alpha -Latrocrustatoxin Increases Neurotransmitter Release by Activating a Calcium Influx Pathway at Crayfish Neuromuscular Junction

Donald B. Elrick and Milton P. Charlton

Department of Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Elrick, Donald B. and Milton P. Charlton. alpha -Latrocrustatoxin Increases Neurotransmitter Release by Activating a Calcium Influx Pathway at Crayfish Neuromuscular Junction. J. Neurophysiol. 82: 3550-3562, 1999. alpha -latrocrustatoxin (alpha -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. alpha -LCTX had no effect when applied in Ca2+-free saline, but subsequent addition of Ca2+ caused an immediate enhancement of mEPSP frequency even when alpha -LCTX was previously washed out of the bath with Ca2+-free saline. Furthermore removal of Ca2+ from the saline after alpha -LCTX had elicited an effect immediately blocked the action on mEPSP frequency. Thus alpha -LCTX binding is insensitive to Ca2+, but toxin action requires extracellular Ca2+ ions. Preincubation with wheat germ agglutinin prevented the effect of alpha -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 alpha -latrotoxin (alpha -LTX) found in vertebrates. alpha -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 alpha -LCTX. In nerve terminals filled with the Ca2+ indicator, calcium green 1, alpha -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 omega -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 alpha -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 alpha -LCTX on both spontaneous and evoked transmitter release at crayfish neuromuscular junctions.


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INTRODUCTION
METHODS
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DISCUSSION
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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 alpha -latrocrustatoxin (alpha -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 alpha -latroinsectotoxin (alpha -LIT) and alpha -latrotoxin (alpha -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 alpha -LCTX at the crayfish neuromuscular junction in greater detail, focusing on the role of Ca2+ in alpha -LCTX action.

The latrotoxins act via two classes of presynaptic receptors, namely neurexins and latrophilins/calcium-independent receptors for latrotoxin (CIRLs). The binding of alpha -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 alpha -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 1alpha 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 alpha -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 alpha -LCTX on crustacean synapses (Burmistrov et al. 1997) have been studied relatively little. alpha -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 alpha -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 alpha -LCTX action shares many features in common with that of alpha -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 alpha -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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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 MOmega ). 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 MOmega ) 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 alpha -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 alpha -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 alpha -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 alpha -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 alpha -LCTX induced high-frequency mEPSP discharge, both mEPSP amplitudes and the intervals between mEPSPs were plotted as cumulative frequency distributions for control and alpha -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 alpha -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 alpha -LCTX, and data collection continued at 30-s intervals for 60-120 min after addition of the toxin. Data are presented graphically, expressed as Delta 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 Delta 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: alpha -LCTX and omega -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). alpha -LCTX was prepared for Latoxan by Professor E. V. Grishin according to the protocol of Krasnoperov et al. (1992). An identical preparation of alpha -LCTX has been subjected to N-terminal sequencing and chymotryptic digest sequence analysis, and a fragment of cDNA from the central domain of alpha -LCTX has been cloned and sequenced (Volynksii et al. 1999), showing alpha -LCTX to be structurally related to, but distinct from, other alpha toxins from black widow spider venom. alpha -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 alpha -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 alpha -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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Effect of alpha -LCTX on synaptic transmission at crayfish neuromuscular junction

We examined the effects of alpha -LCTX on crustacean synaptic transmission using intracellular microelectrode recordings from the opener neuromuscular junction preparation of the crayfish. Bath application of alpha -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 alpha -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 alpha -LCTX. The alpha -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 alpha -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 alpha -LTX at vertebrate synapses.



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Fig. 1. Effects of alpha -latroctrustatoxin (alpha -LCTX) on miniature excitatory postsynaptic potential (mEPSP) frequency and amplitude. A and B: traces showing membrane potential recordings in a single muscle cell during control (A) or after >30 min exposure to 2 µg/ml alpha -LCTX (B). Five consecutive 500-ms sweeps are shown. alpha -LCTX caused periods of high-frequency mEPSP discharge. C: cumulative frequency distributions of mEPSP interevent interval showing that during a period of alpha -LCTX-induced high-frequency mEPSP discharge (open circle ), the intervals between mEPSPs were dramatically less than those during control periods () (Kolmogorov-Smirnoff test, alpha  = 0.001, n = 89 intervals for both samples). D: cumulative frequency distributions of mEPSP amplitudes from the same populations of events used to calculate the interevent intervals in C showing that mEPSP amplitudes are not significantly different between control () and alpha -LCTX treatments (open circle ) (Kolmogorov-Smirnoff test, alpha  > 0.1, n = 90 events for both samples).



