Modulation of calcium currents is eliminated after cleavage of a strategic component of the mammalian secretory apparatus
1 Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611, USA
Abstract
Adenosine inhibits neurotransmitter secretion from motor nerves by an effect on the secretory apparatus in amphibia. In contrast, the inhibitory effect of adenosine is associated with decreases in calcium currents at mouse motor nerve endings. To determine if the action of adenosine in the mouse is mediated thorough a direct effect on calcium channels or through the secretory machinery, the effects of cleavage of the SNARE proteins on the action of adenosine were examined. Cleavage of the SNARE syntaxin with botulinum toxin type C (Botx/C) prevented the inhibitory effect of adenosine on nerve terminal calcium currents. Cleavage of the other SNAREs (synaptobrevin with Botx/D or SNAP-25 with Botx/A) failed to affect the inhibitory action of adenosine. The results provide evidence for an intimate coupling of nerve terminal calcium channels with a plasma membrane component of the SNARE complex, such that modulation of calcium currents by a G-protein coupled receptor cannot occur when syntaxin is cleaved.
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Introduction
Modulation of neurotransmitter secretion by endogenous substances released together with the neurotransmitter is an important control mechanism to fine tune the secretory apparatus (for reviews see Scanziani et al. 1995; Miller, 1998; Silinsky et al. 2001). One important modulator at cholinergic synapses is adenosine, which is a major mediator of prejunctional neuromuscular depression at amphibian (Ribeiro & Sebastiao, 1987; Meriney & Grinnell, 1991; Redman & Silinsky, 1994) and mammalian synapses (Hamilton & Smith, 1991; Nagano et al. 1992; Hirsh & Silinsky, 2002; Hirsh et al. 2002). At amphibian neuromuscular junctions, adenosine derived from neurally released ATP is the mediator of neuromuscular depression at low frequencies of nerve stimulation (Redman & Silinsky, 1994).
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Traditionally, inhibitory effects of neuromodulators had been ascribed to effects on presynaptic ionic channels, i.e. decreases in calcium currents or increases in potassium currents (Miller, 1998). At amphibian nerve endings, it was found that A1 adenosine receptor activation inhibits neurotransmitter secretion from motor nerve endings by an effect on a strategic component of the secretory apparatus and not on membrane ionic channels (Silinsky, 1984; Silinsky & Solsona, 1992; Redman & Silinsky, 1994; Robitaille et al. 1999). This result, whereby neurotransmitter secretion was inhibited downstream of calcium entry, was subsequently confirmed at other vertebrate synapses as well (Scanziani et al. 1995; Trudeau et al. 1998; Miller, 1998; Blackmer et al. 2001).
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In contrast to the results in amphibia, A1 receptor activation in mammals is associated with decreases in nerve terminal calcium currents (Hamilton & Smith, 1991; Silinsky, 2004). Indeed, at the mouse neuromuscular junction, simultaneous decreases in both P/Q-type Ca2+ currents and evoked ACh release were observed (Silinsky, 2004). Whilst these differences between mammalia and amphibia may indicate that prejunctional depression mediated by adenosine is due to different mechanisms in the two species, alternative interpretations are possible. For example, due to a more intimate coupling between Ca2+ channels and the core complex of nerve teminal protein (the SNAREs) in mammals, the effects of adenosine on a SNARE in mammals may be reflected as decreases in Ca2+ currents whilst those in amphibia are not.
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In order to test this hypothesis, the effects of SNARE cleavage on the action of adenosine were examined at mouse neuromuscular junctions. To perform these studies, botulinum toxins, a family of zinc-dependent metalloendopeptidases that block neurotransmitter release by cleaving at highly specific regions of the secretory machinery (Jahn et al. 1995), were used as tools to inactivate specific SNAREs. The results suggest that modulation of Ca2+ currents by adenosine receptor activation is mediated via an interaction with the SNARE, syntaxin.
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Methods
General
Experiments were made on isolated mouse phrenic nerve hemidiaphragm preparations at room temperature (21–23°C) in accordance with the guidelines of the Northwestern University Animal Care and Use Committee and the National Institutes of Health of the US Public Health Service. Specifically, mice (20–30 g) were humanely anaesthetized with 5–10 ml of diethyl ether for 3–5 min. Once the mice were unresponsive to touch, they were exsanguinated. Electrophysiological recordings were made of voltage changes in the perineural space using the perineural recording method (Brigant & Mallart, 1982; Mallart, 1985; Anderson et al. 1988; Silinsky & Solsona, 1992; Protti & Uchitel, 1993; Xu & Atchison, 1996; Silinsky, 2004). For complete details see Silinsky (2004).
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Treatment with botulinum toxins
Preparations were incubated with a specific botulinum toxin serotype and gently rocked in a shaker bath for 1 h. Preparations were then pinned in a tissue bath and stimulated at 1 Hz for approximately 1 h. The period for paralysis of neuromuscular transmission ranged from 40 min to 1 h and 50 min; this reflects the time required to eliminate preformed SNARE complexes at this frequency of stimulation (Raciborska et al. 1998; Kalandakanond & Coffield, 2001).
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With respect to the specific fractions employed, treatment with Botx/C (56 μg ml–1) selectively affects syntaxin immunoreactivity in the mouse phrenic nerve hemidiaphragm preparation and blocks evoked neurotransmitter release (Molgo et al. 1989; Raciborska et al. 1998; Kalandakanond & Coffield, 2001). In these experiments, the 1 Hz stimulation protocol was sufficient to eliminate all residual ACh release from uncleaved SNAREs in times ranging from 55 min and 1 h 20 min (n = 5 experiments). In contrast to the other fractions of Botx, ACh release eliminated by Botx/C could not be retrieved even after adding potassium channel blockers in tubocurarine-free solution in either normal or elevated (8 mM) Ca2+ solutions (n = 20–30 cells in 5 different preparations).
