Department of Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada
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ABSTRACT |
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Elrick, Donald B. and
Milton P. Charlton.
-Latrocrustatoxin Increases Neurotransmitter Release by
Activating a Calcium Influx Pathway at Crayfish Neuromuscular Junction.
J. Neurophysiol. 82: 3550-3562, 1999.
-latrocrustatoxin (
-LCTX), a component of black widow spider
venom (BWSV), produced a 50-fold increase in the frequency of
spontaneously occurring miniature excitatory postsynaptic potentials (mEPSPs) at crayfish neuromuscular junctions but did not alter their
amplitude distribution. During toxin action, periods of high-frequency
mEPSP discharge were punctuated by periods in which mEPSP frequency
returned toward control levels. EPSPs were increased in amplitude
during periods of enhanced mEPSP discharge.
-LCTX had no effect when
applied in Ca2+-free saline, but subsequent addition of
Ca2+ caused an immediate enhancement of mEPSP frequency
even when
-LCTX was previously washed out of the bath with
Ca2+-free saline. Furthermore removal of Ca2+
from the saline after
-LCTX had elicited an effect immediately blocked the action on mEPSP frequency. Thus
-LCTX binding is insensitive to Ca2+, but toxin action requires
extracellular Ca2+ ions. Preincubation with wheat germ
agglutinin prevented the effect of
-LCTX but not its binding. These
binding characteristics suggest that the toxin may bind to a crustacean
homologue of latrophilin/calcium-independent receptor for latrotoxin, a
G-protein-coupled receptor for
-latrotoxin (
-LTX) found in
vertebrates.
-LCTX caused "prefacilitation" of EPSP amplitudes,
i.e., the first EPSP in a train was enhanced in amplitude to a greater
degree than subsequent EPSPs. A similar alteration in the pattern of
facilitation was observed after application of the Ca2+
ionophore, A23187, indicating that influx of Ca2+ may
mediate the action of
-LCTX. In nerve terminals filled with the
Ca2+ indicator, calcium green 1,
-LCTX caused increases
in the fluorescence of the indicator that lasted for several minutes
before returning to rest. Neither fluorescence changes nor toxin action
on mEPSP frequency were affected by the Ca2+ channel
blockers
-agatoxin IVA or Cd2+, demonstrating that
Ca2+ influx does not occur via Ca2+ channels
normally coupled to transmitter release in this preparation. The
actions of
-LCTX could be reduced dramatically by intracellular application of the Ca2+ chelator,
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic
acid. We conclude that induction of extracellular Ca2+
influx into nerve terminals is sufficient to explain the action of
-LCTX on both spontaneous and evoked transmitter release at crayfish
neuromuscular junctions.
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INTRODUCTION |
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The venom of the black widow spider
(Latrodectus mactans tredecimguttatus) contains a family of
related neurotoxins, known as latrotoxins, which cause dramatic
stimulation of exocytosis at synapses and from endocrine cells
(Grishin 1998; Rosenthal and Meldolesi
1989
). Latrotoxins have been described showing differential selectivity for vertebrate (Frontali et al. 1976
;
Tzeng and Siekevitz 1978
, but see also Umbach et
al. 1998
), insect (Dulubova et al. 1996
;
Grishin 1998
; Krasnoperov et al. 1992
;
Magazanik et al. 1992
), or crustacean synapses
(Burmistrov et al. 1997
; Krasnoperov et al.
1992
), and these toxins share a similar domain structure (Grishin 1998
; Kiyatkin et al. 1990
,
1993
). A fragment of the cDNA for
-latrocrustatoxin
(
-LCTX), a latrotoxin that is effective at crustacean synapses
(Burmistrov et al. 1997
; Krasnoperov et al.
1992
), has been cloned and sequenced and showed 68 and 31% homology with the corresponding regions of
-latroinsectotoxin (
-LIT) and
-latrotoxin (
-LTX), respectively (Volynskii
et al. 1999
). The similarity of the latrotoxins in action and
domain structure makes their taxonomic specificity all the more
striking and emphasizes the need for detailed comparative studies of
their actions before using them as tools for examining the molecular mechanisms controlling exocytosis. The aim of the current study was to
characterize the actions of
-LCTX at the crayfish neuromuscular junction in greater detail, focusing on the role of Ca2+ in
-LCTX action.
The latrotoxins act via two classes of presynaptic receptors, namely
neurexins and latrophilins/calcium-independent receptors for latrotoxin
(CIRLs). The binding of -LTX to neurexins is absolutely dependent on
the presence of Ca2+ ions in the medium (Davletov et
al. 1995
; Sugita et al. 1999
), whereas the
binding of
-LTX to latrophilin is unaffected by divalent cations
(Davletov et al. 1996
, 1998
). Structurally, latrophilin belongs to the seven-transmembrane domain, G-protein-coupled class of
cell surface receptors and is thought to be coupled to phospholipase C
and subsequent phosphoinositide metabolism (Davletov et al. 1998
; Krasnoperov et al. 1997
; Lilianova
et al. 1997
). In addition, both Ca2+-independent
and -dependent mechanisms have been described downstream of
toxin-receptor binding interactions. Thus in different systems, latrotoxin action may not be associated with an alteration in intracellular Ca2+ concentration (Lang et al.
1998
), or intracellular Ca2+ levels may be elevated
by influx of Ca2+ from the extracellular fluid
(Barnett et al. 1996
), mobilization of intracellular
Ca2+ stores, or both (Davletov et al. 1998
).
As well as interactions with specific cell surface receptors,
latrotoxins are known to cause formation of channels or pores in
artificial and cellular membranes that could provide a route for
Ca2+ influx from the extracellular fluid (Davletov
et al. 1998; Dulubova et al. 1996
; Liu
and Misler 1998b
; and references therein). Furthermore the
direct interaction of latrotoxin receptors with elements of the
synaptic vesicle release machinery, suggested by the copurification of
either neurexin 1
or latrophilin with critical synaptic proteins (Hata et al. 1993
; Krasnoperov et al.
1997
; O'Connor et al. 1993
), may have
functional implications for exocytosis under physiological conditions
as well as during toxin action.
Most of our understanding of the actions of latrotoxins comes from the
work on -LTX effects on vertebrate systems; the actions of
latroinsectotoxins (LITXs) on insect synapses (Dulubova et al.
