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The Journal of Neuroscience, December 1, 2000, 20(23):8685-8692

-Latrotoxin Releases Calcium in Frog Motor Nerve Terminals

Christopher W. Tsang, Donald B. Elrick, and Milton P. Charlton

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

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

-Latrotoxin (-LTX) is a neurotoxin that accelerates spontaneous exocytosis independently of extracellular Ca²⁺. Although -LTX increases spontaneous transmitter release at synapses, the mechanism is unknown. We tested the hypothesis that -LTX causes transmitter release by mobilizing intracellular Ca²⁺ in frog motor nerve terminals. Transmitter release was measured electrophysiologically and with the vesicle marker FM1-43; presynaptic ion concentration dynamics were measured with fluorescent ion-imaging techniques. We report that -LTX increases transmitter release after release of a physiologically relevant concentration of intracellular Ca²⁺. Neither the blockade of Ca²⁺ release nor the depletion of Ca²⁺ from endoplasmic reticulum affected Ca²⁺ signals produced by -LTX. The Ca²⁺ source is likely to be mitochondria, because the effects on Ca²⁺ mobilization of CCCP (which depletes mitochondrial Ca²⁺) and of -LTX are mutually occlusive. The release of mitochondrial Ca²⁺ is partially attributable to an increase in intracellular Na⁺, suggesting that the mitochondrial Na⁺/Ca²⁺ exchanger is activated. Effects of -LTX were not blocked when Ca²⁺ increases were reduced greatly in saline lacking both Na⁺ and Ca²⁺ and by application of intracellular Ca²⁺ chelators. Therefore, although increases in intracellular Ca²⁺ may facilitate the effects of -LTX on transmitter release, these increases do not appear to be necessary. The results show that investigations of Ca²⁺-independent -LTX mechanisms or uses of -LTX to probe exocytosis mechanisms would be complicated by the release of intracellular Ca²⁺, which itself can trigger exocytosis.

Key words: -latrotoxin; presynaptic toxin; mitochondria; calcium; sodium; exocytosis; frog neuromuscular junction/motor nerve terminal

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Neurotoxins are important tools for studying synaptic physiology. -Latrotoxin (-LTX) is a neurotoxin isolated from the venom of the black widow spider, Latrodectus mactans tredecimguttatus. At the frog neuromuscular junction (NMJ) -LTX increases the frequency of spontaneous transmitter release independently of extracellular Ca²⁺ (Longenecker et al., 1970) despite the fact that Ca²⁺ influx is required for nerve-evoked transmitter release (Bennett, 1999). This implies that there might be a mechanism of transmitter release that could bypass the requirement for Ca²⁺.

Two theories have been proposed to explain how -LTX could increase transmitter release independently of extracellular Ca²⁺. The first suggests that -LTX forms pores in nerve terminals and that changes in ion conductance could mediate its effect on transmitter release. This idea is supported by the fact that -LTX can form nonselective cation pores in lipid bilayer membranes (Finkelstein et al., 1976) by oligomerizing into homotetrameric structures (Orlova et al., 2000). However, this mechanism alone cannot explain the specificity of -LTX for presynaptic nerve terminals (Valtorta et al., 1984). A second theory suggests that -LTX interacts with a membrane receptor and that activation of a signal transduction mechanism triggers vesicle release. This theory was strengthened when two distinct receptors with nanomolar affinity for -LTX were isolated and cloned. One is the single-transmembrane-domain cell surface receptor neurexin-I (Ushkaryov et al., 1992), and the other is a seven-transmembrane-domain G-protein-coupled receptor, latrophilin/CIRL (Krasnoperov et al., 1997; Lelianova et al., 1997), expressed here as CL1. The latter is thought to mediate the actions of -LTX in the absence of extracellular Ca²⁺ because -LTX binding to neurexin is Ca²⁺-dependent (Davletov et al., 1995), but binding to CL1 does not require Ca²⁺ (Davletov et al., 1996). Studies with truncated CL1 mutants transfected into chromaffin cells, however, have demonstrated that receptor activation is not required for transmitter release by -LTX (Sugita et al., 1998). Therefore, it seems likely that -LTX is targeted to presynaptic nerve terminals by the receptor, where it then proceeds to act independently of the receptor; this function could include pore formation.

The mechanism of -LTX action has been difficult to resolve, and inconsistent results are seen in different cell types. For example, transmitter release by -LTX is dependent on Ca²⁺ mobilization in rat brain synaptosomes (Davletov et al., 1998; Rahman et al., 1999), whereas in secretory cell lines such as PC12 cells or -pancreatic cells no changes in intracellular Ca²⁺ are observed in the presence of -LTX (Meldolesi et al., 1984).

-LTX has been used widely under the assumption that it is a Ca²⁺-independent secretagogue at the frog NMJ. However, this assumption has not been tested with Ca²⁺ detection methods. Release of intracellular Ca²⁺ easily could explain the actions of -LTX in the absence of extracellular Ca²⁺. Therefore, we decided to test the hypothesis that at frog motor nerve terminals -LTX causes Ca²⁺ mobilization from intracellular stores and that this triggers transmitter release.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals and experimental treatment. Rana pipiens (leopard) frogs (4-5 cm body length; Wards Scientific, St. Catherine's, Ontario) were housed at 15°C in cages with a flow-through water system. Frogs were double-pithed, and the cutaneous pectoris muscles with the innervating pectoralis propius nerve were dissected out (Dreyer and Peper, 1974). Excised muscles were pinned down in a Sylgard-coated (Dow Corning, Midland, MI) preparation dish and maintained at room temperature (20-22°C) in normal physiological saline (NPS) containing (in mM) 120 NaCl, 2 KCl, 1 NaHCO₃, 1.8 CaCl₂, and 5 HEPES, pH-adjusted to 7.2 with NaOH.

Experimental solutions. Ca²⁺-free saline (CFS) containing (in mM) 120 NaCl, 2 KCl, 1 NaHCO₃, 5 MgCl₂, 10 HEPES, and 5 EGTA, pH-adjusted to 7.2 with NaOH, was used to study the actions of -LTX in the absence of extracellular Ca²⁺. When Na⁺ and Ca²⁺ were not required, a Na⁺- and Ca²⁺-free saline (NCFS) was made containing (in mM) 120 choline-Cl, 5 MgCl₂, 10 HEPES, and 5 BAPTA tetrapotassium salt (Molecular Probes, Eugene, OR), pH-adjusted to 7.2 with KOH. Before the start of any experiment that required the removal of an ion, preparations were washed (in CFS or NCFS) with four to five bath changes every 10 min for 1 hr. All saline salts and buffers were purchased from Sigma (St. Louis, MO).