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Treatment with Botx/A (45–56 μg ml–1) selectively cleaves SNAP-25 in mouse phrenic nerve endings (Kalandakanond & Coffield, 2001). The 1 Hz stimulation protocol eliminated ACh release between 1 h and 1 h and 50 min. Both evoked and spontaneous release could be retrieved in TEA/DAP solution but, in contrast to amphibia, the effect was not sustainable. This is possibly due to an order of magnitude lower quantal content in mouse than frog (Hirsh et al. 2002) coupled to a depletion of ACh quanta in the presence of potassium channel blockers (Anderson et al. 1988).
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Botx/D (7.8 μg ml–1) selectively cleaves synaptobrevin at phrenic motor nerve endings (Kalandakanond & Coffield, 2001); complete block of neuromuscular transmission was observed after 40 min to 1 h 10 min of 1 Hz stimulation (n = 5 experiments). Whilst synchronous ACh release reflected as endplate potentials (EPPs) could not be retrieved, increasing the extracellular Ca2+ concentration to 8 mM (in 100 μM TEA) and stimulating at 5–10 Hz, produced sustained increases in asynchronous evoked ACh release reflected as miniature EPP (MEPP) frequencies (evoked MEPP frequencies ranged from 0.92 to 1.9 s–1, n = 4 experiments). These described effects are characteristic of the specific reported effects of the various Botx fractions both electrophysiologically and morphologically (Molgo et al. 1989; Raciborska et al. 1998; Kalandakanond & Coffield, 2001).
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In control preparations incubated with normal, toxin free physiological salt solutions and stimulated at 1 Hz, suprathreshold EPPs (with superimposed action potentials) and MEPPs were detectable for periods as long as 20 h of 1 Hz stimulation (n = 5 experiments).
Physiological saline solutions
Ca2+ currents were measured in a solution consisting of (mM): NaCl 137, KCl 5, CaCl2 2, MgCl2 0–2, NaH2PO4 1, NaH2HCO3 24, dextrose 11, and potassium channel blockers 3,4-diaminopyridine (DAP, 300 μM) and tetraethylammonium (TEA, 10 mM) (pH to 7.4 gassed with 95% O2 and 5% CO2 or buffered with 30 mM Hepes and gassed with 100% oxygen in a phosphate and bicarbonate free solution – see Silinsky, 2004). Botulinum toxin fractions were obtained from Wako Chemicals USA Inc. All other chemicals were purchased from Sigma, St Louis, USA. Paralysis of neuromuscular transmission was achieved by tubocurarine (50 μM) in the control preparations (e.g. Figs 1 and 3) and by botulinum toxins in all other experiments.
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For this figure, neuromuscular transmission was completely blocked by 50 μM tubocurarine (see Silinsky, 2004). A, trace showing prejunctional Na+ and K+ currents (downward deflections) in normal Ca2+ solution prior to application of K+ channel blockers (average response to 15 stimuli, 0.1 Hz). B, superfusion with a solution containing potassium channel blockers produces the blockade of K+ currents and the development of stable prejunctional Ca2+ currents within 10 min of the beginning of application superfusion with standard Ca2+ current solution (this trace represents the average response to 15 stimuli, 0.017 Hz). The first upward deflection that precedes the Na+ and K+ currents in A and the Ca2+ currents in B represent the longitudinal current flowing towards the nerve terminal and passively discharging the membrane capacitance in the region of the recording electrode. Traces in A and B are unfiltered records. C, the typical effect of supramaximal concentrations of adenosine (10 mM) in normal preparations. Control peak 1.37 ± 0.03 mV, average response to n = 16 stimuli (0.017 Hz); adenosine = 1.05 ± 0.03, average response to n = 16 stimuli). Traces in C are filtered at 200 Hz. For details of current polarities and description of the perineural currents, which reflect voltage changes in the extracellular space and are thus calibrated in mV, see Methods and Silinsky (2004).
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In control preparations, neuromuscular transmission was completely blocked by 50 μM tubocurarine whilst for the Botx fractions, transmission was blocked prejunctionally by cleavage of the SNAREs with the particular Botx serotype. No significant differences were observed between the inhibitory effects of adenosine in the control conditions or after Botx/D or Botx/A treatment. Each bar represents the average inhibition produced by adenosine (as a percentage of control) for 5 different experiments on 5 different preparations. The dashed line (no adenosine) shows that supramaximal concentrations of adenosine failed to inhibit the P/Q Ca2+ current after Botx/C treatment. For further details, see text.
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The perineural ionic currents
Details of these recordings in mouse have been published previously (Silinsky, 2004). Briefly, the inward Ca2+ current in the nerve terminals produces a proportional current in the perineural space which flows upstream to the recording electrode (Brigant & Mallart, 1982; Mallart, 1985; Anderson et al. 1988; Protti & Uchitel, 1993; Xu & Atchison, 1996; Silinsky, 2004); the recorded waveform is thus an inverted likeness of the events occurring in the nerve ending. The upward-going perineural voltage change that is antagonized by P/Q-type Ca2+ channel blockers will thus be termed prejunctional Ca2+ current or the presynaptic Ca2+ current as it is produced by Ca2+ currents in the nerve ending flowing across the perineurial resistance.
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Adenosine concentrations and statistical methods
The important aspects of the adenosine dose–response relationships Ca2+ currents and neurotransmitter release have been published previously from this laboratory (Hirsh et al. 2002; Silinsky, 2004). For the sake of comparison with our earlier papers, in all experiments, the effect of a supramaximal concentration of adenosine (10 mM) on nerve terminal Ca2+ currents was examined (see, e.g. Fig. 7 in Silinsky, 2004). Data are presented ± 1 S.E.M. For complete statistical methods, see Silinsky (2004).