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Fig. 2. EPSP amplitudes are potentiated by alpha -LCTX. Simultaneous plots of mEPSP frequency (A) and the amplitude of the 1st EPSP in a train (B) against time in the same cell demonstrate that EPSP amplitude is potentiated by 2 µg/ml alpha -LCTX () during periods of toxin-induced increased mEPSP frequency. C: action potentials elicited during intracellular microelectrode recording from the excitor motor axon, in control (1), or during a toxin-induced mEPSP burst (2). Records are superimposed for comparison in trace (3).

During periods of increased mEPSP frequency induced by alpha -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 alpha -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 alpha -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 alpha -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 alpha -LCTX.



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Fig. 3. alpha -LCTX and calcium ionophore cause "prefacilitation" of EPSP amplitudes. EPSPs were recorded in response to trains of 3 pulses at 100 Hz before (control) or after either alpha -LCTX (A, 2 µg/ml) or the Ca2+ ionophore, A23187 (B, 50 µM). Bottom: control responses were scaled such that the amplitude of the 3rd EPSP of each trace approximately matched (heavy line is control). Scale bars are 1 mV and 10 ms in each case and apply to the top 2 traces. Stimulus artifacts were digitally removed for clarity.

alpha -LCTX causes fluctuating elevations in intraterminal [Ca2+]

To determine whether or not alpha -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 alpha -LCTX (2 µg/ml) in normal crayfish saline. In three preparations, although resting fluorescence intensity remained at a relatively constant level before exposure to alpha -LCTX, we observed fluctuating elevations in calcium green 1 fluorescence after alpha -LCTX treatment, suggesting that intraterminal Ca2+ is elevated by alpha -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 alpha -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 alpha -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 alpha -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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Fig. 4. alpha -LCTX induces spontaneous increases in intracellular calcium levels. Nerve terminals were filled with the Ca2+ indicator, calcium green 1 and the fluorescence of the indicator was monitored before and after addition of alpha -LCTX (2 µg/ml) at time 0. A: false color images (raw pixel values) showing alpha -LCTX induced Ca2+ signals in individual crayfish motor nerve terminals at various times after application of the toxin. Scale bar = 10 µm. B: plots of Delta F/F throughout the experiment for the boutons labeled 1-3 in A. C: plots of Delta F/F in response to nerve stimulation (, 10 Hz for 10 s) before addition of alpha -LCTX in the same boutons as A.

The increase in intraterminal Ca2+ elicited by alpha -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 alpha -LCTX action.

Extracellular calcium is required for alpha -LCTX action but not for receptor binding

By analogy to other latrotoxins, it is likely that alpha -LCTX acts at crustacean nerve terminals by binding to cell surface receptors located at or near synapses, which are the functional homologs of neurexin Ialpha 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 alpha -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 alpha -LCTX (2 µg/ml) for 60 min (Fig. 5A, n = 4). This suggests that extracellular Ca2+ ions are important in the action of alpha -LCTX but does not discriminate between requirement of Ca2+ for receptor binding or for subsequent action of the toxin. To ask whether alpha -LCTX binding requires extracellular Ca2+ or not, we then washed the preparations in several bath volumes of zero Ca2+ saline to remove unbound alpha -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 alpha -LCTX applied in normal crayfish saline [12.8 ± 1.9 (n = 14), P > 0.5]. This observation clearly suggests not only that alpha -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 alpha -LCTX dissociates from its cell surface receptors over the time course of washing, as is the case for alpha -LTX (Meldolesi 1982). Because binding of alpha -LCTX can take place in the absence of extracellular Ca2+, it is likely that alpha -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 alpha -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 alpha -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 alpha -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 alpha -LCTX on mEPSP frequency.



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Fig. 5. Extracellular calcium ions are required for toxin action but not binding. A: application of alpha -LCTX, 2 µg/ml () in the absence of extracellular Ca2+ ions (, "-Ca2+") does not affect the frequency of mEPSP discharge for <= 60 min after exposure to the toxin. alpha -LCTX was removed from the bath by washing in Ca2+-free saline to remove any excess, unbound toxin from the preparation. Subsequent reintroduction of Ca2+ ions in the saline (, "+Ca2+") caused an immediate increase in mEPSP frequency, similar to that evoked by alpha -LCTX application in the presence of normal Ca2+ saline. B: preparation was exposed to alpha -LCTX, 2 µg/ml () in normal crayfish saline, 13.5 mM Ca2+, (, "+Ca2+") until a definite effect on mEPSP frequency was manifest. Subsequent periods during which Ca2+ ions were removed from the bath saline (, "-Ca2+") caused an immediate abolition of the action of alpha -LCTX on mEPSP frequency, an effect which was reversed by return to normal crayfish saline (, "+Ca2+").

Calcium entry via VOCCs is not required for alpha -LCTX effect on mEPSPs

To ask whether alpha -LCTX acts via voltage-operated Ca2+ channels (VOCCs), we tested the effects of Ca2+ channel blockers on the action of alpha -LCTX.