For BAPTA-AM experiments a 5 mM stock concentration of BAPTA-AM was made up in dimethylsulfoxide (DMSO; Sigma) and diluted 1:50 to get a working concentration of 100 µM. A 1 M stock concentration of probenecid (Sigma) was made up in ethanol and diluted 1:1000 to get a working concentration of 1 mM. Pluronic acid (Molecular Probes) was added to assist in the solubilization of probenecid and BAPTA-AM. A working concentration of 2 µM pluronic acid was achieved by diluting a 1 mM stock in DMSO to 1:1000. The final solution was mixed by sonication for several seconds.

Dye loading. Nerve terminals were loaded with the fluorescent dyes Oregon green 488 BAPTA-1-dextran or sodium green-dextran (Molecular Probes; 10,000 molecular weight) for measuring changes in presynaptic Ca²⁺ or Na⁺, respectively, by forward-filling the dye through the cut end of the innervating motor nerve. The muscles were washed first in a Petri dish with CFS for 10 min to remove excess Ca²⁺. With a pair of sharp scissors the motor nerve was cut ~1 cm proximal to the muscle in a CFS bath. Then the preparation was transferred to a 1.5 ml rectangular well (containing CFS) that was cut out of a Sylgard-coated Petri dish. An adjacent small well contained 1 µl of the dye indicator at a concentration of 5 mM (in distilled water). The freshly cut end of the nerve was drawn into the dye-filled well, and a Vaseline border was made to isolate the contents of the two wells. Once the CFS was replaced with NPS, the dish was sealed with Parafilm (American National Can, Greenwich, CT) and stored at 15°C for 12-20 hr. During the incubation period the indicators were taken up by the axons and carried to the nerve terminals.

For FM1-43 (Molecular Probes) imaging the muscles were incubated with the dye (2 µM) in NPS for 15 min. During this incubation period the nerve was stimulated with 100 pulses (5 sec at 20 Hz) every 30 sec to induce vesicle recycling and uptake of the dye. Once the vesicles were loaded with FM1-43, the preparation was washed thoroughly with NPS to remove any extracellular and nonspecifically bound dye.

Blockade of spontaneous action potentials and postjunctional receptors. In CFS, spontaneous firing of action potentials can cause muscle fibers to twitch and also can load nerve terminals with Na⁺. The latter is known to have a physiological effect on transmitter release (Zengel et al., 1994). Therefore, the firing of spontaneous action potentials was blocked by the addition of 4 µM tetrodotoxin (TTX; Sigma) to block Na⁺ channels.

When recording miniature endplate potentials (MEPPs) and intracellular Ca²⁺ fluorescence in NPS, we used µ-conotoxin GIIIA (10 µg/ml; Bachem, Torrance, CA) to block muscle Na⁺ channels (Sosa and Zengel, 1993). This allowed us to block muscle contractions but preserved nerve action potentials and MEPPs. When only fluorescence imaging was required, -bungarotoxin (5 µg/ml), which blocks nicotinic receptors, was used instead to block muscle contractions. TTX was added to the bath when, subsequently, the muscles were transferred to CFS.

Electrophysiology. Transmitter release was monitored by intracellular recordings in a muscle fiber via 5-15 M glass microelectrodes (World Precision Instruments, Everett, WA) filled with 3 M KCl. Transmitter release was evoked by stimulating the motor nerve (0.2 Hz) at twice the threshold voltage that was required for muscle contraction in NPS. Responses were amplified (Neuroprobe amplifier, AM Systems, Carlsborg, WA), digitized (10 kHz, 12 bit; Labmaster interface, Scientific Solutions, Solon, OH), and averaged in groups of three to five by TOMAHACQ (T. A. Goldthorpe, University of Toronto), a program for PC data acquisition systems. Concurrently, a digital recording of the experiment (VR-10 digital data recorder, Instrutech, Great Neck, NY) was made for later analysis of MEPP frequency.

MEPP records stored on tapes were digitized by a Digidata 1200 Interface A/D Converter (Axon Instruments, Foster City, CA) that used Axoscope (Axon Instruments) data acquisition software and were analyzed with Mini Analysis software v4.0.1 (Synaptosoft, Leonia, NJ). MEPPs were counted by hand, and the frequency was calculated from the time that was required to record 100 MEPPs. During the height of -LTX action the frequency of quantal release is so high that it is difficult to count MEPPs accurately. Therefore, MEPPs were counted when there was a positive inflection of the membrane potential that was greater than the level of noise. Although this method underestimated the frequency of spontaneous transmitter release during toxin action, the absolute frequency was not essential for any of the hypotheses that were tested here. Any treatment that attenuated the action of -LTX by >50% was considered to have a significant effect.

Fluorescence imaging. Dye-loaded nerve terminals located on surface fibers were chosen for all experiments. Fluorescence (F) was measured with a Bio-Rad 600 (Hercules, CA) confocal laser-scanning microscope that used 1% of the maximum laser intensity for the ion indicators. Oregon green and sodium green dyes were excited by using the 488 nm line of the argon ion laser, and the emitted fluorescence was detected via a low-pass filter with a 515 nm cutoff. Confocal images were acquired by using a 40× water-dipping objective (0.55 numerical aperture; Nikon) and were averaged in groups of three.

Confocal images were acquired digitally with data acquisition software provided by Bio-Rad. Image files were analyzed later with BFOCAL, a program for PC analysis of Bio-Rad images written by T. A. Goldthorpe (University of Toronto). Changes in fluorescence were measured from a region of interest on the nerve terminal, which displayed the greatest dynamic range after nerve stimulation, and were expressed as:

Fluorescence images also were captured by using a Nikon Optiphot microscope equipped with a 40× (0.55 numerical aperture; Nikon) water-dipping objective, xenon lamp, and CCD camera (Cohu 4915) for FM1-43 experiments. Changes in fluorescence were processed with Axon Imaging Workbench software (Axon Instruments) and expressed as %F/F (see above). Measurements were made from several clusters of vesicles at 2 min intervals and adjusted by background subtraction.

Chemicals. CCCP (carbonyl cyanide m-chlorophenylhydrazone) and thapsigargin were purchased from Calbiochem (San Diego, CA). BAPTA-AM was purchased from Molecular Probes, and -LTX was bought from Latoxan (Valence, France).