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Results
General observations on prejunctional ionic currents and the effects of adenosine in the absence of botulinum toxins
Figure 1A and B depicts the typical presynaptic ionic currents recorded from the perineurium in a curarized preparation in the absence of botulinum toxin treatment. The first downward waveform (Fig. 1A, Na+) is the Na+ current produced by Na+ influx in the nodes of Ranvier and the terminations of the myelin sheaths (a region termed the heminode). The second downward deflection (Figs 1A, K+) represents the delayed rectifier, the voltage-dependent current K+ in the nerve endings (Brigant & Mallart, 1982; Mallart, 1985; Anderson et al. 1988; Xu & Atchison, 1996; Silinsky, 2004). Application of K+ channel blockers eliminates the potassium component of the waveform and reveals the upward going nerve terminal P/Q-type Ca2+ current that triggers evoked ACh release (Fig. 1B, Ca2+). The upward going deflections such as those shown in Fig. 1B are due to the movement of Ca2+ through P/Q-type calcium channels as they are blocked by -agatoxin IVA or Cd2+ (Brigant & Mallart, 1982; Mallart, 1985; Anderson et al. 1988; Protti & Uchitel, 1993; Xu & Atchison, 1996; Silinsky, 2004).
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Depending upon the concentrations of potassium channel blockers and the presence or absence of procaine (which is frequently used to block repetitive firing in the presence of potassium channel blockers – see Xu & Atchison, 1996), the Ca2+ currents may have one or two distinct phases. Figure 1B shows the phasic peak followed by a slower, more sustained second phase. For quantification in this paper, the early phasic peak of the calcium current was employed; this phase is present in all experiments and, when measured simulataneously with evoked ACh release, is the component of the Ca2+ current that is temporally related to promoting the synchronous release of ACh (see Silinsky, 2004; Fig. 3).
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In experiments such as those shown in Fig. 1, preparations were blocked with the nicotinic receptor blocker tubocurarine to prevent muscle twitching in response to nerve stimulation. The data in Fig. 1A and B are also representative of the presynaptic currents observed in preparations blocked by the prejunctional blocking actions of all of botulinum toxin serotypes used in this study (see also Fig. 2). For further details concerning the polarities of these currents and technical details of current measurements, which reflect voltage changes in the perineural space, see Silinsky (2004).
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A, control Ca2+ current after complete blockade of neurotransmitter release by Botx/C (average response to 5 stimuli, 0.017 Hz). B, 10 mM adenosine fails to inhibit Ca2+ currents after Botx/C (average response to 5 stimuli, 0.017 Hz). C, effects of 1 mM Cd2+ to block Ca2+ currents after Botx/C treatment (average response to 16 stimuli, 0.017Hz). For a discussion of the residual outward current, which is not associated with the P/Q Ca2+ current that mediates evoked ACh release see Xu & Atchison (1996) and Silinsky (2004).
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Figure 1C shows the typical effect of supramaximal adenosine concentrations (10 mM) in preparations treated as described in the Methods but in the absence of toxin treatment; in this experiment an inhibition of the Ca2+ current to 76.7% of control is observed. In all experiments in normal Ca2+ solutions, supramaximal concentrations of adenosine inhibited Ca2+ currents to approximately 75% of the control level (75.4 ± 3.9%, mean ± 1 S.E.M. n = 5 experiments, P << 0.05).
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Effects of botulinum toxins on the inhibitory effects of adenosine (10 mM)
Syntaxin is an integral membrane protein SNARE that is linked to presynaptic Ca2+ channels (Catterall, 2000; Jarvis et al. 2000; Spafford & Zamponi, 2003; Dolphin, 2003). Botulinum toxin type C (Botx/C) cleaves syntaxin between residues K253 and A254 near the transmembrane region of syntaxin (Jahn et al. 1995; Sutton et al. 1998). Treating the preparation with Botx/C produced complete blockade of neurotransmitter release at the mouse neuromuscular junction (see Kalandakanond & Coffield, 2001 and Methods for the electrophysiological assessment of SNARE cleavage).
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Figure 2 illustrates the typical experimental result on the action of adenosine after cleavage of syntaxin by Botx/C. In this experiment, the perineural calcium current was unchanged from the control level (1.28 ± 0.07 mV, n = 5) after application of 10 mM adenosine (1.35 ± 0.06 mV, n = 4, P = 0.36). In contrast, the Ca2+ channel blocker Cd2+ produced its typical inhibitory effect on Ca2+ currents after Botx/C treatment (Fig. 2C; see also Silinsky, 2004). Similar results were obtained in experiments on four other preparations. In summary, in all five experiments on five different preparations made after complete blockade of ACh release by Botx/C treatment, no significant inhibitory effect of supramaximal adenosine concentrations on the Ca2+ currents was observed in any experiment. (P values ranged from 0.43 to 0.94 in these individual experiments; the mean Ca2+ current during adenosine treatment as a percentage of control is 101 ± 0.02%.) Figure 3 shows the composite data on the effects of adenosine on normalized Ca2+ currents in the absence of botulinum toxin poisoning (Control) and after cleavage of syntaxin with Botx/C (filled bar).
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To determine if the blockade of the effects of adenosine is due to a non-selective effect of botulinum toxins, or specific to cleavage of syntaxin by Botx/C, the effects of cleavage of SNAP-25 with Botx/A or synaptobrevin with Botx/D were investigated. Botx/A irreversibly cleaves a nine-amino-acid segment of SNAP-25 between amino acids N197 and R198 near the C terminus of the molecule (Jahn et al. 1995; Sutton et al. 1998). This causes a simultaneous elimination of evoked and spontaneous ACh release at neuromuscular junctions due presumably to an alteration of the calcium sensitivity of the secretory apparatus (Molgo et al. 1989; Kalandakanond & Coffield, 2001). After treatment with Botx/A and the elimination of all evoked release the effects of adenosine were examined. After Botx/A, adenosine produced a statistically significant inhibition of Ca2+ currents in each individual experiment (P < 0.05). The mean percentage inhibition after Botx/A treatment was to 81.4 ± 5.1% of the control value (n = 5 experiment). The results of the Botx/A experiments are also summarized in Fig. 3.