1996
; Magazanik et al. 1992
; Shatursky et
al. 1995
) and
-LCTX on crustacean synapses
(Burmistrov et al. 1997
) have been studied relatively
little.
-LCTX has been shown to increase transmitter release from
crustacean nerve terminals and earlier studies described the actions of
either whole venom or a 65-kDa crustacean specific fraction (the
relationship of which to the currently described 120-kDa
-LCTX is
unknown) on either stretch receptor or neuromuscular junction
preparations (Fritz and Mauro 1982
; Fritz et al.
1980a
,b
; Grasso and Paggi 1967
; Kawai et
al. 1972
), finding that these agents had profound
electrophysiological and morphological consequences (including dramatic
enhancement of transmitter release followed by block of transmission,
synaptic vesicle depletion, and mitochondrial swelling).
We have found that -LCTX action shares many features in common with
that of
-LTX on vertebrate synapses, such as
Ca2+-independent binding, dramatic enhancement of
spontaneous release, and inhibition of toxin action by lectin. Using
both electrophysiological and calcium imaging approaches, we show that
the effects of
-LCTX can be explained by an increase in nerve
terminal Ca2+ levels due to the influx of extracellular
Ca2+, via a pathway distinct from the voltage-operated
Ca2+ channels (VOCCs) normally coupled to synaptic
transmission in this preparation.
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METHODS |
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Electrophysiology
Crayfish (Procambarus clarkii) were obtained from
Atchafalya Biological Supply (Raceland, LA), housed in aerated,
dechlorinated tap water at 15°C, and fed a diet of carrots and
lentils ad libitum. Experiments were conducted on the opener muscle of
the first walking leg at room temperature (21-23°C)
(Wojtowicz and Atwood 1984). In this preparation, the
entire muscle is innervated by a single excitatory and a single
inhibitory axon (Florey and Cahill 1982
). In all
experiments, the excitatory axon was isolated in the meropodite section
of the leg, and all other nerves were cut to ensure selective stimulation of only the excitatory input to the muscle. Nerve stimulation was achieved by applying square wave current pulses (500-µs duration) across bipolar platinum electrodes. Normal crayfish saline contained (in mM): 205 NaCl, 5.4 KCl, 13.5 CaCl2, 2.7 MgCl2, 10 D-glucose, and 10 HEPES. In
Sr2+-substituted saline, all
CaCl2 was replaced with equimolar
SrCl2 and in
Ba2+-substituted saline, all
CaCl2 was replaced with equimolar
BaCl2. In Ca2+-free saline,
equinormal NaCl was substituted for CaCl2 and 1 mM EGTA was included to chelate free Ca2+. The pH
of all solutions was adjusted to 7.4 with NaOH and all solutions,
unless otherwise stated, contained 40 µM picrotoxin to block
GABAergic spontaneous inhibitory junction potentials (SIJPs)
(Takeuchi and Takeuchi 1969
). When solution changes were made, four to five bath volume changes were made to ensure complete exchange into the new solution.
Preparations were mounted on an upright epifluorescence microscope
(Optiphot-2, Nikon, Mississauga, ON), and transmitter release was
monitored by membrane potential recordings from individual muscle
fibers penetrated with 3 M KCl filled glass microelectrodes (2-4
M). In the absence of stimulation, spontaneous excitatory postsynaptic potentials (mEPSPs) were recorded and in response to
trains of nerve stimuli (3-6 pulses, 100 Hz), EPSPs were
recorded. Because these muscle fibers are almost isopotential, the
postsynaptic events at all synapses on a given muscle fiber can be
recorded with a single intracellular electrode (Wojtowicz and
Atwood 1986
). In some experiments, the excitatory axon was
penetrated with a glass microelectrode (20-50 M
) for monitoring
presynaptic action potentials and injecting substances into the axon.
For pressure injection of
1,2-bis(o-aminophenoxy)ethane-N,
N,N',N'-tetraacetic acid (BAPTA), electrodes were filled with a
solution containing 200 mM BAPTA tetrapotassium salt. Rhodamine
dextran (100 µM, 10 kDa) was included in the solution to allow
visualization and estimation of the amount of substance injected.
Pressure injections (5- to 20-ms pulses, 0.05-0.1 Hz and
480 kPa)
were controlled by a Picospritzer II (General Valve, Fairfield, NJ).
Stimulation was controlled by Tomahacq, an IBM-PC-compatible
electrophysiology data collection and analysis program (Tom Goldthorpe, University of Toronto). Presynaptic and postsynaptic membrane potentials were amplified 100 and 500 times, respectively, and subjected to low-pass filtering (4 pole Bessel) at 3 and 1 kHz, respectively (Warner Instrument, Hamden, CT), and recorded continuously on video tape through a VR-10 digital data recorder (Instrutech, Great
Neck, NY). Stimulation-evoked signals were digitized (12 bits) at
50-µs intervals (TL-1 DMA interface, Axon Instruments, Foster City,
CA) and averaged in groups of 12 for computer collection via Tomahacq.
For analysis of mEPSPs, membrane potential recordings stored on video
tape were digitized, 12 bits at 50-µs intervals, through a Digidata
1200 interface (Axon Instruments) and collected and analyzed using
Axoscope 1.1 (Axon Instruments) in gap-free acquisition mode. To
quantify mEPSP frequency, mEPSPs were counted using one of two methods,
determined by their rate. At low frequencies, all mEPSPs occurring
during a 30-s time bin were counted manually and expressed as an
average frequency over this period in hertz. At high frequencies (3
Hz), mEPSPs were counted manually within a given 30-s bin until 100 mEPSPs had been counted, and then the time elapsed during these 100 events was determined and used to express the average frequency during
that time frame, in hertz. For graphic representation, plots were made
of mEPSP frequency versus time.
Because the pattern of changes in mEPSP frequency in the presence of
-LCTX was nonstationary and unpredictable, with variations in both
the maximum frequency and duration of periods of enhanced mEPSP
discharge occurring randomly (for example, see Fig. 2A), data from individual experiments were quantified by measuring the area
under mEPSP frequency versus time plots for each experiment [computed
as the area under a curve (AUC) transform function in Sigmaplot for
Windows 2.0, Jandel Scientific Software, San Rafael, CA]. The duration
of periods during which AUC values were calculated varied from 10 to 40 min depending on the nature of the experiment. Therefore these values
were expressed as an average AUC value per minute by dividing the AUC
value by the duration of the period during which it was calculated.
