in pyramidal cells (Fig.
1C). Because the expression levels of GFP and myc-tagged
SNAP-25 constructs qualitatively appeared to be correlated, we targeted
for study (see below) only those pyramidal cells that strongly
expressed GFP.
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Figure 1.
Expression of toxin-resistant SNAP-25 in
organotypic hippocampal slices. A, Schematic of SNARE
interactions leading to synaptic vesicle fusion. VAMP
(red), syntaxin (green), and
SNAP-25 (blue) are shown. Only one coil of SNAP-25 is
shown for purposes of illustration. B, CA3 pyramidal
cells were transfected with pEGFP by biolistic gene transfer and
visualized 5 d later. C, Coexpression of GFP and
myc-tagged SNAP-25 mutants in axon-like processes in 15 µm cryostat
sections of fixed slices 7-9 d in vitro after
transfection.
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After transfection with DK and GFP, slices were treated with BoNtE (40 µg/ml BoNtE holotoxin; 24-72 hr) to eliminate the activity of native
SNAP-25. Synaptic transmission subsequently was monitored by making
simultaneous whole-cell recordings from two CA3 neurons (Debanne et
al., 1996; Pavlidis and Madison, 1999). When two cells are connected by
active synapses in a nontoxin-treated slice (~40% of all pairs), a
presynaptic action potential is followed at short latency by an inward
AMPA receptor-mediated EPSC (Debanne et al., 1996; Pavlidis and
Madison, 1999). In a BoNtE-treated slice, when the presynaptic cell was
not transfected (GFP-negative), paired recordings revealed no synaptic
transmission (Fig.
2A,B,
2, 3), indicating that the toxin had effectively
cleaved native SNAP-25. However, we did observe synaptic transmission
when the presynaptic cell was transfected with DK and GFP (Fig.
2A,B, 1). In 38% (23 of
61) of presynaptic cells transfected with DK, rescue of transmission occurred with at least one postsynaptic partner. In cells transfected with GFP alone, synaptic transmission was never observed after BoNtE
treatment (n = 18 pairs, 6 GFP-positive/DK-negative
presynaptic cells; data not shown). In addition, cells transfected with
wild-type SNAP-25 (i.e., lacking the D179K mutation) exhibited no
transmission in BoNtE-treated slices (n = 16 pairs;
five GFP-positive/wild-type SNAP-25-transfected presynaptic cells; data
not shown).
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Figure 2.
Toxin-resistant SNAP-25 can restore transmitter
release in the presence of BoNtE. A, In a typical
experiment, a putative presynaptic pyramidal cell can be tested with a
series of potential postsynaptic partners. APs were stimulated at 0.2 Hz for 3-5 min in the putative presynaptic cell at the same time that
the peak current of the potential postsynaptic cell (in
picoamperes;  ) was measured within a short (1-10 msec)
window after the peak of the AP. In this experiment, the GFP+
presynaptic cell (cell 2) evoked monosynaptic EPSCs in
the second postsynaptic partner tested. GFP presynaptic cells
(cells 1 and 3) from the same slice as
the rescue were tested with three putative postsynaptic partners each
and serve as negative controls. No EPSCs were observed in these pairs.
Calibration: 5 min. B, Consecutive postsynaptic currents
(20 traces;  indicates corresponding amplitudes in A)
from the rescue pair are shown (1). EPSCs are
evident as downward deflections of the current trace occurring shortly
after the AP peak. Negative control consecutive traces show no EPSCs in
response to either a single presynaptic AP (2) or
paired-pulse stimulation (3) from the third
putative postsynaptic partner of presynaptic cell 3 (see
corresponding amplitudes in A, ). Calibration: 50 pA,
25 msec.
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Synaptic transmission rescued by DK showed similar variability in the
EPSC amplitudes as seen in nontransfected, nontoxin-treated cell pairs
(average 1/CV2 = 7.5; range from 2.2 to
24.5; average peak amplitude of EPSCs ranged from 9 to 47 pA, 22 pA
average) (Fig.
3A,D)
(cf. Pavlidis and Madison, 1999). There was also a considerable
trial-to-trial failure rate, ranging from 0 to 93% (average = 41 ± 6%; n = 25 synaptically connected pairs),
comparable to the failure rates observed in native pairs, those in
nontransfected slices not treated with BoNtE (average = 34 ± 7%; n = 16; p > 0.05) (Fig.
3D).
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Figure 3.
Mutations that affect the interaction of SNAP-25 C
terminus with other SNAREs reduce transmitter release.