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Botx/D cleaves synaptobrevin (VAMP) between K59 and L60 (Jahn et al. 1995) and eliminates the normal evoked release of ACh at vertebrate neuromuscular junctions (Kalandakanond & Coffield, 2001; Molgo et al. 1989). After treatment with Botx/D and complete elimination of all ACh release, the effects of adenosine were examined. As shown in Fig. 3, the mean percentage inhibition after Botx/D treatment was to 83 ± 2.3% of the control level (n = 5 experiments). In these experiments, no statistically significant differences existed between the control preparations, preparations treated with Botx/A or those treated with Botx/D with respect to the maximal inhibitory effects of adenosine (analysis of variance followed by Student's t test using the Bonferroni correction see Glantz, 1992; P values ranged from 0.11 to 0.47).
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Discussion
The results provide evidence that cleavage of the presynaptic membrane SNARE syntaxin by Botx/C, selectively eliminates the inhibitory effects of adenosine but not traditional Ca2+ channel antagonists (e.g. Cd2+). In contrast, no significant differences were observed between the inhibitory effects of adenosine on non-toxin-treated preparations and those completely blocked with Botx/A or Botx/D. These data provide the first evidence from an adult mammalian synapse that an endogenous mediator of presynaptic depression can no longer inhibit Ca2+ currents when a specific SNARE is cleaved.
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It is of interest in this regard that syntaxin has been implicated as an important protein involved in presynaptic modulation via G protein-coupled receptors at non-mammalian synapses; specifically the effects of GTPS to inhibit presynaptic Ca2+ currents in chick ciliary ganglia are blunted after cleavage of syntaxin with Botx/C (Stanley & Mirotznik, 1997).
A model for the interaction of adenosine with mammalian nerve terminals is shown in Fig. 4 which also depicts the cleavage sites of the three fractions of Botx used in this study (for a primitive prelude of this model, see Silinsky, 1986). In Fig. 4A, the voltage-gated Ca2+ channel is depicted with its known interactions with the SNARE syntaxin (red) (Catterall, 2000; Jarvis et al. 2000, 2002; Spafford & Zamponi, 2003; Dolphin, 2003). Binding of exogenous or endogenous adenosine to the A1 adenosine receptor (Figs 1, 4A), produces GTP/GDP exchange on the G subunit (not shown) and leads to the dissociation of the G complex from the GTP-bound G subunit (Figs 2, 4A). The G complex then proceeds to interact with HABC domain of syntaxin (for evidence see Blackmer et al. 2001; Jarvis et al. 2002). This interaction of the G-protein subunits with the N terminus of syntaxin influences the helical core region of this SNARE to interact with the synprint site (pink region) on the P/Q-type Ca2+ channel, which in turn reduces the Ca2+ channel activity and neurotransmitter release. When syntaxin is cleaved with Botx/C (Fig. 4B), the target for G is eliminated, thus preventing adenosine from inhibiting calcium currents and neurotransmitter release. This model is supported both by studies in which GTPS is injected into the presynaptic element of the chick ciliary ganglion (Stanley & Mirotznik, 1997) and by quantitative immunocolocalization of a complex between calcium channels, syntaxin and G-proteins (Li et al. 2004).
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A, sketches of syntaxin (red), SNAP-25 (light green) and synaptobrevin (blue) based upon the crystal structures of these proteins (Sutton et al. 1998). The G-protein cartoon, includes the propeller region of G that is believed to interact with effectors (Ford et al. 1998); this is likely to occur via the N terminal HABC domain of syntaxin (Jarvis et al. 2002). In turn, the Hcore region of syntaxin has been shown to interact with the synprint region of P/Q- and N-type Ca2+ channels (pink). The synprint region and the P/Q Ca2+ channel structure were drawn in accordance with Catterall (2000), Jarvis et al. (2000), Spafford & Zamponi (2003) and Dolphin (2003). B, after cleavage of syntaxin with Botx/C, the target for inhibition by adenosine is no longer present, thus precluding an inhibitory effect of adenosine. B also shows the approximate sites of cleavage by Botx/D on synaptobrevin and Botx/A on SNAP-25.
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These results thus suggest that the presence of intact syntaxin is essential for the modulation of Ca2+ currents by adenosine, rather than merely being a facilitator of G protein-coupled receptor activation. Whilst these data do not exclude the possibility that adenosine receptor activation inhibits ACh release downstream of Ca2+ entry in the mouse, the effects of adenosine on evoked and spontaneous ACh release are eliminated in the presence of Ca2+ channel blockers, making this possibility less likely (De Lorenzo et al. 2004). Furthermore, although the results of the present study do not exclude the possibility that syntaxin helps to localize G protein-coupled receptors to Ca2+ channels, the results of Blackmer et al. (2001) would argue against such a role for syntaxin as a general mechanism for presynaptic modulation.
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Regardless of the precise mode of G-protein receptor coupling, these results suggest that the apparent difference in presynaptic targets for adenosine between mammalian and frog motor nerve endings might reflect a differential coupling between the SNAREs and presynaptic calcium channels rather than a different presynaptic target for neuromodulation (Bezprozvanny et al. 2000; Lu et al. 2001). The differential coupling is unlikely to be due to differences in the types of motor nerve ending calcium channels between amphibia (N type) and mammalia (P/Q type), as presynaptic N-type Ca2+ channels are inhibited by adenosine in chick preganglionic nerve endings (Yawo & Chuhma, 1993). It is more likely that these differences involve intrinsic differences in the relationships between the secretory machinery and the presynaptic calcium channels in the two species. For example, structural studies have suggested that vesicle docking and fusion sites at mouse motor nerve endings are embedded between paired rows of particles believed to be Ca2+ channels, thus providing a morphological correlate of the high apparent degree of coupling between the SNAREs and Ca2+ channels in mammals (Stanley, 1997). In contrast, the vesicle docking and fusion sites in amphibia appear at the edges of the Ca2+ channel zones, possibly reflecting a less intimate coupling of the SNAREs with the depolarization–secretion coupling process in amphibia and providing a morphological correlate for an effect of adenosine that is independent of changes in Ca2+ currents at frog motor nerve endings (Silinsky & Solsona, 1992; Stanley, 1997).