These values provided a convenient method by which to summarize the
data from different experiments, taking into account both the magnitude
and duration of mEPSP bursts.
For experiments comparing the ability of Sr2+ or
Ba2+ substitution for Ca2+ to support toxin
action, preparations first were incubated in the appropriate test
divalent saline before addition of -LCTX (2 µg/ml). The
preparation was incubated with toxin for a further 60 min and then the
bath was washed with 4-5 volumes of the test divalent saline to wash
out excess, unbound
-LCTX before finally incubating the preparation
in normal crayfish saline. Continuous recordings of membrane potential
were made throughout the duration of these manipulations, and plots
were made of mEPSP frequency over time for each experiment. AUC per
minute values were determined for three periods during such
experiments: a control period (period 1) in test
divalent ion solution before addition of toxin; a period 30-60 min
after addition of toxin in the test divalent ion solution (period
2); a period after washout of the test divalent ion and return to
normal crayfish saline (i.e., >60 min after exposure to
-LCTX),
this period being designated as the maximum response possible
(period 3). For comparisons among these three divalent cations, AUC/min data for each experiment were expressed as a percentage of the maximum possible response, i.e., (period
2/period 3) × 100%. Statistical
comparisons of data derived from AUC analyses were performed using
the Mann-Whitney rank sum test. Numerical values are given as
means ± SE.
To determine whether mEPSP amplitudes were altered during periods of
-LCTX induced high-frequency mEPSP discharge, both mEPSP amplitudes
and the intervals between mEPSPs were plotted as cumulative frequency
distributions for control and
-LCTX recording periods and the
distributions tested for statistically significant differences using
the Kolmogorov-Smirnoff test.
Calcium imaging
In calcium imaging experiments, the excitatory axon was
penetrated with a microelectrode containing 500 µM calcium green 1 dextran (10 kDa) in 100 mM KCl, and this solution was pressure injected
into the nerve until the indicator could be seen to have diffused to
the boutons. For Ca2+ imaging, the preparation
was scanned on a Bio-Rad 600 confocal microscope (Bio-Rad Laboratories,
Mississauga, Ontario) under a ×40, 0.55 NA, water-immersion objective
(Nikon, Mississauga, Ontario) and the free radical scavenger, trolox
(100 µM), was included in the bath saline (Scheenen et al.
1996). Calcium green 1 was excited using the 488-nm line, and
emitted light >515 nm was detected. In all preparations studied,
control responses to trains of nerve stimuli (5-10 s at 5-20 Hz) were
collected first. To detect any
-LCTX-induced increases in
intracellular Ca2+, single scans were collected
every 30 s for a period of 10-15 min (control fluorescence)
before exposure to 2 µg/ml
-LCTX, and data collection continued at
30-s intervals for 60-120 min after addition of the toxin. Data are
presented graphically, expressed as
F/F
values, where F was calculated as the average pixel
intensity in the region of interest during 10 scans within the control
recording and
F represents the difference between this
value and the pixel intensity at a given time. For illustrative
purposes, a false color scale was applied to images, with progression
from blue through green to red representing increasing pixel values.
Materials
Reagents used were from the following sources: -LCTX and
-Aga-IVA, Latoxan (Rosans, France); joro spider toxin-3, Natural Product Sciences (Salt Lake City, UT); thapsigargin and BAPTA-AM, Calbiochem-Novabiochem (San Diego, CA); calcium green 1 dextran (10 kDa) and rhodamine dextran (10 kDa), Molecular Probes. All other
reagents were obtained from Sigma-Aldrich (Oakville, Ontario).
-LCTX
was prepared for Latoxan by Professor E. V. Grishin according to
the protocol of Krasnoperov et al. (1992)
. An identical
preparation of
-LCTX has been subjected to N-terminal sequencing and
chymotryptic digest sequence analysis, and a fragment of cDNA from the
central domain of
-LCTX has been cloned and sequenced
(Volynksii et al. 1999
), showing
-LCTX to be
structurally related to, but distinct from, other alpha toxins
from black widow spider venom.
-LCTX, received in a lyophylized
state, was reconstituted as a stock solution (1 µg/µl) in distilled
water and stored at 4°C for
6 wk, during which period we observed
no decrement in the activity of the toxin. We used a concentration of 2 µg/ml
-LCTX throughout, as this gave a reliable and reproducible
effect which was sustained over a long-enough period to allow
subsequent experimental manipulations. Because we could not be sure
that the lyophylized
-LCTX retained 100% activity after
reconstitution (compared with whole venom or freshly purified toxin),
using this minimally active concentration (which acted in ~30 min and
the effect of which could be maintained for
2-3 h) essentially
defined an internal bioassay to control for the activity of the toxin
by which different batches of toxin could be titrated in the future.
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RESULTS |
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Effect of -LCTX on synaptic transmission at crayfish
neuromuscular junction
We examined the effects of -LCTX on crustacean synaptic
transmission using intracellular microelectrode recordings from the opener neuromuscular junction preparation of the crayfish. Bath application of
-LCTX (2 µg/ml) to preparations mounted in normal crayfish saline (13.5 mM Ca2+) caused a profound
increase in the frequency of spontaneous miniature excitatory
postsynaptic potentials (mEPSPs) in all preparations examined
(n = 14; Fig. 1). Mean
AUC/min values (see METHODS) increased significantly 30-60
min after exposure to 2 µg/ml
-LCTX, from 0.2 ± 0.02 in
control recordings to 12.8 ± 1.9 (P < 0.0001, Mann-Whitney test). This effect occurred after a delay of ~30 min
(29.2 ± 2.9 min) and always <50 min. This increased mEPSP
frequency was characterized by irregular oscillations in mean frequency
(recorded during 30-s bins) such that high-intensity bursts of mEPSP
discharge of variable duration were punctuated by periods during which
mEPSP frequency returned to near control levels (see Fig.
