The C2A Domain of Synaptotagmin Alters the Kinetics of Voltage-gated Ca2+ Channels Cav1.2 (Lc-type) and Cav2.3 (R-type)*

Roy CohenDagger , Lisa A. Elferink§, and Daphne AtlasDagger

From the Dagger  Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel and the § Department of Physiology & Biophysics and the Marine and Biomedical Institute, University of Texas Medical Branch, Galveston, Texas 77555-1069

Received for publication, October 8, 2002, and in revised form, December 18, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Biochemical and genetic studies implicate synaptotagmin (Syt 1) as a Ca2+ sensor for neuronal and neuroendocrine neurosecretion. Calcium binding to Syt 1 occurs through two cytoplasmic repeats termed the C2A and C2B domains. In addition, the C2A domain of Syt 1 has calcium-independent properties required for neurotransmitter release. For example, mutation of a polylysine motif (residues 189-192) reverses the inhibitory effect of injected recombinant Syt 1 C2A fragment on neurotransmitter release from PC12 cells. Here we examined the requirement of the C2A polylysine motif for Syt 1 interaction with the cardiac Cav1.2 (L-type) and the neuronal Cav2.3 (R-type) voltage-gated Ca2+ channels, two channels required for neurotransmission. We find that the C2A polylysine motif presents a critical interaction surface with Cav1.2 and Cav2.3 since truncated Syt 1 containing a mutated motif (Syt 1*1-264) was ineffective at modifying the channel kinetics. Mutating the polylysine motif also abolished C2A binding to Lc753-893, the cytosolic interacting domain of Syt 1 at Cav1.2 alpha 1 subunit. Syt 1 and Syt 1* harboring the mutation at the KKKK motif modified channel activation, while Syt 1* only partially reversed the syntaxin 1A effects on channel activity. This mutation would interfere with the assembly of Syt 1/channel/syntaxin into an exocytotic unit. The functional interaction of the C2A polylysine domain with Cav1.2 and Cav2.3 is consistent with tethering of the secretory vesicle to the Ca2+ channel. It indicates that calcium-independent properties of Syt 1 regulate voltage-gated Ca2+ channels and contribute to the molecular events underlying transmitter release.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The synaptic vesicle protein Synaptotagmin I (Syt 1),1 is proposed to function as a Ca2+ sensor for neurotransmitter release (1, 2). Consistent with its proposed role as a calcium sensor protein, Syt 1 binds calcium via two repeating structures termed C2A and C2B domains (3).

A role for the C2A and C2B domains of Syt 1 in calcium-triggered neurosecretion is well established (4-7). For example, Ca2+ binding to the C2A domain enhances the association of Syt 1 with several proteins required for neurotransmission including syntaxin 1A (8, 9), SNAP-25 (10, 11), and AP2 (9, 12). Furthermore, Ca2+ binding to the C2A domain promotes its insertion into membranes via an interaction with the acidic phospholipids (8, 9, 13, 14) consistent with the Ca2+ requirements of neurosecretion. Microinjection of recombinant C2A domains and antibodies specific for this region impair neurotransmitter release from neuroendorine PC12 cells (15) and giant squid axons (16). Interestingly, the inhibitory effect of recombinant C2A fragments in PC12 cells occurs independently of its calcium binding properties and is mediated through a novel polybasic motif (17). Thus, the Syt 1 C2A domain contains calcium-dependent and -independent activities, which mediate Syt 1 function during neurotransmitter release. Furthermore, Syt 1 and Syt 4 were recently shown to promote transmitter release independently of Ca2+ binding to the C2A domain (18).

Interactions through the Syt 1 C2B domain are also functionally important for neurosecretion (19-26). Several studies have demonstrated that the activity of Ca2+ channels is modified by syntaxin 1A, Syt 1, and SNAP-25 (27-30). The syntaxin 1A or SNAP-25 inhibitory effects of Cav1.2, Cav2.2, and Cav2.3 activity are reversed by co-expression of Syt 1 (31-34). Recovery of channel activity by Syt 1 was directly proportional to the ratio of Syt 1 and syntaxin 1A, indicating that Syt 1 and syntaxin 1A regulate the Ca2+ channel directly (32, 33). Consistent with this, recombinant proteins comprising the C2A and C2B domains of Syt I bind to the II-III cytosolic domain of the a11.2, a12.1, and alpha 12.2 channel subunits (31-33, 35, 36).