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Yawo H & Chuhma N (1993). Preferential inhibition of omega-conotoxin sensitive presynaptic Ca2+ channels by adenosine autoreceptors. Nature 365, 256–258., 百拇医药(Eugene M Silinsky)
Abstract
Adenosine inhibits neurotransmitter secretion from motor nerves by an effect on the secretory apparatus in amphibia. In contrast, the inhibitory effect of adenosine is associated with decreases in calcium currents at mouse motor nerve endings. To determine if the action of adenosine in the mouse is mediated thorough a direct effect on calcium channels or through the secretory machinery, the effects of cleavage of the SNARE proteins on the action of adenosine were examined. Cleavage of the SNARE syntaxin with botulinum toxin type C (Botx/C) prevented the inhibitory effect of adenosine on nerve terminal calcium currents. Cleavage of the other SNAREs (synaptobrevin with Botx/D or SNAP-25 with Botx/A) failed to affect the inhibitory action of adenosine. The results provide evidence for an intimate coupling of nerve terminal calcium channels with a plasma membrane component of the SNARE complex, such that modulation of calcium currents by a G-protein coupled receptor cannot occur when syntaxin is cleaved.
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Introduction
Modulation of neurotransmitter secretion by endogenous substances released together with the neurotransmitter is an important control mechanism to fine tune the secretory apparatus (for reviews see Scanziani et al. 1995; Miller, 1998; Silinsky et al. 2001). One important modulator at cholinergic synapses is adenosine, which is a major mediator of prejunctional neuromuscular depression at amphibian (Ribeiro & Sebastiao, 1987; Meriney & Grinnell, 1991; Redman & Silinsky, 1994) and mammalian synapses (Hamilton & Smith, 1991; Nagano et al. 1992; Hirsh & Silinsky, 2002; Hirsh et al. 2002). At amphibian neuromuscular junctions, adenosine derived from neurally released ATP is the mediator of neuromuscular depression at low frequencies of nerve stimulation (Redman & Silinsky, 1994).
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Traditionally, inhibitory effects of neuromodulators had been ascribed to effects on presynaptic ionic channels, i.e. decreases in calcium currents or increases in potassium currents (Miller, 1998). At amphibian nerve endings, it was found that A1 adenosine receptor activation inhibits neurotransmitter secretion from motor nerve endings by an effect on a strategic component of the secretory apparatus and not on membrane ionic channels (Silinsky, 1984; Silinsky & Solsona, 1992; Redman & Silinsky, 1994; Robitaille et al. 1999). This result, whereby neurotransmitter secretion was inhibited downstream of calcium entry, was subsequently confirmed at other vertebrate synapses as well (Scanziani et al. 1995; Trudeau et al. 1998; Miller, 1998; Blackmer et al. 2001).
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In contrast to the results in amphibia, A1 receptor activation in mammals is associated with decreases in nerve terminal calcium currents (Hamilton & Smith, 1991; Silinsky, 2004). Indeed, at the mouse neuromuscular junction, simultaneous decreases in both P/Q-type Ca2+ currents and evoked ACh release were observed (Silinsky, 2004). Whilst these differences between mammalia and amphibia may indicate that prejunctional depression mediated by adenosine is due to different mechanisms in the two species, alternative interpretations are possible. For example, due to a more intimate coupling between Ca2+ channels and the core complex of nerve teminal protein (the SNAREs) in mammals, the effects of adenosine on a SNARE in mammals may be reflected as decreases in Ca2+ currents whilst those in amphibia are not.
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In order to test this hypothesis, the effects of SNARE cleavage on the action of adenosine were examined at mouse neuromuscular junctions. To perform these studies, botulinum toxins, a family of zinc-dependent metalloendopeptidases that block neurotransmitter release by cleaving at highly specific regions of the secretory machinery (Jahn et al. 1995), were used as tools to inactivate specific SNAREs. The results suggest that modulation of Ca2+ currents by adenosine receptor activation is mediated via an interaction with the SNARE, syntaxin.
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Methods
General
Experiments were made on isolated mouse phrenic nerve hemidiaphragm preparations at room temperature (21–23°C) in accordance with the guidelines of the Northwestern University Animal Care and Use Committee and the National Institutes of Health of the US Public Health Service. Specifically, mice (20–30 g) were humanely anaesthetized with 5–10 ml of diethyl ether for 3–5 min. Once the mice were unresponsive to touch, they were exsanguinated. Electrophysiological recordings were made of voltage changes in the perineural space using the perineural recording method (Brigant & Mallart, 1982; Mallart, 1985; Anderson et al. 1988; Silinsky & Solsona, 1992; Protti & Uchitel, 1993; Xu & Atchison, 1996; Silinsky, 2004). For complete details see Silinsky (2004).
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Treatment with botulinum toxins
Preparations were incubated with a specific botulinum toxin serotype and gently rocked in a shaker bath for 1 h. Preparations were then pinned in a tissue bath and stimulated at 1 Hz for approximately 1 h. The period for paralysis of neuromuscular transmission ranged from 40 min to 1 h and 50 min; this reflects the time required to eliminate preformed SNARE complexes at this frequency of stimulation (Raciborska et al. 1998; Kalandakanond & Coffield, 2001).
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With respect to the specific fractions employed, treatment with Botx/C (56 μg ml–1) selectively affects syntaxin immunoreactivity in the mouse phrenic nerve hemidiaphragm preparation and blocks evoked neurotransmitter release (Molgo et al. 1989; Raciborska et al. 1998; Kalandakanond & Coffield, 2001). In these experiments, the 1 Hz stimulation protocol was sufficient to eliminate all residual ACh release from uncleaved SNAREs in times ranging from 55 min and 1 h 20 min (n = 5 experiments). In contrast to the other fractions of Botx, ACh release eliminated by Botx/C could not be retrieved even after adding potassium channel blockers in tubocurarine-free solution in either normal or elevated (8 mM) Ca2+ solutions (n = 20–30 cells in 5 different preparations).