A, Two representative examples (not including the
example in Fig. 2) of DK rescue of synaptic transmission. Consecutive
postsynaptic responses from each rescue are shown. B,
Consecutive traces from one MAIA rescue. Responses to single AP
(1) or a brief burst of APs (at 60 Hz;
2) are shown. Superimposed consecutive traces (~20)
from three different putative postsynaptic partners of a single GFP
presynaptic pyramidal cell stimulated to fire 60 Hz bursts of APs
(3). Cells are from the same slice as the rescue.
C, Consecutive traces from two different
(1, 2) STOP rescues recorded in Ringer's
solution containing either 2.5 or 5 mM extracellular
Ca2+. Paired-pulse stimulation was used to observe
responses. Negative control (GFP presynaptic cell, 3)
from the same slice as 1 showing no responses to
paired-pulse stimulation in the presence of 5 mM
Ca2+ Ringer's solution in any of its three
postsynaptic partners (30 consecutive traces superimposed for each).
D, An average of mean peak amplitudes (±SE; the mean
for each rescue was determined from 5-40 EPSCs); E,
percentage failures (100 × number of failures/total number of
stimulations at 0.2 Hz; ~50 trials per rescue) for all rescues of the
given mutation (n = 5, 8, 9, 23, and 16 pairs for
STOP, QA, MAIA, DK, and native pairs, respectively). MAIA, QA, and STOP
mutant rescues each have significantly higher failure rates compared
with DK rescues and native pairs (one-way between-subjects ANOVA;
p < 0.0001; with all pair-wise contrasts using
Tukey error protection at 99% confidence interval; * indicates
significance when compared with DK or native). Average peak amplitudes
tended to be higher for DK and native pairs relative to the other
mutants, although not significantly (p = 0.2; one-way between-subjects ANOVA). Note that values for STOP mutants
were taken from responses during paired-pulse stimulation and not
single APs as for native, DK, MAIA, and QA (see Results).
F, Expression of STOP in the absence of toxin acts to
reduce transmission. Consecutive traces from a control cell
(1) and a STOP-expressing cell
(2) from the same slice. Note that this STOP
expressing cell shows responses (*) only with a train of action
potentials. Calibration (A-C,
F): 50 pA, 25 msec.
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Next, we tested the effects of further mutations of SNAP-25 on synaptic
transmission. CA3 pyramidal cells transfected with SNAP-25 mutant MAIA
or QA showed rescue of transmission with at least one postsynaptic
partner in 7 of 24 (29%) or 8 of 21 (38%) of cell pairs tested,
respectively, within the range of that observed for DK (see above).
However, synaptic transmission mediated by MAIA or QA was characterized
by a dramatic reduction in synaptic transmission [(Fig.
3B,D,E) and data not
shown]. Single action potentials typically evoked few responses in
MAIA or QA rescues, demonstrating a significantly higher failure rate
relative to DK rescues (Fig. 3E). To observe an increased
number of EPSCs in MAIA and QA rescues, we applied brief high-frequency
bursts of APs to the presynaptic cell (Fig. 3B).
Brief bursts of APs can increase calcium entry into the presynaptic
terminal and increase the probability of neurotransmitter release
(Kreitzer and Regehr, 2000). For both MAIA and QA, stimulating the
presynaptic cell in this manner resulted in an increase in the number
of responses, whereas these bursts did not tend to change the failure
rate for DK rescues [(Fig. 3B, 2) and data not
shown; failures changed from an average of 85 to 79% for MAIA,
p = 0.07, n = 9; from 88 to 75% for
QA, p = 0.02, n = 6; from 57 to 59%
for DK, p = 0.8, n = 7; paired
t tests compared the failure rate at 0.2 Hz with the failure
rate during brief pulses of three to five APs at 20-60 Hz,
respectively). STOP-mediated rescue displayed an even more severe
reduction in neurotransmission. To observe any EPSCs with this mutant,
paired-pulse stimulation or application of high external calcium
concentrations, or both, was almost always necessary (Fig. 3C) (cf. Capogna et al., 1997; Trudeau et al., 1998).
Although DK rescues typically showed paired-pulse depression, STOP
rescues typically displayed facilitation with paired-pulse stimulation [average log (EPSC2/EPSC1) = 1.3 ± 0.4 for STOP and = 0.15 ± 0.2 for DK; p < 0.01; independent
samples t test; n = 5 and 10, respectively]. The average peak amplitudes of EPSCs for MAIA, QA, and
STOP rescues were each reduced relative to DK rescues and native
recordings (Fig. 3D) (Native indicates data from
paired recordings from nontransfected, nontoxin-treated slices). The trial-to-trial failure rate was significantly increased in MAIA, QA,
and STOP rescues in comparison to DK rescue and native transmission (Fig. 3E), suggesting that fewer neurotransmitter release
events were stimulated by each presynaptic action potential.