Here we studied the functional interaction of Syt 1 with Cav1.2 and Cav2.3 by examining the relative contribution of the C2A polylysine motif on channel activity and binding to the cytosolic domain Lc753-893 of the alpha 11.2 of Cav1.2. Our data indicate that the C2A domain of Syt 1 modulates the activation kinetics of Cav1.2 and Cav2.3. Mutation of the C2A polylysine motif abolished the binding to the cytosolic interaction domains of the channel. Moreover, this mutation altered the modulatory effect of Syt 1 on Cav1.2 and Cav2.3 activity, impairing the ability of Syt 1 to reverse the syntaxin 1A inhibition of channel activity.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
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alpha 11.2 (dN60-del1773; X15539) rat beta 2A (m80545); alpha 12.3 subunit cloned into pHBE239 (L27745) were obtained from Dr. L. Birnbaumer; alpha 2/delta rabbit skeletal (M86621) from A. Schwartz . Rat Syt 1 was obtained from M. L Bennett. The in vitro transcription kit was from Stratagene. Anti-syntaxin antibody was a kind gift of M. Takahashi and was prepared by us; anti-Syt 1 antibody was from Sigma; Anti-Lc753-893 antibody (32). CM5 sensor chip, N-hydroxysuccinimide, N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), and ethanolamine-HCl were purchased from Biacore, AB (Uppsala, Sweden). Glutathione-agarose 4B beads were from Amersham Biosciences.

Syt 1 Mutants-- Syt 1* (K189A/K190A/K191A/K192A) was prepared by insertion of the Eco47III-PflM fragment into Syt 1. Syt 11-264* (K189A/K190A/K191A/K192A) was prepared by excising the C2B domain (amino acids 265-421) and religating the PflMI-XbaI fragment in directional cloning.

cRNA Injection and Protein Expression in Xenopus Oocytes-- Stage V-VI oocytes were removed and defolliculated by collagenase (type I) treatment as described (37). Oocytes were injected with cRNA of alpha 11.2 or alpha 12.3 (5 ng/oocyte), alpha 2delta 1 (5 ng/oocyte), beta 2a (10 ng/oocyte), and a day later with Syt 1, Syt 1*, Syt 11-264, or Syt-1*1-264 (5 ng/oocyte). After cRNA injection, oocytes were maintained for 6 days at 19 °C in ND96 solution (mM): 96 NaCl/2 KCl/1 MgCl2/1.8 CaCl2/2.5 sodium pyruvate/5 HEPES, pH 7.4, with antibiotics. Plasmid DNA for the channel subunits, alpha 11.2 (alpha *1C dN60-del1773), alpha 12.3 (alpha 1E), alpha 2delta 1alpha , beta 2a, syntaxin 1A, Syt 1, Syt 1*, and Syt 11-264, were linearized and transcribed in vitro using T7 or T3 polymerase (Stratagene kit) in the presence of the cap analog G (5') ppp (5') G (Amersham Biosciences). The in vitro transcribed capped cRNAs were injected into oocytes at a final volume of 40 nl per oocyte. Channel subunits alpha 11.2 or alpha 12.3, alpha 2delta 1, and beta 2A were injected 1 day prior to injection of synaptic proteins.

Electrophysiological Assays-- Whole cell currents were recorded at room temperature (20-24 °C) by applying a standard two-microelectrode voltage clamp using a Dagan 8500 amplifier. Voltage and current agar-cushioned electrodes (0.3-0.6 MOmega ) were filled with 3 M KCl (32). Current-voltage relationships were determined by voltage steps as indicated in the legend to figures, in Ba2+ solution (mM): 5 Ba(OH)2/50 N-methyl-D-glucamine/1 KOH/40 tetraethylammonium/5 HEPES, pH 7.5 and titrated to pH 7.5 with (CH3)2SO4. The activation kinetics was determined from leak-subtracted current traces by a mono-exponential fit of the pClamp8 software (Axon Inst.). The activation time constants were determined by fitting the raw current data with the equation: I(t) = Imax [1-exp(t/tau act)], where I(t) indicates the amplitude of current at time t, Imax is the maximum amplitude, and tau act is the time constant for activation. Each trace was fitted separately according to Boltzmann, and the averaged values were plotted. Cav1.2 activation was fitted to single exponential function, while a two exponential function nicely described the data of Cav2.3 time course. Data presentation was done using Origin 6 software (Microcal). All quantitative results are given as the mean ± S.E. (n = 6-10)

Protein Expression-- Protein expression in oocytes was tested for by Western analysis 5-7 days after cRNA injection. Oocytes were homogenized in buffer containing (in mM): 1 EDTA/250 sucrose/10 Tris-HCl, pH 7.0, and addition of a mixture of protease:aprotinin, phenylmethylsulfonyl fluoride, iodoacetamide, pepstatin A, and leupeptin at 4 °C. Homogenates were centrifuged (12,000 × g, 10 min); the pellet was discarded and supernatant was collected. Protein was determined by a micro-Bradford assay in enzyme-linked immunosorbent assay-reader plate using bovine serum albumin as standard (38). Protein samples (30 µg) mixed with 100 mM dithiothreitol and 2% SDS, boiled 3 min, applied to 10% SDS-PAGE, transferred to nitrocellulose, and probed using affinity-purified monoclonal anti-syntaxin 1A (Sigma) followed by a horseradish peroxidase-conjugated anti-mouse antibody. Syntaxin 1A expression was detected by enhanced chemiluminescence (ECL system).