Application of the P-type Ca2+ channel blocker, omega -Aga-IVA (1 µM), blocked nerve evoked EPSPs but had no effect on alpha -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 alpha -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 alpha -LCTX action, we applied the general VOCC blocker, Cd2+ (50 µM). As with omega -Aga-IVA, Cd2+ had no effect on the action of alpha -LCTX on mEPSP frequency (Fig. 6, n = 4). In Ca2+ green 1 imaging experiments, we also found no effect of omega -Aga-IVA (1 µM) on alpha -LCTX-induced elevations of calcium green fluorescence (Fig. 6C, n = 2).



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Fig. 6. Ca2+ channel blockers do not affect the action of alpha -LCTX on mEPSP frequency. A and B: plots of average mEPSP frequency and EPSP amplitude, respectively, vs. time, measured simultaneously in a single cell in normal crayfish saline (13.5 mM Ca2+). alpha -LCTX (2 µg/ml, ) was applied until a definite effect on mEPSP frequency and EPSP amplitude was manifest. Subsequent application of the Ca2+ channel blockers omega -Aga-IVA, 1 µM, () or Cd2+, 50 µM, () completely abolished EPSPs but did not affect the ability of alpha -LCTX to enhance mEPSP frequency. C: plots of Delta F/F in 3 boutons, throughout a calcium imaging experiment conducted in the presence of 1 µM omega -Aga-IVA (which blocked nerve evoked Ca2+ signals elicited by 10 s trains of stimuli at 15 Hz). alpha -LCTX (2 µg/ml) was applied at time = 0 min. Notice that omega -Aga-IVA does not affect the ability of alpha -LCTX to induce increases in calcium green 1 fluorescence (cf. Fig. 4B).

These data demonstrate that the extracellular Ca2+ influx triggered by alpha -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 alpha -LCTX action but not for receptor binding are reminiscent of the action of alpha -LTX on spontaneous catecholamine release from adrenal chromaffin cells (Liu and Misler 1998a). In addition to alpha -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 alpha -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 alpha -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 alpha -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 alpha -LCTX action on mEPSP frequency. Microinjection of BAPTA blocked the effect of alpha -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 alpha -LCTX action on mEPSP frequency, the action of alpha -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 alpha -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 alpha -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 alpha -LCTX (data not shown).



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Fig. 7. Intracellular bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)blocks the action of alpha -LCTX. A: incubation of the preparation in 50 µM BAPTA-AM () before application of alpha -LCTX (2 µg/ml, ) strongly attenuates the ability of the toxin to increase the frequency of mEPSP discharge. B: injection of the excitatory motor nerve terminals with BAPTA (200 mM in the electrode solution, ) abolishes the ability of previously applied alpha -LCTX (2 µg/ml, ) to elicit increases in mEPSP frequency. Similar results were obtained in 3 experiments in each case.

Sr2+ and Ba2+ are less effective than Ca2+ in supporting alpha -LCTX action

Because alpha -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 alpha -LCTX action. alpha -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 alpha -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 alpha -LCTX in the test divalent cation, expressed as the percentage of maximum response possible (in normal crayfish saline >60 min after exposure to alpha -LCTX) are shown for all three ions in Fig. 8C. The percentage of maximum response obtained in test divalent 30-60 min after alpha -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 alpha -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.



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Fig. 8. Sr2+ and Ba2+ are less effective than calcium in supporting alpha -LCTX action but not binding. Either Sr2+ (A) or Ba2+ (B) was substituted for Ca2+ in the saline () before application of alpha -LCTX (2 µg/ml, ) for >= 60 min. After this time, excess, unbound alpha -LCTX was removed by washing in either Sr2+ (A)- or Ba2+ (B)-substituted saline before returning to normal crayfish saline (13.5 mM Ca2+). C: summary data from 7, 4, and 6 cells respectively in Ba2+-, Sr2+-, or Ca2+-containing medium. Average area under a curve (AUC)/minute values measured for each cell during the period 30-60 min after application of alpha -LCTX in the test divalent cation were expressed as a percentage of the maximum toxin response achieved in that cell. Maximum toxin response for each cell was defined as the average AUC/min value after returning the preparation to normal crayfish saline (13.5 mM Ca2+) at the end of the experiment, >= 60 min after exposure to alpha -LCTX. Both Sr2+ and Ba2+ were significantly less effective than Ca2+ in supporting the action of alpha -LCTX (Mann-Whitney rank sum test, P < 0.025 and P < 0.002), respectively. D: maximum toxin response (i.e., average AUC/min after returning the preparation to normal crayfish saline at the end of the experiment, >= 60 min after exposure to alpha -LCTX) was not significantly different when either Ba2+ or Sr2+ was substituted for Ca2+ in the bath saline before exposure to alpha -LCTX (Mann-Whitney rank sum test, P > 0.5 and P > 0.9, respectively, same experiments as those analyzed in C).