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Treatment with Botx/A (45–56 μg ml–1) selectively cleaves SNAP-25 in mouse phrenic nerve endings (Kalandakanond & Coffield, 2001). The 1 Hz stimulation protocol eliminated ACh release between 1 h and 1 h and 50 min. Both evoked and spontaneous release could be retrieved in TEA/DAP solution but, in contrast to amphibia, the effect was not sustainable. This is possibly due to an order of magnitude lower quantal content in mouse than frog (Hirsh et al. 2002) coupled to a depletion of ACh quanta in the presence of potassium channel blockers (Anderson et al. 1988).
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Botx/D (7.8 μg ml–1) selectively cleaves synaptobrevin at phrenic motor nerve endings (Kalandakanond & Coffield, 2001); complete block of neuromuscular transmission was observed after 40 min to 1 h 10 min of 1 Hz stimulation (n = 5 experiments). Whilst synchronous ACh release reflected as endplate potentials (EPPs) could not be retrieved, increasing the extracellular Ca2+ concentration to 8 mM (in 100 μM TEA) and stimulating at 5–10 Hz, produced sustained increases in asynchronous evoked ACh release reflected as miniature EPP (MEPP) frequencies (evoked MEPP frequencies ranged from 0.92 to 1.9 s–1, n = 4 experiments). These described effects are characteristic of the specific reported effects of the various Botx fractions both electrophysiologically and morphologically (Molgo et al. 1989; Raciborska et al. 1998; Kalandakanond & Coffield, 2001).
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In control preparations incubated with normal, toxin free physiological salt solutions and stimulated at 1 Hz, suprathreshold EPPs (with superimposed action potentials) and MEPPs were detectable for periods as long as 20 h of 1 Hz stimulation (n = 5 experiments).
Physiological saline solutions
Ca2+ currents were measured in a solution consisting of (mM): NaCl 137, KCl 5, CaCl2 2, MgCl2 0–2, NaH2PO4 1, NaH2HCO3 24, dextrose 11, and potassium channel blockers 3,4-diaminopyridine (DAP, 300 μM) and tetraethylammonium (TEA, 10 mM) (pH to 7.4 gassed with 95% O2 and 5% CO2 or buffered with 30 mM Hepes and gassed with 100% oxygen in a phosphate and bicarbonate free solution – see Silinsky, 2004). Botulinum toxin fractions were obtained from Wako Chemicals USA Inc. All other chemicals were purchased from Sigma, St Louis, USA. Paralysis of neuromuscular transmission was achieved by tubocurarine (50 μM) in the control preparations (e.g. Figs 1 and 3) and by botulinum toxins in all other experiments.
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For this figure, neuromuscular transmission was completely blocked by 50 μM tubocurarine (see Silinsky, 2004). A, trace showing prejunctional Na+ and K+ currents (downward deflections) in normal Ca2+ solution prior to application of K+ channel blockers (average response to 15 stimuli, 0.1 Hz). B, superfusion with a solution containing potassium channel blockers produces the blockade of K+ currents and the development of stable prejunctional Ca2+ currents within 10 min of the beginning of application superfusion with standard Ca2+ current solution (this trace represents the average response to 15 stimuli, 0.017 Hz). The first upward deflection that precedes the Na+ and K+ currents in A and the Ca2+ currents in B represent the longitudinal current flowing towards the nerve terminal and passively discharging the membrane capacitance in the region of the recording electrode. Traces in A and B are unfiltered records. C, the typical effect of supramaximal concentrations of adenosine (10 mM) in normal preparations. Control peak 1.37 ± 0.03 mV, average response to n = 16 stimuli (0.017 Hz); adenosine = 1.05 ± 0.03, average response to n = 16 stimuli). Traces in C are filtered at 200 Hz. For details of current polarities and description of the perineural currents, which reflect voltage changes in the extracellular space and are thus calibrated in mV, see Methods and Silinsky (2004).
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In control preparations, neuromuscular transmission was completely blocked by 50 μM tubocurarine whilst for the Botx fractions, transmission was blocked prejunctionally by cleavage of the SNAREs with the particular Botx serotype. No significant differences were observed between the inhibitory effects of adenosine in the control conditions or after Botx/D or Botx/A treatment. Each bar represents the average inhibition produced by adenosine (as a percentage of control) for 5 different experiments on 5 different preparations. The dashed line (no adenosine) shows that supramaximal concentrations of adenosine failed to inhibit the P/Q Ca2+ current after Botx/C treatment. For further details, see text.
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The perineural ionic currents
Details of these recordings in mouse have been published previously (Silinsky, 2004). Briefly, the inward Ca2+ current in the nerve terminals produces a proportional current in the perineural space which flows upstream to the recording electrode (Brigant & Mallart, 1982; Mallart, 1985; Anderson et al. 1988; Protti & Uchitel, 1993; Xu & Atchison, 1996; Silinsky, 2004); the recorded waveform is thus an inverted likeness of the events occurring in the nerve ending. The upward-going perineural voltage change that is antagonized by P/Q-type Ca2+ channel blockers will thus be termed prejunctional Ca2+ current or the presynaptic Ca2+ current as it is produced by Ca2+ currents in the nerve ending flowing across the perineurial resistance.
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Adenosine concentrations and statistical methods
The important aspects of the adenosine dose–response relationships Ca2+ currents and neurotransmitter release have been published previously from this laboratory (Hirsh et al. 2002; Silinsky, 2004). For the sake of comparison with our earlier papers, in all experiments, the effect of a supramaximal concentration of adenosine (10 mM) on nerve terminal Ca2+ currents was examined (see, e.g. Fig. 7 in Silinsky, 2004). Data are presented ± 1 S.E.M. For complete statistical methods, see Silinsky (2004).