Statistical analysis and figures. All values are reported as the mean ± SEM. An independent Student's t test was used to determine statistical significance at a 95.0% confidence level. N,n refers to the number of muscles (i.e., preparations) and the number of endplates, respectively. SigmaPlot 4 graphing software (Jandel Scientific, San Rafael, CA) and Corel Draw 8 (Corel, Ottawa, Canada) were used to graph and display the data.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

-LTX increases intracellular Ca²⁺ and transmitter release

In normal physiological saline, stimulation of the motor nerve at 10 Hz for 5 sec produced a rapid rise in Ca²⁺ fluorescence of 40 ± 2% above baseline (N,n = 7,7; Fig. 1A). Increasing the stimulation frequency to 20 and 40 Hz produced larger Ca²⁺ signals (72 ± 1 and 107 ± 1%, respectively; N,n = 7,7 for both) because of the more frequent opening of voltage-gated Ca²⁺ channels (Robitaille and Charlton, 1992). This control was performed for every nerve terminal that we examined to demonstrate the dynamic range of the indicator and detection system.

View larger version (29K): [in this window] [in a new window]   Figure 1.   Effect of -LTX on transmitter release and presynaptic Ca2+ in Ca2+-free saline. A, Left, Presynaptic intracellular Ca2+ signals in NPS in response to 10, 20, and 40 Hz nerve stimulation. A, Right, Absence of presynaptic Ca2+ signals in CFS in response to the same stimulation frequencies. The motor nerve was stimulated for 5 sec at twice the voltage that was required for muscle contraction. Inset pictures show indicator fluorescence in the presynaptic terminal at the peak of the response at each frequency of stimulation. Similar results were obtained in seven preparations. B, The bottom graph shows measurements from a simultaneous recording of spontaneous quantal transmitter release frequency (MEPP frequency, blue) and Ca2+ fluorescence (red) in the same motor nerve terminal as in A after treatment with 0.5 nM -LTX (arrow). Pictures of nerve terminal fluorescence and MEPP recordings are given at three time points during the experiment (1-3, top panels). The intracellular Ca2+ concentration increased before the increase in transmitter release. All of the data in this figure are from a single endplate. Similar results were obtained in two other experiments that recorded MEPPs and intracellular Ca2+ simultaneously.

Ca²⁺ was removed by washing the preparations with CFS (see Materials and Methods). When the nerve was restimulated with 10, 20, and 40 Hz stimulation in CFS (Fig. 1A), no Ca²⁺ signals were produced (N,n = 7,7 for each stimulation frequency). This suggests that the bath was nominally free of unchelated Ca²⁺ and that any changes in intracellular Ca²⁺ by -LTX could not be attributable to Ca²⁺ entry.

When -LTX (0.5 nM) was applied to nerve terminals bathed in CFS, the average change in Ca²⁺ fluorescence was 55% (Table 1). Figure 1B shows a typical result in which there was ~62% increase in fluorescence. The increase in Ca²⁺ fluorescence with the application of 0.5 nM -LTX was similar to that produced by 10 Hz nerve stimulation in NPS (Fig. 1A). Ca²⁺ signals were not significantly different when a 10-fold higher concentration of -LTX (5 nM) was applied (50 ± 6%; N,n = 6,6). Unlike nerve stimulation, which increased intracellular Ca²⁺ by Ca²⁺ entry, -LTX increased intracellular Ca²⁺ by Ca²⁺ mobilization from intracellular stores. The time course of Ca²⁺ elevation by -LTX is much slower than that obtained with nerve stimulation (several minutes compared with a few seconds).

View this table: [in this window] [in a new window]   Table 1.   Summary of drug effects on the actions of -LTX

Slightly after elevating the intracellular Ca²⁺ concentration, -LTX also caused a gross increase in spontaneous transmitter release as MEPP frequency increased from 1-2 to ~3-400 MEPPs/sec (Fig. 1B). Then, over the course of 10 min, the frequency of spontaneous transmitter release declined to low levels (<1 MEPP/sec). The run-down in MEPP frequency is attributable to synaptic vesicle depletion (Clark et al., 1970, 1972), because vesicle recycling does not occur in CFS after treatment with -LTX (Ceccarelli and Hurlbut, 1980; Henkel and Betz, 1995).

-LTX does not mobilize Ca²⁺ from endoplasmic reticulum (ER)

CL1 has been classified as a seven-transmembrane receptor coupled to the G-protein, G_q/11 (Rahman et al., 1999). This G-protein can activate phospholipase C (PLC) to produce inositol trisphosphate (IP₃), which mobilizes Ca²⁺ from the ER. Therefore, the PLC inhibitor U-73122 was used to determine whether the activation of PLC was responsible for the action of -LTX on transmitter release and Ca²⁺ mobilization. Nerve-muscle preparations were incubated with U-73122 (50 µM) for 1 hr in CFS, and then -LTX (0.5 nM) was applied. In the presence of U-73122, -LTX still increased MEPP frequency and intracellular Ca²⁺ similar to controls (Fig. 2A, Table 1).

View larger version (16K): [in this window] [in a new window]   Figure 2.   The effect of U73122 and thapsigargin on -LTX-induced Ca2+ mobilization. Nerve terminals were bathed first in CFS containing 4 µM TTX for 1 hr. Then changes in intracellular Ca2+ were measured after the addition of 50 µM U-73122 (A) or 20 µM thapsigargin (B), followed by 0.5 nM -LTX 1 hr later. Similar results were observed from six other U-73122-treated nerve terminals and two other thapsigargin-treated nerve terminals (see Table 1). Neither drug prevented the release of intracellular Ca2+ by -LTX.

We next tried to inhibit the release of Ca²⁺ from ER by first depleting the store with thapsigargin. When thapsigargin (20 µM) was applied to nerve terminals bathed in CFS for 1 hr, there was very little change in the intracellular Ca²⁺ concentration (Fig. 2B) or transmitter release (Table 1). However, when -LTX (0.5 nM) was applied after thapsigargin, a significant increase in Ca²⁺ fluorescence (Fig. 2B) and an acceleration of spontaneous transmitter release were still observed (Table 1). Although Ca²⁺ in ER stores can have subtle physiological effects at the frog NMJ (Narita et al., 1998), our data suggest that it is not sufficient to support the actions of -LTX in CFS. Therefore, ER is probably not the primary Ca²⁺ pool affected by -LTX.