The effects of expression of STOP and MAIA in the absence of toxin
treatment were also tested. Pairs of CA3 cells in which the presynaptic
cell expressed STOP showed an impairment of synaptic transmission
relative to native (untransfected) pairs in the absence of BoNtE
treatment. (Fig. 3F) (failure rates of 34 ± 7%
and 82 ± 9% for native and STOP, respectively; p < 0.01; n = 16 and 5, respectively). In contrast, when
we tested MAIA in the absence of BoNtE treatment, we were unable to
detect a significant reduction in transmission (failure rate of 31 ± 18%; n = 5 pairs).
Although the probability of release is reduced with MAIA, QA, and STOP
rescues relative to DK, we sought to determine whether the nature of
transmission, when it did occur, was altered by these mutations. We
compared kinetic parameters of EPSCs from DK, QA, MAIA, and STOP
transfected cells and those of native pairs, including average 10-90%
rise time and the  decay. We found no differences in these kinetic parameters among any of these conditions (Fig.
4A,B).
In addition, there was no significant difference in the latency to
onset of the EPSC (Fig. 4C).
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Figure 4.
Kinetics of EPSCs are not significantly different
among SNAP-25 mutation rescues. Comparisons of average 10-90% rise
time (A), decay (for single
exponential fit) (B), and latency
(C) given in milliseconds for DK, STOP, MAIA, and
QA rescues and native recordings (n = 23, 5, 9, 8, and 10 pairs, respectively). EPSC kinetics were analyzed using Mini
Analysis Program (Synaptosoft Inc., Leona, NJ). The 10-90% rise time
and the decay were determined for each rescue by
analyzing an average waveform composed of 7-47 EPSCs. Latency was
determined by measuring the delay between the peak of the AP and the
onset of the EPSC using custom software developed in Labview (National
Instruments) (Pavlidis and Madison, 1999). No statistical difference
was found among any of the SNAP-25 mutant rescues or native pairs in
latency (p = 0.6), 10-90% rise time
(p = 0.75), or decay
(p = 0.64) using a one-way between subjects
ANOVA.
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DISCUSSION |
Our study directly investigates, for the first time, the effects
of specific amino acid mutations in SNAP-25 on neurotransmitter release
in a functional mammalian CNS synapse. Critical to this study was the
use of a BoNtE-resistant form of SNAP-25, namely the D179K mutation.
Previous studies have demonstrated that SNAP-25 rendered resistant to
cleavage by BoNtA (O'Sullivan et al., 1999) and BoNtE (Washbourne et
al., 1999) can support neurosecretion in chromaffin and PC12 cells,
respectively. Similarly, neither GFP alone nor wild-type SNAP-25
overexpression was able to restore transmission in our study,
indicating that expression of BoNtE-resistant SNAP-25 is solely
responsible for restoring synaptic transmission in the presence of
nonfunctional native SNAP-25. In addition, our observations
demonstrated that synaptic transmission supported by DK shares the same
basic properties of transmission as observed under native conditions in
this preparation.
Previous studies in chromaffin cells or PC12 cells have tested MAIA
(Chen et al., 1999), mutations of Q174 (Chen et al., 1999; Wei et al.,
2000), and K201STOP (Criado et al., 1999). Our results are consistent
with those observations in that we saw an effective reduction of
transmission with these mutants (Fig. 3B-D).
Most of these studies draw conclusions based on the fusion of many (tens to hundreds) vesicles over the course of seconds to tens of
minutes, where it is difficult to directly observe whether release of
individual vesicles is impaired by a reduction in the probability of
vesicle fusion or by a slowing of release kinetics, or both [but see
Criado et al. (1999)]. One of the greatest advantages of our approach
is the ability to measure the kinetics of transmission at a limited
population of CNS synapses with millisecond resolution in response to a
precise stimulus. Our data suggest that mutations that disrupt, even
subtly (e.g., MAIA), interactions between the SNARE coiled coils, have
profound effects on the probability of vesicle fusion (Fig. 3) but do
not alter the kinetics of transmitter release or the time to onset of
the fusion event (Fig. 4).