2A). During mEPSP bursts, the
mean frequency of mEPSP discharge was elevated from a control level of
around 0.1 Hz to frequencies in the range 10-100 Hz. The duration of
mEPSP bursts and interburst intervals varied from several seconds to
several minutes, and there was no obvious pattern in the oscillations
in mEPSP frequency elicited by
-LCTX. The
-LCTX -evoked increase
in mEPSP frequency was not due to induction of spontaneous action
potential firing in the motor axons because spontaneous action
potentials were never observed in experiments in which the motor axon
membrane potential was recorded by an intracellular microelectrode and
because in the absence of nerve stimulation, we never observed a
synchronized discharge of multiple quanta. During periods of
significantly enhanced mEPSP discharge frequency there was no change in
the amplitude distribution of mEPSPs (Fig. 1, C and
D). The increase in spontaneous event frequency was blocked
entirely by application of JSTX-3 (30 µg/ml), a glutamate receptor
blocker (Kawai et al. 1991
), suggesting that the
increased activity was indeed due to an increase in the frequency of
spontaneous release of glutamate containing synaptic vesicles (data not
shown). In some experiments, picrotoxin was omitted from, and JSTX-3
included in, the bath saline so that SIJPs could be recorded
selectively (confirmed by their blockade by picrotoxin, 40 µM). In
such experiments, SIJP frequency was also potentiated by
-LCTX (data
not shown) in a similar manner to mEPSPs, suggesting that the action of
the toxin is not specific for glutamatergic terminals but acts more generally at crustacean synapses, independent of the neurotransmitter involved, as is the case with
-LTX at vertebrate synapses.
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During periods of increased mEPSP frequency induced by -LCTX, there
was a concomitant increase in EPSP amplitudes in response to short
trains of nerve stimuli (4 pulses at 100 Hz, Fig. 2). This effect
generally mirrored the action on mEPSP frequency in that there were
oscillations in the degree to which EPSP amplitudes were enhanced,
punctuated by periods in which EPSP amplitudes returned toward control
levels, suggesting that a common mechanism might be responsible for
both effects. The effects on both mEPSP frequency and EPSP amplitude
continued for the duration of the recording (
3.5 h after onset of
toxin action). Exposure to
-LCTX did not cause failure of action
potential generation or changes in action potential shape or amplitude
in response to nerve stimulation (Fig. 2C), in intracellular
microelectrode recordings of motor axon membrane potential.
During periods in which EPSP amplitudes were potentiated by exposure to
-LCTX, there was a change in the pattern of short-term facilitation
of EPSPs such that potentiation of EPSP amplitude was associated with a
decrease in the degree of facilitation during a train of three nerve
stimuli at 100 Hz (Fig. 3A).
This was due to a "prefacilitation" of EPSP amplitudes, i.e., EPSP
amplitudes were enhanced to a greater degree early in the train. A
similar prefacilitation was seen in three experiments where EPSP
amplitudes were enhanced by increasing intraterminal
Ca2+ concentration using the
Ca2+ ionophore, A23187 (50 µM, Fig.
3B). Because short-term facilitation in this preparation is
thought to reflect an accumulating action of intraterminal
Ca2+ ions during successive stimuli
(Blundon et al. 1993
; Kamiya and Zucker
1994
; Ravin et al. 1999
; Winslow et al.
1994
; Wright et al. 1996
), the prefacilitation
seen during
-LCTX action is suggestive of an increase in
intraterminal Ca2+ levels as a result of exposure
to the toxin. To test this possibility further, we used
Ca2+ imaging experiments to ask whether
intracellular Ca2+ ion concentration is increased
by exposure to
-LCTX.
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-LCTX causes fluctuating elevations in intraterminal
[Ca2+]
To determine whether or not -LCTX action was associated with an
increase in intraterminal Ca2+ levels, we
microinjected the excitatory motor axon with the fluorescent Ca2+ indicator, calcium green 1 dextran (10 kDa)
and monitored the intensity of fluorescence in individual boutons at
30-s intervals, before and during exposure to
-LCTX (2 µg/ml) in
normal crayfish saline. In three preparations, although resting
fluorescence intensity remained at a relatively constant level before
exposure to
-LCTX, we observed fluctuating elevations in calcium
green 1 fluorescence after
-LCTX treatment, suggesting that
intraterminal Ca2+ is elevated by
-LCTX (Fig.
4). Elevations in calcium green 1 fluorescence were not observed in time-matched control experiments without exposure to the toxin (data not shown). Elevations in calcium
green 1 fluorescence after
-LCTX treatment varied in their
characteristics between boutons but generally lasted up to several
minutes and often were followed by a return to baseline fluorescence
values. In some boutons, a more sustained elevation in
Ca2+ eventually was achieved (e.g., Fig.
6C), and this is likely to correspond to the progressive
increase in baseline mEPSP frequency or EPSP amplitude seen in some
experiments. Individual boutons were capable of multiple rounds of
elevated fluorescence, and intervals between periods of increased
fluorescence varied widely between individual boutons of the same motor
neuron (generally in the range <5-20 min). Interestingly, the
magnitudes of
-LCTX-induced fluorescence changes were comparable
with those evoked by trains of nerve stimuli in the same boutons (Fig.
4C). The pulsatile increases in calcium green 1 fluorescence
elicited by
-LCTX suggest that periods of elevated
Ca2+ concentration in individual boutons might
underlie the oscillations in mEPSP frequency and EPSP amplitude
observed in electrophysiological recordings, which reflect the summed
activity of a large number of synapses on a single muscle fiber.
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The increase in intraterminal Ca2+ elicited by
-LCTX could arise as a result of either Ca2+
influx or release of Ca2+ from intraterminal
stores. Therefore we next asked whether extracellular Ca2+ was required for
-LCTX action.
Extracellular calcium is required for -LCTX action but not for
receptor binding
By analogy to other latrotoxins, it is likely that -LCTX acts
at crustacean nerve terminals by binding to cell surface receptors located at or near synapses, which are the functional homologs of
neurexin I
and latrophilin. Because it is known that these latrotoxin receptors differ in their divalent cation requirements for
receptor binding (Davletov et al. 1998
), it was
important to examine the influence of extracellular
Ca2+ on
-LCTX action.
When preparations were preincubated in saline with no added
Ca2+ and 1 mM EGTA (zero
Ca2+ saline), there was no change in mEPSP
frequency during application of -LCTX (2 µg/ml) for 60 min (Fig.
5A, n = 4).
This suggests that extracellular Ca2+ ions are
important in the action of
-LCTX but does not discriminate between
requirement of Ca2+ for receptor binding or for
subsequent action of the toxin. To ask whether
-LCTX binding
requires extracellular Ca2+ or not, we then
washed the preparations in several bath volumes of zero
Ca2+ saline to remove unbound
-LCTX from the
preparation before returning to normal crayfish saline, containing 13.5 mM Ca2+. On return to normal crayfish saline,
there was an immediate increase in mEPSP frequency (Fig.