-LTX mobilizes Ca²⁺ from mitochondria

Another major Ca²⁺-storing organelle found in nerve terminals is the mitochondrion. Unlike ER, these stores may not be depleted readily in CFS because of the large internally negative membrane potential (~150-200 mV) opposing the efflux of Ca²⁺. Several drugs, such as CCCP, are well known to interfere with mitochondrial metabolism and can cause mitochondria to lose their Ca²⁺. When CCCP (10 µM) was applied to nerve terminals bathed in CFS, a significant rise in intracellular Ca²⁺ (50 ± 12%; N,n = 5,5) and transmitter release (102 ± 4 MEPPs/sec; N,n = 3,3) was produced (Table 1). The amount of Ca²⁺ mobilized by CCCP on average was not significantly different from that mobilized by -LTX (Table 1), suggesting that mitochondria are likely to be the Ca²⁺ source.

To determine whether -LTX mobilizes Ca²⁺ from mitochondria, we first used CCCP (10 µM) to deplete mitochondrial Ca²⁺ stores from nerve terminals bathed in CFS. This was done in the presence of oligomycin (10 µg/ml), which prevents the reverse action of the mitochondrial ATPase from consuming ATP (Budd and Nicholls, 1996). Oligomycin on its own had no effect on Ca²⁺ homeostasis (data not shown). Once the Ca²⁺ signal had stabilized after the addition of CCCP, the addition of -LTX (0.5 nM) did not produce any further increase in intracellular Ca²⁺ (2.4 ± 1%; N,n = 3,3; Fig. 3A). Similarly, when nerve terminals bathed in CFS were pretreated with -LTX (0.5 nM), the addition of CCCP (10 µM) produced no further change in intracellular Ca²⁺ (1.3 ± 1%; N,n = 4,4; Fig. 3B). Because the effects of CCCP and -LTX on Ca²⁺ mobilization were mutually occlusive, this suggests that -LTX targets mitochondrial Ca²⁺ pools. In both cases, further increases in the Ca²⁺ signal were not prevented as a result of dye saturation because the dynamic range of the dye, determined before the experiment by nerve stimulation in NPS (see Fig. 1A), was on average at least two times larger than the Ca²⁺ signal produced by -LTX or CCCP. Furthermore, replacing the bath with NPS at the end of the experiment rapidly produced a much larger Ca²⁺ signal than that produced by any combination of CCCP and -LTX (Fig. 3A,B). Both of these observations indicate that larger Ca²⁺ signals could have been detected in these occlusion experiments. The Ca²⁺ signal produced by adding Ca²⁺ back to the bath was insensitive to the Ca²⁺ channel blocker Cd²⁺ (100 µM CaCl₂ added to saline; data not shown). This suggests that Ca²⁺ must have entered through toxin-induced pores and not through Ca²⁺ channels.

View larger version (30K): [in this window] [in a new window]   Figure 3.   CCCP and -LTX release Ca2+ from the same store. A, Left, Change in Ca2+ fluorescence when -LTX was applied after 10 µM CCCP, followed by 0.5 nM -LTX to a nerve terminal bathed in CFS with 4 µM TTX and 10 µg/ml oligomycin. There was a 10 min wash period (Wash) with CFS between the application of CCCP and -LTX. When NPS (i.e., containing 1.8 mM Ca2+) was applied (Ca2+) after -LTX, there was a large increase in Ca2+ fluorescence. A, Right, Bar graph compares the additional average peak Ca2+ fluorescence achieved when -LTX was applied after CCCP [i.e., (F/F)-LTX + CCCP = ([F-LTX+CCCP]/Frest)  ([FCCCP  Frest]/Frest)] with the average peak Ca2+ fluorescence achieved when -LTX was applied alone in other experiments [i.e., F/F-LTX = ([F-LTX  Frest]/Frest)]. The value for F/F-LTX was normalized to 100%. An asterisk indicates a significant difference in Ca2+ fluorescence relative to control. B, Change in Ca2+ fluorescence when CCCP was applied after -LTX. The bar graph comparisons are the same as in A.

It is possible that stimulus-dependent Ca²⁺ entry during the experiment may have caused mitochondria to accumulate Ca²⁺. Similarly, it is possible that, during the long incubation to allow dye transport to terminals, mitochondria accumulated Ca²⁺ to the extent that the results are an artifact of the incubation time. To examine these possibilities, we avoided the long incubation time by loading the dye for only 3 hr at room temperature into nerves cut close to the muscle. Furthermore, the nerve was left unstimulated for the duration of the experiment, and 4 µM TTX was added to prevent spontaneous nerve activity. When 10 µM CCCP was applied to nerve terminals bathed in CFS, a large Ca²⁺ signal was produced (155 ± 1%; N,n = 1,5). Because this signal is greater than the signal produced by the terminals incubated overnight and by stimulation during the experiment, we conclude that Ca²⁺ release by mitochondria is not an artifact of incubation time or nerve-evoked activity.

Ca²⁺ mobilization by -LTX is Na⁺-dependent

We next asked how -LTX signals the mitochondria to release Ca²⁺. Because -LTX forms a pore in frog nerve terminals (Davletov et al., 1998), we tested the hypothesis that Na⁺ entry through this pore causes mitochondria to lose Ca²⁺. It has been shown previously that methods that increase intracellular Na⁺ at nerve terminals also cause an increase in transmitter release (Baker and Crawford, 1975; Meiri et al., 1981; Atwood et al., 1983). To determine whether -LTX increases intracellular Na⁺, we detected changes in the intracellular Na⁺ concentration with the fluorescent indicator sodium green-dextran loaded in nerve terminals that were bathed in CFS. Application of -LTX (0.5 nM) caused the Na⁺ signal to increase by 42 ± 4% (N,n = 5,5). Similar responses were observed with 5 nM -LTX (Fig. 4A). Entry of Na⁺, however, was not attributable to the opening of voltage-gated Na⁺ channels because these were blocked by 4 µM TTX.

View larger version (53K): [in this window] [in a new window]   Figure 4.   The role of Na+ in the action of -LTX. A, -LTX increases intracellular Na+. The graph shows measurements from a simultaneous recording of spontaneous transmitter release (black dots) and Na+ fluorescence (white dots) in a motor nerve terminal after treatment with 5 nM -LTX (bar). Similar results were obtained in two other experiments. B, -LTX-induced Na+ and Ca2+ signals (0.5 nM) in CFS (black) and NCFS (white) with 4 µM TTX. Values were normalized to the effects of the -LTX in CFS and were displayed as a percentage of control. The left and right pairs of bar graphs show the change in intracellular Na+ and Ca2+, respectively, after the application of -LTX in CFS and NCFS. Both results in NCFS were significantly different from results in CFS (*). C, -LTX causes exocytosis in the absence of extracellular Na+ and Ca2+. The graph shows the changes in vesicular FM1-43 fluorescence after the application of 5 nM -LTX to nerve terminals bathed in NCFS containing 4 µM TTX. Similar results were obtained in two other experiments. The insets show pictures of the terminal when -LTX first was applied (5 min) and then 15 and 30 min later. Note the disappearance of fluorescent spots that correspond to clusters of labeled vesicles. In these images the contrast has been reversed so that bright areas appear dark.