Either overexpression of SNAP-25 lacking the last nine amino acids in
chromaffin cells (Wei et al., 2000) or treatment of dissociated
hippocampal neurons with botulinum neurotoxin A (but see Capogna et
al., 1997; Trudeau et al., 1998), which removes the last nine amino
acids of SNAP-25, resulted in a delay to onset of vesicle release in
addition to significantly reduced levels of vesicle fusion. Our results
with rescue mediated by the STOP mutant (lacking the last six amino
acids), where we detected a severe reduction in vesicle fusion events
(Fig. 3B,E) but no change in onset
to fusion (Fig. 4C), may suggest that the three additional amino acids (198-200) are enough to keep the fusion event time-locked to the presynaptic trigger.
Interestingly, overexpression of STOP in the absence of BoNtE resulted
in reduced transmission (Fig. 3F) relative to
transmission between native (untransfected) pairs, whereas MAIA was
unable to inhibit release in this manner. The MAIA mutation occurs in the N-terminal half of the C-terminal coil, whereas the STOP mutation deletes the most C-terminal amino acids of this coil, closest to the
vesicle (Poirier et al., 1998; Sutton et al., 1998). In a zippering
model of fusion (Lin and Scheller, 1997), the SNARE coils come together
at their N-terminal ends first, then "zipper up" to the C termini.
MAIA may be less able to form initial "loose" SNARE complexes,
which may require N-terminal interactions, and is therefore unable to
compete with functional, native SNAP-25 to get into SNARE complexes. In
contrast, STOP can enter SNARE complexes in the presence of functional,
native SNAP-25, but it does not zipper completely because of the
C-terminal deletion.
Our results indicate that the stability of the SNARE coiled-coil
interactions is important in determining the probability that an action
potential will promote neurotransmitter release, but that this complex
does not regulate the onset or the kinetics of the EPSC once it is
initiated. In the context of our data, the following models are
plausible. First, the mutants (MAIA, QA, and STOP) may reduce the
ability of the SNAP-25 C terminus to nucleate with the other SNAREs,
leading to reduced zippering/fusion events or a reduction of, at any
given time, the number of complexes available to zipper in response to
an action potential. Second, the mutants may not interfere with
nucleation, but instead may impair the ability of the SNARE coiled coil
to zipper stably. When complete zippering does occur, fusion occurs at
the same rate. Third, a final rate-limiting step before fusion may be
downstream of zippering. In this case, the mutants may provide fewer
zippered complexes and/or may increase the energy barrier for the
zippered complexes to transition to this final rate-limiting step.
Our study may also have interesting implications for the potential role
of SNAP-25 (and the SNARE complex in general) in postsynaptic delivery
of AMPA receptors. Recent studies have indicated that either disruption
of NSF interactions (Lüthi et al., 1999) or cleavage of VAMP
postsynaptically with botulinum neurotoxin B (Lüscher et al.,
1999) can reduce the basal AMPA receptor-mediated EPSC on the time
scale of minutes, reaching a lower steady-state level. A continual
supply of AMPA receptors seems to be necessary for a sizable portion of
the basal AMPA synaptic current (Lüscher et al., 1999;
Lüthi et al., 1999; Kim and Lisman, 2001). In our study, the
postsynaptic cell has been exposed to botulinum neurotoxin over the
course of 1-2 d. The fact that we see any rescue of transmission at
all is consistent with the idea that there exists a pool of AMPA
receptors that is either stable in the membrane or does not require
SNAREs for movement into or out of the postsynaptic membrane. In our
study, peak conductances (used to correct for differences in holding
potential) of DK rescues showed no significant difference from those of
native pairs (0.26 ± 0.03 nS, n = 26 pairs, and 0.33 ± 0.09 nS, n = 15 pairs, respectively;
p  0.05; independent sample t test). This
observation may suggest that compensatory mechanisms exist to keep the
"stable" receptor pool at a certain size over the long term.
| |
FOOTNOTES |
Received June 27, 2001; revised Oct. 11, 2001; accepted Oct. 22, 2001.
This work was sponsored by the Howard Hughes Medical Institute (R.H.S.)
and the Silvio Conte-National Institute of Mental Health Center for
Neuroscience Research Grant MH48108 (D.V.M.). We thank T. F. J. Martin for providing the pcDNA3.1-SNAP-25bD179K construct, Yu Chen,
Johanna Montgomery, Murali Prakrya, Suzie Scales, Audrey Ettinger, and
members of the Scheller and Madison labs for helpful discussions, and
Eric Schaible for slice preparation.
Correspondence should be addressed to Daniel V. Madison,
Associate Professor, Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Beckman Center, B003, Stanford,
CA 94305. E-mail: madison{at}stanford.edu.
R. H. Scheller's present address: Genentech, Inc., 1 DNA Way,
South San Francisco, CA 94080.
| |
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ABSTRACT
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