5A), and the mean AUC/min value of 9.9 ± 1.9 (n = 4) was not significantly different from that obtained with
-LCTX applied in normal crayfish saline [12.8 ± 1.9 (n = 14), P > 0.5]. This
observation clearly suggests not only that
-LCTX can bind to cell
surface binding sites/receptors in the absence of extracellular
Ca2+ but that the mechanism by which the toxin
enhances transmitter release in this preparation is absolutely
dependent on extracellular Ca2+. In addition,
little
-LCTX dissociates from its cell surface receptors over the
time course of washing, as is the case for
-LTX (Meldolesi
1982
). Because binding of
-LCTX can take place in the
absence of extracellular Ca2+, it is likely that
-LCTX interacts with a crustacean latrophilin/CIRL-like receptor
rather than a neurexin homologue. To confirm the necessity of
extracellular Ca2+ ions in the action of
-LCTX, we examined the effect of removing extracellular
Ca2+ after the toxin had been allowed to bind to
the cell surface, and the onset of the effect on mEPSP frequency had
occurred. Replacing the bathing solution with zero
Ca2+ saline immediately abolished the effect of
-LCTX on mEPSP frequency, and no further mEPSP bursts were seen
while the preparation was incubated in zero Ca2+
saline (Fig. 5B, n = 2). Returning to normal crayfish
saline (13.5 mM Ca2+) immediately restored the
action of
-LCTX. Multiple exchanges between
Ca2+-free saline and normal crayfish saline could
be made in the same preparation, always with the result that removing
Ca2+ immediately quenched the toxin effect and
returning Ca2+ to the extracellular immediately
restored toxin-induced enhancement of mEPSP frequency. This experiment
clearly demonstrates a requirement for extracellular
Ca2+ ions in the action of
-LCTX on mEPSP
frequency.
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Calcium entry via VOCCs is not required for -LCTX effect on
mEPSPs
To ask whether -LCTX acts via voltage-operated
Ca2+ channels (VOCCs), we tested the effects of
Ca2+ channel blockers on the action of
-LCTX.
Application of the P-type Ca2+ channel
blocker, -Aga-IVA (1 µM), blocked nerve evoked EPSPs but had no
effect on
-LCTX induced increases in mEPSP frequency (Fig.
6, n = 4). This suggests
that although evoked transmitter release in this preparation is
dependent on Ca2+ influx via P-type
Ca2+ channels (Araque et al.
1994
), these channels do not play a role in the action of
-LCTX on spontaneous release. In some preparations, one type of VOCC
controls transmitter release during low-intensity stimulation but
additional subtypes of Ca2+
channels may come into play during periods of intense stimulation (Gonzalez Burgos et al. 1995
; Smith and Cunnane
1996
, 1997
). To exclude the possibility that other VOCCs not
normally linked to synaptic transmission in this preparation might play
a part in
-LCTX action, we applied the general VOCC blocker,
Cd2+ (50 µM). As with
-Aga-IVA,
Cd2+ had no effect on the action of
-LCTX on
mEPSP frequency (Fig. 6, n = 4). In
Ca2+ green 1 imaging experiments, we also found
no effect of
-Aga-IVA (1 µM) on
-LCTX-induced elevations of
calcium green fluorescence (Fig. 6C, n = 2).
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These data demonstrate that the extracellular
Ca2+ influx triggered by -LCTX does not occur
through the VOCCs coupled to synaptic vesicle exocytosis in this
preparation but must occur via some other Ca2+
entry pathway.
Increased intraterminal calcium concentration is sufficient to explain toxin action
Our results showing that extracellular Ca2+
is required for -LCTX action but not for receptor binding are
reminiscent of the action of
-LTX on spontaneous catecholamine
release from adrenal chromaffin cells (Liu and Misler
1998a
). In addition to
-LTX-induced Ca2+ influx causing a massive increase in
spontaneous catecholamine release, chromaffin cells show an enhancement
of depolarization-evoked catecholamine secretion at toxin
concentrations insufficient to increase intracellular
Ca2+ levels (Liu and Misler
1998b
). We tested whether increased intraterminal Ca2+ levels evoked by
-LCTX were sufficient to
account for the entire action of the toxin on transmitter release at
crayfish neuromuscular junction by attempting to block the toxin action
using the Ca2+ chelator, BAPTA. In three
experiments, preincubation in BAPTA-AM (50 µM), much reduced the
effect of
-LCTX compared with control experiments in the absence of
BAPTA-AM. However, the effect of the toxin was not completely abolished
by BAPTA-AM pretreatment. One possible explanation for this result is
simply that insufficient BAPTA molecules accumulate within the nerve
terminal to completely chelate the influx of Ca2+
ions elicited by
-LCTX. This was likely the case here, because EPSPs
could be evoked in these experiments during prolonged trains of
stimulation. It is known that the effects of BAPTA-AM on transmitter release can be overcome by sufficient Ca2+ influx
(Winslow et al. 1994
). To ensure sufficient
intraterminal BAPTA concentrations, we injected the excitatory motor
axon with BAPTA salt after the onset of
-LCTX action on mEPSP
frequency. Microinjection of BAPTA blocked the effect of
-LCTX (Fig.
7), and it was no longer possible to
elicit EPSPs even during prolonged trains. This effect of BAPTA
reversed very slowly after cessation of injection and withdrawal of the
axonal electrode. This occurred in parallel with a decline in the
fluorescence of coinjected rhodamine dextran, presumably reflecting the
diffusion of the dye (and, we assume, the chelator) away from the
terminals, into the large motor axon, leading to gradual saturation of
the remaining BAPTA in the boutons. Because the intracellular
Ca2+ chelator BAPTA powerfully inhibits
-LCTX
action on mEPSP frequency, the action of
-LCTX is likely to be
entirely dependent on a rise in intraterminal
Ca2+ concentration. Although this observation,
taken in conjunction with the absolute requirement for extracellular
Ca2+ for toxin action, suggests that
Ca2+ influx is the primary mechanism involved in
the enhancement of mEPSP frequency by
-LCTX, it is possible that the
Ca2+ signal initially triggered by
Ca2+ influx is amplified or maintained by a
secondary release of Ca2+ from intracellular
stores, perhaps by way of a Ca2+-induced
Ca2+ release (CICR) pathway. However, we could
find no evidence of a role for such a mechanism contributing to the
action of
-LCTX at these synapses because neither thapsigargin (10 µM, n = 2), an inhibitor of the endoplasmic reticulum
Ca2+ ATPase, nor the ryanodine receptor ligand,
dantrolene (50 µM, n = 2), had any effect on the
increased mEPSP frequency evoked by
-LCTX (data not shown).
|
Sr2+ and Ba2+ are less effective than
Ca2+ in supporting -LCTX action
Because -LCTX-induced elevation of intracellular
Ca2+ appeared to be a sufficient mechanism to
explain the action of the toxin, we investigated the ability of other
divalent cations to substitute for Ca2+ in
supporting the action of the toxin, in an effort to provide clues about
the nature of the Ca2+ influx pathway and/or
Ca2+-sensitive molecules involved in
-LCTX
action.