To determine whether the increase in intracellular Na⁺ caused the increase in intracellular Ca²⁺, we removed extracellular Na⁺ by choline substitution (Fig. 4B). When -LTX was applied to nerve terminals bathed in NCFS (i.e., no Ca²⁺ or Na⁺), the Na⁺ fluorescence decreased (16.7 ± 2%; N,n = 3,3). The decrease in intracellular Na⁺ was not attributable to dye loss because the Na⁺ signal increased when extracellular Na⁺ was reintroduced. Thus, unlike results in CFS, increases in the intracellular Na⁺ concentration do not occur when nerve terminals are treated with -LTX in NCFS. When changes in intracellular Ca²⁺ were measured in NCFS, the effect of -LTX on Ca²⁺ mobilization was reduced by 70% (N,n = 4,13; Fig. 4B). This suggests that Na⁺ influx is necessary for Ca²⁺ mobilization by -LTX.

Is Na⁺ entry required for -LTX-dependent exocytosis?

To determine whether transmitter release by -LTX still occurred in the absence of extracellular Na⁺ and Ca²⁺, we could not use electrophysiological techniques because there would have been no Na⁺-dependent postsynaptic current. Therefore, we measured changes in fluorescence from terminals for which the vesicles had been loaded with FM1-43 by nerve stimulation (see Materials and Methods; Cochilla et al., 1999). In this manner, FM1-43 was taken up into vesicles during endocytosis and was released during exocytosis. When -LTX was applied in NCFS, the nerve terminals, which had accumulated FM1-43 previously, lost most of their fluorescence in 40 min (Fig. 4C). Similar results were obtained in two other experiments. This suggests that Na⁺ entry is not required by -LTX to stimulate the fusion of synaptic vesicles.

Relationship between Ca²⁺ and -LTX-induced transmitter release

To examine the role of Ca²⁺ in mediating the effects of -LTX on spontaneous transmitter release, we used the cell-permeant Ca²⁺ chelator BAPTA-AM to quell changes in intracellular free Ca²⁺. To maximize the effect of BAPTA-AM at the time of -LTX action, (1) we added an anion pump inhibitor, probenecid (1 mM), to minimize the loss of BAPTA from the cytosol (Ouanounou et al., 1996); (2) we gave a second treatment of BAPTA-AM 15 min after the first (final concentration 200 µM) to get a longer-lasting effect of the chelator; and (3) we added -LTX at 10 times the normal concentration to hasten the action of the toxin (time to onset, <2 min) 15 min after the second BAPTA-AM addition. Following these criteria ensured that the effects of -LTX were observed when Ca²⁺ buffering was at its strongest. In CFS, BAPTA-AM significantly reduced the toxin-induced increase in Ca²⁺ fluorescence by ~94% (N,n = 3,3) but had no effect on the acceleration of transmitter release (>300 MEPPs/sec; N,n = 2,2; Fig. 5). The data suggest that Ca²⁺ released by -LTX from intracellular stores does not play a major role in toxin-induced exocytosis.

View larger version (17K): [in this window] [in a new window]   Figure 5.   -LTX causes transmitter release with minimal change in the intracellular Ca2+ concentration. Shown are peak Ca2+ fluorescence and peak MEPP frequency achieved by 5 nM -LTX from terminals treated with (white) or without (black) 200 µM BAPTA-AM in CFS supplemented with 4 µM TTX and 1 mM probenecid. Values for Ca2+ fluorescence and MEPP frequency were normalized and expressed as a percentage of control (5 nM -LTX in CFS). -LTX-induced Ca2+ fluorescence after BAPTA-AM was reduced significantly as compared with -LTX control (*).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

-Latrotoxin releases intracellular Ca²⁺

The first finding here is that -LTX causes an increase in the intracellular Ca²⁺ concentration to physiologically significant levels that are sufficient to trigger exocytosis. Because these experiments were performed in CFS, there must have been a release of intracellular Ca²⁺ in the motor nerve terminals. It is clear from these results that presynaptic terminals do not lose all of their organelle Ca²⁺ during a 1 hr incubation in CFS.

The source of released Ca²⁺

The next set of experiments revealed some details about the source of this released Ca²⁺. In nerve terminals, Ca²⁺ is found in ER, mitochondria, and synaptic vesicles (Meldolesi et al., 1988). High-resolution electron spectroscopic imaging showed that Ca²⁺ in frog motor nerve terminals was found predominantly in synaptic vesicles and the lumen of smooth ER cisternae (Grohovaz et al., 1996; Pezzati and Grohovaz, 1999). Parts of mitochondria also appear to contain Ca²⁺ but at a lower concentration than vesicles. In rat brain synaptosomes, -LTX binding to CL1 stimulates PLC that mobilizes Ca²⁺ from intracellular stores (Davletov et al., 1998). Although -LTX stimulates the breakdown of phosphoinositides (Vicentini and Meldolesi, 1984), this is not critical to the toxin mechanism because an -LTX mutant, which on binding still triggers the breakdown of phosphoinositides, cannot stimulate exocytosis (Ichtchenko et al., 1998). Furthermore, activation of PLC by -LTX in synaptosomes is dependent on the presence of extracellular Ca²⁺ (Davletov et al., 1998). At the NMJ the source of released Ca²⁺ by -LTX is unlikely to be the ER, because a PLC inhibitor and a blocker of ER Ca²⁺ uptake both failed to affect -LTX-induced Ca²⁺ signals.

Our occlusion experiments showed that CCCP, which is known to release mitochondrial Ca²⁺, released the same pool of Ca²⁺ as that released by -LTX. The amount of Ca²⁺ released by CCCP is similar to that released by -LTX and is sufficient to cause a sustained increase in spontaneous transmitter release from a resting value of ~1 to 102 MEPP/sec (Table 1; Alnaes and Rahamimoff, 1975; Zengel et al., 1994). Therefore, the release of Ca²⁺ by -LTX is likely to reach physiologically significant concentrations.

Mitochondria in lizard motor nerve terminals acquire and release Ca²⁺ during physiological stimulation (David et al., 1998; David, 1999). In contrast to our results, David (1999) did not report that CCCP could release mitochondrial Ca²⁺ although the uptake of Ca²⁺ was blocked. However, his study used the Ca²⁺ indicator Oregon green BAPTA-5N, which has much lower affinity than the Oregon green BAPTA-1-dextran (60 µM vs 170 nM) used in our experiments. In addition to species differences, another difference between our study and that of David (1999) is that we used 10-fold more CCCP and applied it for a longer period of time; this enhances the possibility of detecting Ca²⁺ release.