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Results
General observations on prejunctional ionic currents and the effects of adenosine in the absence of botulinum toxins
Figure 1A and B depicts the typical presynaptic ionic currents recorded from the perineurium in a curarized preparation in the absence of botulinum toxin treatment. The first downward waveform (Fig. 1A, Na+) is the Na+ current produced by Na+ influx in the nodes of Ranvier and the terminations of the myelin sheaths (a region termed the heminode). The second downward deflection (Figs 1A, K+) represents the delayed rectifier, the voltage-dependent current K+ in the nerve endings (Brigant & Mallart, 1982; Mallart, 1985; Anderson et al. 1988; Xu & Atchison, 1996; Silinsky, 2004). Application of K+ channel blockers eliminates the potassium component of the waveform and reveals the upward going nerve terminal P/Q-type Ca2+ current that triggers evoked ACh release (Fig. 1B, Ca2+). The upward going deflections such as those shown in Fig. 1B are due to the movement of Ca2+ through P/Q-type calcium channels as they are blocked by -agatoxin IVA or Cd2+ (Brigant & Mallart, 1982; Mallart, 1985; Anderson et al. 1988; Protti & Uchitel, 1993; Xu & Atchison, 1996; Silinsky, 2004).
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Depending upon the concentrations of potassium channel blockers and the presence or absence of procaine (which is frequently used to block repetitive firing in the presence of potassium channel blockers – see Xu & Atchison, 1996), the Ca2+ currents may have one or two distinct phases. Figure 1B shows the phasic peak followed by a slower, more sustained second phase. For quantification in this paper, the early phasic peak of the calcium current was employed; this phase is present in all experiments and, when measured simulataneously with evoked ACh release, is the component of the Ca2+ current that is temporally related to promoting the synchronous release of ACh (see Silinsky, 2004; Fig. 3).
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In experiments such as those shown in Fig. 1, preparations were blocked with the nicotinic receptor blocker tubocurarine to prevent muscle twitching in response to nerve stimulation. The data in Fig. 1A and B are also representative of the presynaptic currents observed in preparations blocked by the prejunctional blocking actions of all of botulinum toxin serotypes used in this study (see also Fig. 2). For further details concerning the polarities of these currents and technical details of current measurements, which reflect voltage changes in the perineural space, see Silinsky (2004).
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A, control Ca2+ current after complete blockade of neurotransmitter release by Botx/C (average response to 5 stimuli, 0.017 Hz). B, 10 mM adenosine fails to inhibit Ca2+ currents after Botx/C (average response to 5 stimuli, 0.017 Hz). C, effects of 1 mM Cd2+ to block Ca2+ currents after Botx/C treatment (average response to 16 stimuli, 0.017Hz). For a discussion of the residual outward current, which is not associated with the P/Q Ca2+ current that mediates evoked ACh release see Xu & Atchison (1996) and Silinsky (2004).
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Figure 1C shows the typical effect of supramaximal adenosine concentrations (10 mM) in preparations treated as described in the Methods but in the absence of toxin treatment; in this experiment an inhibition of the Ca2+ current to 76.7% of control is observed. In all experiments in normal Ca2+ solutions, supramaximal concentrations of adenosine inhibited Ca2+ currents to approximately 75% of the control level (75.4 ± 3.9%, mean ± 1 S.E.M. n = 5 experiments, P << 0.05).
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Effects of botulinum toxins on the inhibitory effects of adenosine (10 mM)
Syntaxin is an integral membrane protein SNARE that is linked to presynaptic Ca2+ channels (Catterall, 2000; Jarvis et al. 2000; Spafford & Zamponi, 2003; Dolphin, 2003). Botulinum toxin type C (Botx/C) cleaves syntaxin between residues K253 and A254 near the transmembrane region of syntaxin (Jahn et al. 1995; Sutton et al. 1998). Treating the preparation with Botx/C produced complete blockade of neurotransmitter release at the mouse neuromuscular junction (see Kalandakanond & Coffield, 2001 and Methods for the electrophysiological assessment of SNARE cleavage).
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Figure 2 illustrates the typical experimental result on the action of adenosine after cleavage of syntaxin by Botx/C. In this experiment, the perineural calcium current was unchanged from the control level (1.28 ± 0.07 mV, n = 5) after application of 10 mM adenosine (1.35 ± 0.06 mV, n = 4, P = 0.36). In contrast, the Ca2+ channel blocker Cd2+ produced its typical inhibitory effect on Ca2+ currents after Botx/C treatment (Fig. 2C; see also Silinsky, 2004). Similar results were obtained in experiments on four other preparations. In summary, in all five experiments on five different preparations made after complete blockade of ACh release by Botx/C treatment, no significant inhibitory effect of supramaximal adenosine concentrations on the Ca2+ currents was observed in any experiment. (P values ranged from 0.43 to 0.94 in these individual experiments; the mean Ca2+ current during adenosine treatment as a percentage of control is 101 ± 0.02%.) Figure 3 shows the composite data on the effects of adenosine on normalized Ca2+ currents in the absence of botulinum toxin poisoning (Control) and after cleavage of syntaxin with Botx/C (filled bar).
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To determine if the blockade of the effects of adenosine is due to a non-selective effect of botulinum toxins, or specific to cleavage of syntaxin by Botx/C, the effects of cleavage of SNAP-25 with Botx/A or synaptobrevin with Botx/D were investigated. Botx/A irreversibly cleaves a nine-amino-acid segment of SNAP-25 between amino acids N197 and R198 near the C terminus of the molecule (Jahn et al. 1995; Sutton et al. 1998). This causes a simultaneous elimination of evoked and spontaneous ACh release at neuromuscular junctions due presumably to an alteration of the calcium sensitivity of the secretory apparatus (Molgo et al. 1989; Kalandakanond & Coffield, 2001). After treatment with Botx/A and the elimination of all evoked release the effects of adenosine were examined. After Botx/A, adenosine produced a statistically significant inhibition of Ca2+ currents in each individual experiment (P < 0.05). The mean percentage inhibition after Botx/A treatment was to 81.4 ± 5.1% of the control value (n = 5 experiment). The results of the Botx/A experiments are also summarized in Fig. 3.