-LCTX was applied to preparations for 60 min with equimolar
substitution of either Sr2+ or
Ba2+ for Ca2+. Neither
Sr2+ nor Ba2+ was as
effective in supporting the action of
-LCTX (Fig.
8, A and B), and
this was manifest as both an increase in the time to onset of toxin
action and a decrease in the intensity of action of the toxin
(reduction in the duration of mEPSP bursts as well as the
magnitude of frequency increases). In normal crayfish saline, 100%
of cells responded to toxin within 50 min, whereas in
Sr2+-substituted saline, 66% of cells responded
within this period and in Ba2+-substituted
saline, only 29% of cells responded in <50 min (n = 7, 4, and 6 experiments respectively). AUC/min values 30-60 min after
exposure to
-LCTX in the test divalent cation, expressed as the
percentage of maximum response possible (in normal crayfish saline >60
min after exposure to
-LCTX) are shown for all three ions in Fig.
8C. The percentage of maximum response obtained in test
divalent 30-60 min after
-LCTX exposure was 75.8 ± 13.7% in
calcium, 21.9 ± 12.8% in Sr2+, and
2.0 ± 0.6% in Ba2+-containing saline
(n = 7, 4, and 6 experiments respectively). There was
no significant difference in the mean AUC/min values obtained in the
absence of
-LCTX in either Ba2+- or
Sr2+-containing saline compared with normal
crayfish saline (0.2 ± 0.02 in calcium, 0.1 ± 0.02 in
Sr2+, and 0.4 ± 0.2 in
Ba2+, respectively). In either
Sr2+ or Ba2+ substitution
experiments, returning the preparation to normal crayfish saline (13.5 mM Ca2+) caused an increase in the magnitude of
the toxin effect such that it was indistinguishable from control
experiments conducted entirely in normal crayfish saline (Fig.
8D), suggesting that neither toxin binding nor subsequent
action was compromised in either Sr2+- or
Ba2+-substituted saline.
|
Trivalent ions such as Gd3+ or
La3+ have been shown to block a variety of
Ca2+ channels and nonspecific cation channels as
well as -LTX induced Ca2+ flux (Boland
et al. 1991
; Inazu et al. 1995
; Rosenthal
et al. 1990
; Scheer 1989
; Yoshii et al.
1987
). We considered whether these trivalent cations might be
able to block the influx of Ca2+ induced by
-LCTX and thereby inhibit the action of the toxin. Neither
Gd3+ nor La3+ (500 µM)
had any effect on the action of
-LCTX, when added to the bath saline
after the toxin had begun to elevate mEPSP frequency in normal crayfish
saline (n = 3 in each case, data not shown).
The lectin, wheat germ agglutinin, inhibits -LCTX effects
Our data showing Ca2+-independent binding of
-LCTX to the cell surface suggests that this toxin may act by
binding to the crustacean homologue of latrophilin, the vertebrate
receptor responsible for Ca2+-independent binding
of
-LTX to cell membranes. The lectin, wheat germ agglutinin (WGA),
from Triticum vulgaris binds tightly to N-acetyl-
-D-glucosaminyl residues and
N-acetyl-
-D-glucosamine oligomer and recognizes such
residues on the latrophilin receptor, a reaction used in the
purification of the receptor (Davletov et al. 1996
). It
has been shown previously that another lectin, concanavalin A,
interferes with the secretagogue action of
-LTX at frog
neuromuscular junction (Rubin et al. 1978
), rat brain synaptosomes (Grasso et al. 1978
; Meldolesi
1982
) and PC12 cells (Meldolesi et al. 1983
),
and this effect is thought to be due to a reduction in toxin binding
after concanavalin A pretreatment. Therefore we asked whether the
latrophilin binding lectin, WGA, would interfere with
-LCTX action
at the crayfish synapse.
Preparations were incubated for 1 h in WGA (300 µg/ml) to
prebind WGA binding sites, followed by washing in normal crayfish saline to remove excess, unbound lectin. Subsequently, -LCTX (2 µg/ml) was applied, and mEPSP frequency was monitored for 60 min.
Preincubation with WGA had no effect on mEPSP frequency after a 60-min
incubation but did block the effect of
-LCTX on mEPSP frequency
(Fig. 9). The average AUC/min value in
the period 30-60 min after exposure to
-LCTX in cells preincubated
with WGA was 0.8 ± 0.3 (n = 5) compared with
11.3 ± 2.7 (n = 7) in time-matched control
experiments (P < 0.005, Mann-Whitney test).
N-acetyl-
-D-glucosamine (10 mM) was applied
after the 1-h toxin incubation. In all cases, this led to a rapid
initiation of high-frequency bursts of mEPSPs, similar to that seen in
control experiments after
-LCTX application, suggesting that removal
of WGA from cell surface binding sites by titration with
N-acetyl-
-D-glucosamine allowed the toxin to act. The mean AUC/min value 20-40 min after exposure to
N-acetyl-
-D-glucosamine was 22.6 ± 5.3 (n = 5) and is not significantly different to the value
for time-matched control preparations exposed only to
-LCTX (16.9 ± 3.9, n = 7). Because the preparations
were washed to remove excess or unbound
-LCTX before addition of
N-acetyl-
-D-glucosamine, it seems that
-LCTX must have been prebound to cell surface binding sites and that
this binding was sufficient to allow a toxin effect indistinguishable
from the control effect, i.e., WGA did not occlude binding of
-LCTX
to the receptors but did interrupt an additional process or interaction
required for the activity of the toxin. Interestingly, WGA was
ineffective if applied after
-LCTX had been allowed to act.