We cannot rule out the possibility that synaptic vesicles, which occupy most of the terminal volume, could release Ca²⁺ also (for review, see Gonçalves et al., 2000). For instance, Gonçalves et al. (1998) showed that synaptic vesicles can acquire Ca²⁺ and that uptake is blocked by CCCP. Release of Ca²⁺ by vesicles was not demonstrated.

Mechanism of -LTX signaling

-LTX elevated the intracellular Na⁺ concentration as expected from the action of a nonspecific cation channel (Finkelstein et al., 1976) inserted in the presynaptic membrane. In the absence of extracellular Na⁺, -LTX caused the loss of Na⁺, and the release of stored Ca²⁺ was reduced greatly. Therefore, it appears that Na⁺ influx caused by -LTX is primarily responsible for the mobilization of Ca²⁺, possibly by activating the mitochondrial Na⁺/Ca²⁺ exchanger. Because removing extracellular Na⁺ did not block completely all of the Ca²⁺ that was released, there may have been an additional mechanism operating.

Black widow spider venom causes nerve terminals to swell in a Na⁺-dependent manner (Gorio et al., 1978). We confirmed, with observations of terminals filled with fluorescent indicators, that swelling with -LTX occurs in CFS but does not occur in NCFS (data not shown). It is unlikely that swelling is responsible for the effects of -LTX on transmitter release, because it has been shown that the frequency of exocytotic fusion events is reduced considerably as terminals swell (Solsona et al., 1998). Moreover, our experiments in NCFS showed that swelling was not required for -LTX effects.

-LTX increases spontaneous exocytosis in the absence of extracellular Ca²⁺ provided that Mg²⁺ or another divalent cation is present (Misler and Hurlbut, 1979; Misler and Falke, 1987). Although our results cannot rule out the possibility that -LTX allows the entry of extracellular Mg²⁺, it is unlikely that an increase in intracellular Mg²⁺ is responsible for the Ca²⁺ signal, because the Ca²⁺ indicator dye is ~200 times less sensitive to Mg²⁺ than Ca²⁺ (Molecular Probes). Because -LTX probably depolarizes the nerve terminal by increasing membrane conductance to Na⁺, the extent to which the intracellular Mg²⁺ concentration could increase would be small. Furthermore, even if -LTX caused the intracellular Mg²⁺ concentration to increase, Mg²⁺ is not a good substitute for Ca²⁺ in triggering transmitter release (Miledi, 1973). The most likely explanation for the requirement of extracellular Mg²⁺ is that it is required for -LTX to form functional pores in the membrane (Orlova et al., 2000).

Release of Ca²⁺ is not necessary for -LTX action

-LTX does not require extracellular Na⁺ to stimulate exocytosis. We showed that -LTX triggers exocytosis of FM1-43-labeled vesicles in the absence of extracellular Na⁺ and Ca²⁺ (see Fig. 4C). Under these conditions the intracellular Ca²⁺ signal is reduced greatly. This supports previous ultrastructural data demonstrating that nerve terminals lose their vesicles after treatment with black widow spider venom in Na⁺- and Ca²⁺-free saline (Gorio et al., 1978). Similarly, Na⁺ is not required by -LTX to stimulate the secretion of radiolabeled neurotransmitters from rat brain synaptosomes (Deri et al., 1993; Storchak et al., 1994).

The Ca²⁺ dependence of -LTX action on exocytosis has been controversial. For instance, in rat brain synaptosomes and adrenal chromaffin cells some studies show that transmitter release by -LTX depends on the presence of extracellular Ca²⁺ and a rise in intracellular Ca²⁺ (Davletov et al., 1998; Liu and Misler, 1998; Rahman et al., 1999). However, other studies in these same systems have shown that -LTX does not require any increase in intracellular Ca²⁺ to stimulate exocytosis (Meldolesi et al., 1984; Michelena et al., 1997). Studies on PC12 cells and -pancreatic cells have reached the latter conclusion (Meldolesi et al., 1984; Lang et al., 1998).

At the frog NMJ, -LTX appears to stimulate vesicular exocytosis independently of extracellular Ca²⁺ and any increase in intracellular Ca²⁺. When the amplitude of Ca²⁺ signals was reduced vastly in NCFS (see Fig. 4C) or after the application of an intracellular Ca²⁺ chelator (see Fig. 5), the effect of -LTX appeared undiminished. Although we cannot prove that the chelator controlled Ca²⁺ signals in microdomains at vesicle fusion sites in these experiments, as little as 25 µM BAPTA-AM can reduce stimulus-evoked transmitter release in this preparation drastically (Robitaille and Charlton, 1992; Robitaille et al., 1993). It therefore appears that acceleration of exocytosis by -LTX can occur by a mechanism different from that used in normal Ca²⁺-regulated secretion. This is a plausible conclusion because, in systems in which synaptotagmin function is impaired by peptide injection or genetic mutation, Ca²⁺-regulated secretion by ionophores and depolarizing agents is impaired, yet acceleration of transmitter release by -LTX remains unaffected (Geppert et al., 1994; Thomas and Elferink, 1998). Similarly, munc13-1, a phorbol ester receptor essential for Ca²⁺-dependent exocytosis in glutamatergic neurons, is not required for exocytosis by -LTX (Augustin et al., 1999). In contrast, it is possible that -LTX increases the sensitivity of transmitter release to Ca²⁺. For instance, in permeabilized cells -LTX causes more transmitter release than Ca²⁺-ionophores or high K⁺ solutions, given the same extracellular Ca²⁺ concentration (Davletov et al., 1998). We also have seen that the frequency of spontaneous transmitter release with -LTX far exceeds that obtained with CCCP, although the Ca²⁺ signals produced by both agents are similar.

The action of -LTX contrasts with that of -latrocrustatoxin (-LCTX), a similar toxin in black widow spider venom. -LCTX also causes increased spontaneous transmitter release in crustacean synapses, but this action requires only the elevation of intracellular Ca²⁺ concentration subsequent to Ca²⁺ entry via a pore (Elrick and Charlton, 1999).