Trivalent ions such as Gd3+ or La3+ have been shown to block a variety of Ca2+ channels and nonspecific cation channels as well as alpha -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 alpha -LCTX and thereby inhibit the action of the toxin. Neither Gd3+ nor La3+ (500 µM) had any effect on the action of alpha -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 alpha -LCTX effects

Our data showing Ca2+-independent binding of alpha -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 alpha -LTX to cell membranes. The lectin, wheat germ agglutinin (WGA), from Triticum vulgaris binds tightly to N-acetyl-beta -D-glucosaminyl residues and N-acetyl-beta -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 alpha -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 alpha -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, alpha -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 alpha -LCTX on mEPSP frequency (Fig. 9). The average AUC/min value in the period 30-60 min after exposure to alpha -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-beta -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 alpha -LCTX application, suggesting that removal of WGA from cell surface binding sites by titration with N-acetyl-beta -D-glucosamine allowed the toxin to act. The mean AUC/min value 20-40 min after exposure to N-acetyl-beta -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 alpha -LCTX (16.9 ± 3.9, n = 7). Because the preparations were washed to remove excess or unbound alpha -LCTX before addition of N-acetyl-beta -D-glucosamine, it seems that alpha -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 alpha -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 alpha -LCTX had been allowed to act.



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Fig. 9. Wheat germ agglutinin (WGA) blocks alpha -LCTX action when applied before, but not after, the toxin. A: data from an experiment examining the role of cell surface glycosylation on the action of alpha -LCTX. Preparation was incubated in WGA (300 µg/ml) for >= 60 min (). After washing off excess WGA, the preparation was exposed to alpha -LCTX (2 µg/ml) for 60 min (, alpha -LCTX). Toxin was washed off and finally N-acetyl-beta -D-glucosamine (10 mM) was added to the bath (N-ac-G, ). B: summary data from 5 experiments following the same protocol as in A, expressed as AUC/min values during 15- to 30-min recording periods. Preparations were exposed in sequence to the following treatments (with washing between treatments): control (), normal crayfish saline; WGA (), >60 min in WGA (300 µg/ml); alpha -LCTX (),30-60 min in 2 µg/ml alpha -LCTX; N-ac-G (), 20-40 min after adding N-acetylglucosamine (10 mM). Control responses to toxin alone from separate experiments, time matched to the responses in N-ac-G, are shown for comparison (, n = 7). C: alpha -LCTX-induced mEPSP discharge () was not significantly reduced when WGA (300 µg/ml) was applied after the toxin (). Data summarize results from 3 experiments expressed as AUC/min values.


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We have found that alpha -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, alpha -LCTX also caused periodic fluctuations in the levels of Ca2+ within nerve terminals, with individual terminals being capable of generating multiple alpha -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 alpha -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 alpha -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 alpha -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 alpha -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 alpha -LCTX may have both Ca2+-influx-dependent and -independent actions, related to toxin concentration, as is the case for alpha -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.

alpha -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 alpha -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 alpha -LCTX to its receptors: applying alpha -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 alpha -LCTX displays a strong interaction with cell surface binding sites that show no marked sensitivity to extracellular divalent cation concentrations, suggesting that alpha -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 alpha -LTX to synaptosomal membranes (see Davletov et al. 1996). WGA does affect the action of alpha -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 alpha -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 alpha -LCTX-induced Ca2+ influx route also may bind this lectin. Additional glycosylated residues are known to be important in the action of alpha -LTX because concanavalin A (a lectin with different selectivity than WGA) affects alpha -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 alpha -LCTX-induced calcium influx generate bursting behavior

Several features of the action of alpha -LCTX in the present study are strikingly similar to observations made in other phyla, with either whole venom or alpha -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 alpha -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 alpha -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 alpha -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 alpha -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 alpha -LCTX-induced Ca2+ influx occurs, but VOCCs do not make a major contribution because pharmacological blockers of these channels did not reduce the effect ofalpha -LCTX. alpha -LTX is known to elicit step-like increases in holding current in whole cell recordings, which are thought to represent insertion of alpha -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 alpha -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 alpha -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 alpha -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 alpha -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 alpha -LTX from receptors is extremely slow at physiological salt concentrations (Meldolesi 1982) suggests that unbinding of alpha -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 alpha -LCTX exist in a dynamic pool that recycles between the plasma membrane and intracellular locations. Clearly, even if alpha -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 alpha -LCTX effects?

The rank order of potency of divalent cations in supporting alpha -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 beta  cells (Barnett and Misler 1995). These observations suggest that the differences in the ability of these three divalent cations to support the effect of alpha -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 alpha -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 alpha -LCTX was its insensitivity to blockade by trivalent cations such as La3+ or Gd3+. It is known that these cations block alpha -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 alpha -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.


    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.


    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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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society