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DISCUSSION |
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We have found that -LCTX produces a profound enhancement of
both spontaneous and evoked transmitter release at the crayfish claw
opener neuromuscular junction preparation. Enhancement of transmitter
release was characterized by periods of burst-like spontaneous
discharge of transmitter quanta with simultaneous enhancement of evoked
transmitter release. In addition,
-LCTX also caused periodic
fluctuations in the levels of Ca2+ within nerve
terminals, with individual terminals being capable of generating
multiple
-LCTX-induced Ca2+ signals. These
data suggest that fluctuating periods of enhanced transmitter release
are caused by periods of increased permeability of the nerve terminal
to extracellular Ca2+ ions, which then reach
sufficient concentration within the nerve terminal to increase the
probability of vesicular fusion. Our observations did not reveal any
Ca2+-independent contribution to the mechanism of
action of
-LCTX at the low concentration used here, although we
cannot exclude the possibility of additional
Ca2+-independent mechanisms coming into play on
exposure to higher toxin concentrations (see following text).
Our data confirm and extend previous studies with this purified venom
fraction (or with whole venom or a 65-kDa crustacean specific
fraction). Thus it has been shown that both spontaneous and evoked
transmitter release are initially enhanced at crustacean neuromuscular
junctions treated with either BWSV or -LCTX, followed by failure of
transmission, at which point drastic morphological changes, including
synaptic vesicle depletion and mitochondrial swelling, ensue
(Burmistrov et al. 1997
; Fritz and Mauro
1982
; Fritz et al. 1980a
,b
; Kawai et al.
1972
). The minimally effective concentration used here was
insufficient to lead to blockade of transmitter release or any obvious
morphological derangement and revealed that the mechanism by which
-LCTX elicits Ca2+ entry and enhancement of
transmitter release is clearly dynamic (even though the rate of
dissociation from the receptors is very slow), a phenomenon that may
not be observed at higher, faster-acting toxin concentrations. In
contrast to the Ca2+-dependent effects reported
here with
-LCTX, BWSV has been reported to cause increased
spontaneous transmitter release at crustacean neuromuscular junction in
the absence of extracellular Ca2+ ions
(Kawai et al. 1972
). These observations suggest that
-LCTX may have both Ca2+-influx-dependent and
-independent actions, related to toxin concentration, as is the case
for
-LTX, (e.g., see Capogna et al. 1996
; Liu and Misler 1998b
) or that an additional component in whole BWSV stimulates Ca2+-independent effects on crustacean synapses.
-LCTX binding site resembles latrophilin/CIRL
It is known that latrotoxins bind to cell surface receptors to
elicit their actions on ionic fluxes and exocytosis. However, the
G-protein-coupled signaling function of the receptors may not be
involved in the mechanism of action of -LTX (Ichtchenko et
al. 1998
; Krasnoperov et al. 1999
; Sugita
et al. 1998
), i.e., receptors may serve only as synaptically
targeted binding sites. We found that there is no obvious
Ca2+ requirement for binding of
-LCTX to its
receptors: applying
-LCTX in either zero Ca2+
saline, or in saline in which Ca2+ ions were
replaced by either Sr2+ or
Ba2+ ions, did not alter the magnitude of the
response obtained on return to normal Ca2+ saline
(see Figs. 5A and 8D). Because neither
Sr2+ nor Ba2+ can
substitute for Ca2+ in supporting the binding
interaction of latrotoxins with neurexin (Davletov et al.
1998
), it appears that
-LCTX displays a strong interaction
with cell surface binding sites that show no marked sensitivity to
extracellular divalent cation concentrations, suggesting that
-LCTX
interacts with a latrophilin-like cell surface receptor.
Latrophilin exhibits a tight binding interaction with the lectin, WGA,
presumably because of the presence of glycosyl residues on the
extracellular portion of the receptor (Davletov et al. 1996). In addition, WGA has been reported to inhibit the
binding of
-LTX to synaptosomal membranes (see Davletov et
al. 1996
). WGA does affect the action of
-LCTX, although
this interaction is more complicated than simple competition between
the lectin and the toxin for a single binding site. Our results suggest
that
-LCTX requires interaction with two sites for activity. A
primary binding interaction, which is not occluded by the presence of WGA, is sufficient to provide stable binding of the toxin to the receptor. However, a secondary interaction with a distinct site is
required for activity of the toxin, and this interaction is blocked by
preincubation with WGA. Although we speculate that this secondary
interaction site represents the WGA binding site(s) on the receptor, it
is possible that endogenous proteins involved in forming the
-LCTX-induced Ca2+ influx route also may bind
this lectin. Additional glycosylated residues are known to be important
in the action of
-LTX because concanavalin A (a lectin with
different selectivity than WGA) affects
-LTX action and binding
(Boehm and Huck 1998
; Filippov et al.
1990
; Grasso et al. 1978
; Hurlbut and
Ceccarelli 1979
; Magazanik et al. 1992
;
Meldolesi 1982
; Meldolesi et al. 1983
; Rubin et al. 1978
).
Properties of -LCTX-induced calcium influx generate bursting
behavior
Several features of the action of -LCTX in the present study
are strikingly similar to observations made in other phyla, with either
whole venom or
-LTX. Specifically, bursting patterns of spontaneous
quantal discharge have been observed at insect neuromuscular junction
with BSWV (Cull-Candy et al. 1973
) and at mammalian CNS
synapses (Auger and Marty 1997
; Capogna et al. 1996
) and adrenal chromaffin cells with
-LTX (Liu and
Misler 1998b
). In addition, bursts of spontaneous transmitter
release have been reported at the frog neuromuscular junction using
venom from a related spider, Latrodectus geometricus
(Pumplin and del Castillo 1975
). The bursts of
spontaneous transmitter release in hippocampal CA3 pyramidal cells
(Capogna et al. 1996
) or cerebellar interneurons
(Auger and Marty 1997
) were sensitive to removal of
extracellular Ca2+, as was the case for
-LCTX
action on the crayfish neuromuscular junction. This obvious similarity
between the effects of two distinct, but related toxins, clearly
suggests that similar substrates for latrotoxin action exist at both
vertebrate and invertebrate synapses.