In conclusion, our results provide a more complete picture of the actions of -LTX at the frog NMJ, the classic preparation in which -LTX action first was described. Our data show that assumptions about Ca²⁺ independence of drug and toxin effects in the absence of extracellular Ca²⁺ must be tested. Experiments that are designed to test hypotheses of Ca²⁺-independent mechanisms of -LTX action would be confused by the exocytosis triggered by intracellular Ca²⁺ release. Furthermore, -LTX is used frequently as a tool to obtain Ca²⁺-independent exocytosis, and the interpretation of these experiments too may be complicated by the release of intracellular Ca²⁺. The data also show that the main effect of -LTX in the NMJ is not via a Ca²⁺-dependent mechanism.

	FOOTNOTES

Received June 15, 2000; revised Sept. 5, 2000; accepted Sept. 6, 2000.

This research work was supported by a grant to M.P.C. from the Medical Research Council of Canada and scholarships to C.W.T. from the Department of Physiology, University of Toronto and the Ontario Ministry of Education.

Correspondence should be addressed to Dr. Milton P. Charlton, Medical Sciences Building, Room 3232, Department of Physiology, University of Toronto, 1 Kings College Circle, Toronto, ON, Canada M5S 1A8. E-mail: milton{at}spine.med.utoronto.ca.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