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Botx/D cleaves synaptobrevin (VAMP) between K59 and L60 (Jahn et al. 1995) and eliminates the normal evoked release of ACh at vertebrate neuromuscular junctions (Kalandakanond & Coffield, 2001; Molgo et al. 1989). After treatment with Botx/D and complete elimination of all ACh release, the effects of adenosine were examined. As shown in Fig. 3, the mean percentage inhibition after Botx/D treatment was to 83 ± 2.3% of the control level (n = 5 experiments). In these experiments, no statistically significant differences existed between the control preparations, preparations treated with Botx/A or those treated with Botx/D with respect to the maximal inhibitory effects of adenosine (analysis of variance followed by Student's t test using the Bonferroni correction see Glantz, 1992; P values ranged from 0.11 to 0.47).
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Discussion
The results provide evidence that cleavage of the presynaptic membrane SNARE syntaxin by Botx/C, selectively eliminates the inhibitory effects of adenosine but not traditional Ca2+ channel antagonists (e.g. Cd2+). In contrast, no significant differences were observed between the inhibitory effects of adenosine on non-toxin-treated preparations and those completely blocked with Botx/A or Botx/D. These data provide the first evidence from an adult mammalian synapse that an endogenous mediator of presynaptic depression can no longer inhibit Ca2+ currents when a specific SNARE is cleaved.
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It is of interest in this regard that syntaxin has been implicated as an important protein involved in presynaptic modulation via G protein-coupled receptors at non-mammalian synapses; specifically the effects of GTPS to inhibit presynaptic Ca2+ currents in chick ciliary ganglia are blunted after cleavage of syntaxin with Botx/C (Stanley & Mirotznik, 1997).
A model for the interaction of adenosine with mammalian nerve terminals is shown in Fig. 4 which also depicts the cleavage sites of the three fractions of Botx used in this study (for a primitive prelude of this model, see Silinsky, 1986). In Fig. 4A, the voltage-gated Ca2+ channel is depicted with its known interactions with the SNARE syntaxin (red) (Catterall, 2000; Jarvis et al. 2000, 2002; Spafford & Zamponi, 2003; Dolphin, 2003). Binding of exogenous or endogenous adenosine to the A1 adenosine receptor (Figs 1, 4A), produces GTP/GDP exchange on the G subunit (not shown) and leads to the dissociation of the G complex from the GTP-bound G subunit (Figs 2, 4A). The G complex then proceeds to interact with HABC domain of syntaxin (for evidence see Blackmer et al. 2001; Jarvis et al. 2002). This interaction of the G-protein subunits with the N terminus of syntaxin influences the helical core region of this SNARE to interact with the synprint site (pink region) on the P/Q-type Ca2+ channel, which in turn reduces the Ca2+ channel activity and neurotransmitter release. When syntaxin is cleaved with Botx/C (Fig. 4B), the target for G is eliminated, thus preventing adenosine from inhibiting calcium currents and neurotransmitter release. This model is supported both by studies in which GTPS is injected into the presynaptic element of the chick ciliary ganglion (Stanley & Mirotznik, 1997) and by quantitative immunocolocalization of a complex between calcium channels, syntaxin and G-proteins (Li et al. 2004).
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A, sketches of syntaxin (red), SNAP-25 (light green) and synaptobrevin (blue) based upon the crystal structures of these proteins (Sutton et al. 1998). The G-protein cartoon, includes the propeller region of G that is believed to interact with effectors (Ford et al. 1998); this is likely to occur via the N terminal HABC domain of syntaxin (Jarvis et al. 2002). In turn, the Hcore region of syntaxin has been shown to interact with the synprint region of P/Q- and N-type Ca2+ channels (pink). The synprint region and the P/Q Ca2+ channel structure were drawn in accordance with Catterall (2000), Jarvis et al. (2000), Spafford & Zamponi (2003) and Dolphin (2003). B, after cleavage of syntaxin with Botx/C, the target for inhibition by adenosine is no longer present, thus precluding an inhibitory effect of adenosine. B also shows the approximate sites of cleavage by Botx/D on synaptobrevin and Botx/A on SNAP-25.
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These results thus suggest that the presence of intact syntaxin is essential for the modulation of Ca2+ currents by adenosine, rather than merely being a facilitator of G protein-coupled receptor activation. Whilst these data do not exclude the possibility that adenosine receptor activation inhibits ACh release downstream of Ca2+ entry in the mouse, the effects of adenosine on evoked and spontaneous ACh release are eliminated in the presence of Ca2+ channel blockers, making this possibility less likely (De Lorenzo et al. 2004). Furthermore, although the results of the present study do not exclude the possibility that syntaxin helps to localize G protein-coupled receptors to Ca2+ channels, the results of Blackmer et al. (2001) would argue against such a role for syntaxin as a general mechanism for presynaptic modulation.
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Regardless of the precise mode of G-protein receptor coupling, these results suggest that the apparent difference in presynaptic targets for adenosine between mammalian and frog motor nerve endings might reflect a differential coupling between the SNAREs and presynaptic calcium channels rather than a different presynaptic target for neuromodulation (Bezprozvanny et al. 2000; Lu et al. 2001). The differential coupling is unlikely to be due to differences in the types of motor nerve ending calcium channels between amphibia (N type) and mammalia (P/Q type), as presynaptic N-type Ca2+ channels are inhibited by adenosine in chick preganglionic nerve endings (Yawo & Chuhma, 1993). It is more likely that these differences involve intrinsic differences in the relationships between the secretory machinery and the presynaptic calcium channels in the two species. For example, structural studies have suggested that vesicle docking and fusion sites at mouse motor nerve endings are embedded between paired rows of particles believed to be Ca2+ channels, thus providing a morphological correlate of the high apparent degree of coupling between the SNAREs and Ca2+ channels in mammals (Stanley, 1997). In contrast, the vesicle docking and fusion sites in amphibia appear at the edges of the Ca2+ channel zones, possibly reflecting a less intimate coupling of the SNAREs with the depolarization–secretion coupling process in amphibia and providing a morphological correlate for an effect of adenosine that is independent of changes in Ca2+ currents at frog motor nerve endings (Silinsky & Solsona, 1992; Stanley, 1997).
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