What are the underlying mechanisms that give rise to this bursting
spontaneous discharge of transmitter quanta? We observed that exposure
to -LCTX caused periodic elevations of intraterminal Ca2+ levels, which we believe to be the
fundamental basis for the action of this toxin on both spontaneous and
evoked transmitter release for the following reasons: the temporal
pattern of Ca2+ elevations within individual
boutons was consistent with the bursting discharges of quanta recorded
electrophysiologically; removal of extracellular
Ca2+ ions abolished
-LCTX-induced bursting
behavior; application of Ca2+ ionophore caused a
similar alteration in the pattern of facilitation of EPSP amplitudes to
the toxin; and chelation of intracellular Ca2+
with BAPTA drastically reduced the occurrence of toxin induced mEPSP
bursts. We infer that the bursting behavior is therefore likely to
represent a finite period of increased intraterminal Ca2+ concentration, via
Ca2+ influx, to
600-700 nM [the minimum
concentration required to elicit asynchronous transmitter release in
this preparation (Ravin et al. 1997
)]. At present we do
not know the route by which the
-LCTX-induced
Ca2+ influx occurs, but VOCCs do not make a major
contribution because pharmacological blockers of these channels did not
reduce the effect of
-LCTX.
-LTX is known to elicit step-like
increases in holding current in whole cell recordings, which are
thought to represent insertion of
-LTX molecules into the membrane
to form a conductance pathway by which Ca2+ could
enter (Auger and Marty 1997
) and endogenous proteins of the synaptic plasma membrane also may participate in the formation large conductance pores (Davletov et al. 1998
). It is
not yet known if
-LCTX exhibits similar pore-forming behavior.
It is not clear why a toxin induced burst of spontaneous quantal
discharge ceases once it has been established. The most obvious possibilities are depletion of a releasable pool of vesicles, some form
of inactivation of the Ca2+ influx pathway or
because the toxin dissociates from the receptor. Furthermore adaptation
of the exocytotic mechanism can occur such that release rates are not
maintained in response to a steady Ca2+
elevation, even though supplies of vesicles are not exhausted (Hsu et al. 1996). However, because
-LCTX-induced
Ca2+ elevations in individual boutons are often
of finite duration, it seems likely that inactivation of the
Ca2+ influx pathway must play a role in
terminating bursting activity. What factors could trigger channels
showing long, stable open states to suddenly enter a closed or
inactivated state? Single-channel conductances for
-LTX-induced
pores estimated in cellular systems vary from 3 to 400 pS
(Filippov et al. 1994
; Liu and Misler
1998b
; Wanke et al. 1986
), possibly due to a
Ca2+-activated synchronization of the activity of
groups of small conductance channels to produce larger, stepwise
ensemble currents (Filippov et al. 1994
). Eventually
such a composite pore might dissociate, abruptly destroying the
Ca2+ influx route and terminating the burst.
Alternatively, internalization of the receptor (and bound toxin) or
endogenous channel components could terminate
Ca2+ influx. Agonist stimulation of the secretin
receptor [which belongs to the same subfamily of G-protein-coupled
receptors as latrophilin (Krasnoperov et al. 1997
;
Lelianova et al. 1997
)] leads to internalization of
both receptor and ligand (Holtmann et al. 1996
;
Izzo et al. 1989
), but this is unlikely to be a major
mechanism for termination of the bursts of enhanced transmitter release
elicited by
-LCTX because prolonged washing of the toxin from the
bath did not diminish the effects on transmitter release once
initiated. The fact that dissociation of
-LTX from receptors is
extremely slow at physiological salt concentrations (Meldolesi
1982
) suggests that unbinding of
-LCTX from the receptors is
also an unlikely mechanism for the termination of a burst. An
alternative possibility is that endogenous proteins that contribute to
the Ca2+ influx pathway elicited by
-LCTX
exist in a dynamic pool that recycles between the plasma membrane and
intracellular locations. Clearly, even if
-LCTX-latrophilin
complexes are stable at the cell surface, internalization of a critical
component of the pore structure could terminate
Ca2+ influx until new molecules are delivered to
the cell surface. Further study is required to investigate this possibility.
Can other divalent cations substitute for calcium in supporting
-LCTX effects?
The rank order of potency of divalent cations in supporting
-LCTX action was Ca2+ > Sr2+ > Ba2+, with
Ba2+ ions providing weak support of the toxin
action (Fig. 8). A similar sequence of effectiveness in supporting
exocytosis has been reported in a variety of systems (where differences
in permeation of the ions has been controlled for), including squid
giant synapse (Augustine and Eckert 1984
), neutrophils
(Boonen et al. 1993
), and pancreatic
cells
(Barnett and Misler 1995
). These observations suggest that the differences in the ability of these three divalent cations to
support the effect of
-LCTX on spontaneous transmitter release can
be accounted for by differences in their ability to trigger exocytosis.
Differences in the permeation of the toxin induced Ca2+ influx pathway may also contribute to their
abilities to support toxin action although, at least for
-LTX
channels in lipid bilayers, permeation is similar for all three ions
(Robello et al. 1987
).
An unexpected feature of the Ca2+ influx pathway
elicited by -LCTX was its insensitivity to blockade by trivalent
cations such as La3+ or
Gd3+. It is known that these cations block
-LTX induced divalent cation influx in synaptosomes, presumably by
blocking the passage of ions through the pore (Scheer
1989
). In PC12 cells both Ca2+-dependent
and -independent dopamine release is inhibited by
La3+, suggesting that a
mechanism other than Ca2+ influx blockade is also
sensitive to La3+ (Rosenthal et al.
1990
). It will be interesting to understand which distinct
features of the
-LCTX-induced Ca2+ influx
pathway convey insensitivity to La3+ blockade.
Clearly, further work is required to enhance our understanding of the physiological role of latrotoxin receptors, both neurexins and latrophilins, in nerve terminal function. The striking similarities that exist across phyla in their responses to related latrotoxins highlight the fundamental conservation of synaptic mechanisms throughout evolution. However, a deeper understanding of the unique features of the toxin-receptor interactions that are responsible for taxonomic specificity may shed light on important aspects of synaptic diversity.
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ACKNOWLEDGMENTS |
---|
We thank Dr. H. L. Atwood for suggestions on the manuscript. This work was supported by a Canadian Medical Research Council Grant to M. P. Charlton.
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FOOTNOTES |
---|
Address for reprint requests: M. P. Charlton, Dept. Physiology,
Medical Sciences Building, University of Toronto, 1 King's College
Circle, Toronto, ON M5S 1A8, Canada.
E-mail:
milton{at}spine.med.utoronto.ca
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.
Received 12 May 1999; accepted in final form 20 August 1999.
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REFERENCES |
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