• Alnaes E, Rahamimoff R (1975) On the role of mitochondria in transmitter release from motor nerve terminals. J Physiol (Lond) 248:285-306[Abstract/Free Full Text].
• Atwood HL, Charlton MP, Thompson CS (1983) Neuromuscular transmission in crustaceans is enhanced by a sodium ionophore, monensin, and by prolonged stimulation. J Physiol (Lond) 335:179-195[Abstract/Free Full Text].
• Augustin I, Rosenmund C, Sudhof TC, Brose N (1999) Munc13-1 is essential for fusion competence of glutamatergic synaptic vesicles. Nature 400:457-461[Medline].
• Baker PF, Crawford AC (1975) A note of the mechanism by which inhibitors of the sodium pump accelerate spontaneous release of transmitter from motor nerve terminals. J Physiol (Lond) 247:209-226[Abstract/Free Full Text].
• Bennett MR (1999) The concept of a calcium sensor in transmitter release. Prog Neurobiol 59:243-277[ISI][Medline].
• Budd SL, Nicholls DG (1996) A reevaluation of the role of mitochondria in neuronal Ca²⁺ homeostasis. J Neurochem 66:403-411[ISI][Medline].
• Ceccarelli B, Hurlbut WP (1980) Ca²⁺-dependent recycling of synaptic vesicles at the frog neuromuscular junction. J Cell Biol 87:297-303[Abstract/Free Full Text].
• Clark AW, Mauro A, Longenecker Jr HE, Hurlbut WP (1970) Effects of black widow spider venom on the frog neuromuscular junction. Effects on the fine structure of the frog neuromuscular junction. Nature 225:703-705[Medline].
• Clark AW, Hurlbut WP, Mauro A (1972) Changes in the fine structure of the neuromuscular junction of the frog caused by black widow spider venom. J Cell Biol 52:1-14[Abstract/Free Full Text].
• Cochilla AJ, Angleson JK, Betz WJ (1999) Monitoring secretory membrane with FM1-43 fluorescence. Annu Rev Neurosci 22:1-10[ISI][Medline].
• David G (1999) Mitochondrial clearance of cytosolic Ca²⁺ in stimulated lizard motor nerve terminals proceeds without progressive elevation of mitochondrial matrix [Ca²⁺]. J Neurosci 19:7495-7506[Abstract/Free Full Text].
• David G, Barrett JN, Barrett EF (1998) Evidence that mitochondria buffer physiological Ca²⁺ loads in lizard motor nerve terminals. J Physiol (Lond) 509:59-65[Abstract/Free Full Text].
• Davletov BA, Krasnoperov V, Hata Y, Petrenko AG, Sudhof TC (1995) High-affinity binding of -latrotoxin to recombinant neurexin I. J Biol Chem 270:23903-23905[Abstract/Free Full Text].
• Davletov BA, Shamotienko OG, Lelianova VG, Grishin EV, Ushkaryov YA (1996) Isolation and biochemical characterization of a Ca²⁺-independent -latrotoxin-binding protein. J Biol Chem 271:23239-23245[Abstract/Free Full Text].
• Davletov BA, Meunier FA, Ashton AC, Matsushita H, Hirst WD, Lelianova VG, Wilkin GP, Dolly JO, Ushkaryov YA (1998) Vesicle exocytosis stimulated by -latrotoxin is mediated by latrophilin and requires both external and stored Ca²⁺. EMBO J 17:3909-3920[ISI][Medline].
• Deri Z, Bors P, Adam-Vizi V (1993) Effect of -latrotoxin on acetylcholine release and intracellular Ca²⁺ concentration in synaptosomes: Na⁺-dependent and Na⁺-independent components. J Neurochem 60:1065-1072[ISI][Medline].
• Dreyer F, Peper K (1974) A monolayer preparation of innervated skeletal muscle fibres of the m. cutaneous pectoris of the frog. Pflügers Arch 348:257-262[ISI][Medline].
• Elrick DB, Charlton MP (1999) -Latrocrustatoxin increases neurotransmitter release by activating a calcium influx pathway at crayfish neuromuscular junction. J Neurophysiol 82:3550-3562[Abstract/Free Full Text].
• Finkelstein A, Rubin LL, Tzeng MC (1976) Black widow spider venom: effect of purified toxin on lipid bilayer membranes. Science 193:1009-1011[Abstract/Free Full Text].
• Geppert M, Goda Y, Hammer RE, Li C, Rosahl TW, Stevens CF, Sudhof TC (1994) Synaptotagmin I: a major Ca²⁺ sensor for transmitter release at a central synapse. Cell 79:717-727[ISI][Medline].
• Gonçalves PP, Meireles SM, Gravato C, Vale MGP (1998) Ca²⁺-H⁺ antiport activity in synaptic vesicles isolated from sheep brain cortex. Neurosci Lett 247:87-90[ISI][Medline].
• Gonçalves PP, Meireles SM, Neves P, Vale MGP (2000) Methods for analysis of Ca²⁺/H⁺ antiport activity in synaptic vesicles isolated from sheep brain cortex. Brain Res Brain Res Protoc 5:102-108[Medline].
• Gorio A, Rubin LL, Mauro A (1978) Double mode of action of black widow spider venom on frog neuromuscular junction. J Neurocytol 7:193-202[ISI][Medline].
• Grohovaz F, Bossi M, Pezzati R, Meldolesi J, Tarelli FT (1996) High resolution ultrastructural mapping of total calcium: electron spectroscopic imaging/electron energy loss spectroscopy analysis of a physically/chemically processed nerve-muscle preparation. Proc Natl Acad Sci USA 93:4799-4803[Abstract/Free Full Text].
• Henkel AW, Betz WJ (1995) Monitoring of black widow spider venom (BWSV) induced exo- and endocytosis in living frog motor nerve terminals with FM1-43. Neuropharmacology 34:1397-1406[ISI][Medline].
• Ichtchenko K, Khvotchev M, Kiyatkin N, Simpson L, Sugita S, Sudhof TC (1998) -Latrotoxin action probed with recombinant toxin: receptors recruit -latrotoxin but do not transduce an exocytotic signal. EMBO J 17:6188-6199[ISI][Medline].
• Krasnoperov VG, Bittner MA, Beavis R, Kuang Y, Salnikow KV, Chepurny OG, Little AR, Plotnikov AN, Wu D, Holz RW, Petrenko AG (1997) -Latrotoxin stimulates exocytosis by the interaction with a neuronal G-protein-coupled receptor. Neuron 18:925-937[ISI][Medline].
• Lang J, Ushkaryov Y, Grasso A, Wollheim CB (1998) Ca²⁺-independent insulin exocytosis induced by -latrotoxin requires latrophilin, a G-protein-coupled receptor. EMBO J 17:648-657[ISI][Medline].
• Lelianova VG, Davletov BA, Sterling A, Rahman MA, Grishin EV, Totty NF, Ushkaryov YA (1997) -Latrotoxin receptor, latrophilin, is a novel member of the secretin family of G-protein-coupled receptors. J Biol Chem 272:21504-21508[Abstract/Free Full Text].
• Liu J, Misler S (1998) -Latrotoxin-induced quantal release of catecholamines from rat adrenal chromaffin cells. Brain Res 799:55-63[ISI][Medline].
• Longenecker Jr HE, Hurlbut WP, Mauro A, Clark AW (1970) Effects of black widow spider venom on the frog neuromuscular junction. Effects on endplate potential, miniature endplate potential, and nerve terminal spike. Nature 225:701-703[Medline].
• Meiri H, Erulkar SD, Lerman T, Rahamimoff R (1981) The action of the sodium ionophore, monensin, on transmitter release at the frog neuromuscular junction. Brain Res 204:204-208[ISI][Medline].
• Meldolesi J, Huttner WB, Tsien RY, Pozzan T (1984) Free cytoplasmic Ca²⁺ and neurotransmitter release: studies on PC12 cells and synaptosomes exposed to -latrotoxin. Proc Natl Acad Sci USA 81:620-624[Abstract/Free Full Text].
• Meldolesi J, Volpe P, Pozzan T (1988) The intracellular distribution of calcium. Trends Neurosci 11:449-452[ISI][Medline].
• Michelena P, de la Fuente MT, Vega T, Lara B, Lopez MG, Gandia L, Garcia AG (1997) Drastic facilitation by -latrotoxin of bovine chromaffin cell exocytosis without measurable enhancement of Ca²⁺ entry or [Ca²⁺]_i. J Physiol (Lond) 502:481-496[ISI].
• Miledi R (1973) Transmitter release induced by injection of calcium ions into nerve terminals. Proc R Soc Lond [Biol] 183:421-425[Medline].
• Misler S, Falke LC (1987) Dependence on multivalent cations of quantal release of transmitter induced by black widow spider venom. Am J Physiol 253:C469-C476[Abstract/Free Full Text].
• Misler S, Hurlbut WP (1979) Action of black widow spider venom on quantized release of acetylcholine at the frog neuromuscular junction: dependence upon external Mg²⁺. Proc Natl Acad Sci USA 76:991-995[Abstract/Free Full Text].
• Narita K, Akita T, Osanai M, Shirasaki T, Kijima H, Kuba K (1998) A Ca²⁺-induced Ca²⁺ release mechanism involved in asynchronous exocytosis at frog motor nerve terminals. J Gen Physiol 112:593-609[Abstract/Free Full Text].
• Orlova EV, Rahman MA, Gowen B, Volynski KE, Ashton AC, Manser C, van Heel M, Ushkaryov YA (2000) Structure of -latrotoxin oligomers reveals that divalent cation-dependent tetramers form membrane pores. Nat Struct Biol 7:48-53[ISI][Medline].
• Ouanounou A, Zhang L, Tymianski M, Charlton MP, Wallace MC, Carlen PL (1996) Accumulation and extrusion of permeant Ca²⁺ chelators in attenuation of synaptic transmission at hippocampal CA1 neurons. Neuroscience 75:99-109[ISI][Medline].
• Pezzati R, Grohovaz F (1999) The frog neuromuscular junction revisited after quick-freezing-freeze-drying: ultrastructure, immunogold labeling, and high resolution calcium mapping. Philos Trans R Soc Lond [Biol] 354:373-378[ISI][Medline].
• Rahman MA, Ashton AC, Meunier FA, Davletov BA, Dolly JO, Ushkaryov YA (1999) Norepinephrine exocytosis stimulated by -latrotoxin requires both external and stored Ca²⁺ and is mediated by latrophilin, G-proteins, and phospholipase C. Philos Trans R Soc Lond [Biol] 354:379-386[ISI][Medline].
• Robitaille R, Charlton MP (1992) Presynaptic calcium signals and transmitter release are modulated by calcium-activated potassium channels. J Neurosci 12:297-305[Abstract].
• Robitaille R, Garcia ML, Kaczorowski GJ, Charlton MP (1993) Functional colocalization of calcium and calcium-gated potassium channels in control of transmitter release. Neuron 11:645-655[ISI][Medline].
• Solsona C, Innocenti B, Fernández JM (1998) Regulation of exocytotic fusion by cell inflation. Biophys J 74:1061-1073[Abstract/Free Full Text].
• Sosa MA, Zengel JE (1993) Use of µ-conotoxin GIIIA for the study of synaptic transmission at the frog neuromuscular junction. Neurosci Lett 157:235-238[ISI][Medline].
• Storchak LG, Pashkov VN, Pozdnyakova NG, Himmelreich NH, Grishin EV (1994) -Latrotoxin-stimulated GABA release can occur in Ca²⁺-free, Na⁺-free medium. FEBS Lett 351:267-270[ISI][Medline].
• Sugita S, Ichtchenko K, Khvotchev M, Sudhof TC (1998) -Latrotoxin receptor CIRL/latrophilin 1 (CL1) defines an unusual family of ubiquitous G-protein-linked receptors. G-protein coupling not required for triggering exocytosis. J Biol Chem 273:32715-32724[Abstract/Free Full Text].
• Thomas DM, Elferink LA (1998) Functional an