Affinity Determination Using the Surface Plasmon Resonance Spectroscopy and GST Binding Assays-- The affinity of Lc753-893, the II-III loop that links domains II-III of the CaV1.2 alpha 1 subunit (32) and GST-C2A wt (31) or GST-C2A* mutant (17) was determined using (i) Biacore 3000 system (Biacore AB) based on surface plasmon resonance methodology and (ii) glutathione-S-transferase (GST) binding assay using GST-agarose beads.

Purified His6-tagged Lc753-893 was immobilized on a research-grade CM5 sensor chip in a flow cell coated with carboxyl-methyl dextran as the surface matrix using activated carboxyl groups and EDC coupling in HBS-EP buffer (150 mM NaCl, 3.4 mM EDTA, and 0.005% (v/v) 10 mM Hepes, pH 7.4, and surfactant P20) at a flow rate of 10 µl/min. The surface was activated for 7 min with a mixture of N-hydroxysuccinimide (0.05 M) and EDC (0.02 M). His6-tagged Lc753-893 was injected at a concentration of 20 µg/ml in 10 mM sodium acetate, pH 3.5, until the desired level of binding was achieved. Ethanolamine (1 M, pH 8.5) was injected for 7 min to block the remaining activated groups. Control flow-cell surface was prepared by activating and then deactivating (blocking) the carboxyl groups as mentioned above. His6Lc753-893 binding studies to wild type and mutant C2A domains were initiated by passing the recombinant fusion proteins GST-C2A wild type, GST C2A* mutant, and GST alone at increasing concentrations as indicated through the flow cells at a rate of 20 µl/min in HBS-EP running buffer. Surface regeneration was carried out after each binding assay by a 10-µl pulse of 1 M NaCl in 10 mM NaOH. The data were analyzed using the Kinetics Wizard of the Biacore control software with automatic corrections for nonspecific binding by subtraction of the responses obtained for the control surface from the data obtained. The kinetics of binding and affinity constants were calculated using the Biaevaluation software.

Binding of the cytoplasmic domain of Lc753-893 (100 nmol) to GST fusion proteins, Syt 1, C2A, C2B, C2A*, and GST alone (100 pmol) using glutathione-agarose 4B beads (25 µl) was performed as described (31, 32). Immunoblots were probed using affinity-purified anti-Lc753-893 antibody and visualized by enhanced chemiluminescence (ECL system).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The C2A Domain of Syt 1 Is Required for Functional Interactions with the Voltage-gated Ca2+ Channels, Cav1.3-- Functional interactions of voltage-gated Ca2+ channels with the full-length Syt 1 have been previously described using the Xenopus oocytes expression system. To assess the role of the C2A polylysine motif (amino acids 189-192) on Syt 1 interactions with the Ca2+ channel, the C2A polylysine motif was substituted with alanine residues in full-length Syt 1 (Syt 1*) or a truncated form of Syt 1 (Syt 11-264) lacking the C2B domain (Fig. 1).


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Fig. 1.   Schematic presentation of Syt 1 and the Syt 1 mutants. Mutations at the C2A polylysine motif of synaptotagmin (Syt 1) were generated by substituting 189KKKK with Ala residues (Syt 1*) as indicated. The two truncated Syt mutants aa 1-164, Syt 11-264, and the truncated mutant bearing the same mutation at 189KKKK to Ala, Syt 1*1-264, are shown.

Cav1.2 currents were elicited in oocytes co-expressing the three-channel subunits alpha 11.2/beta 2a/alpha 2delta 1 with Syt 1 or Syt 1* from a holding potential of -80 mV to test potentials between -30 and +45 mV in response to 160-ms test pulse (Fig. 2). Peak current amplitudes were not affected by Syt 1 (35) or Syt 1, as demonstrated by current-voltage relationship (Fig. 2A). The activation component of Cav1.2 current was measured at each test pulse and was fitted with a single exponential function between the lines marked by asterisks (Fig. 2B). Under these experimental conditions both Syt 1 and Syt 1* slightly reduced activation rate at voltage range of -15 to -5 mV, while at more positive potentials tau act approached control values (Fig. 2C; Table I). Lysates of oocytes co-injected with Syt 1, Syt 1*, and Cav1.2 were prepared and analyzed for Syt 1/Syt *1 expression by Western analysis using anti-Syt 1 antibody (see "Experimental Procedures"). As shown in Fig. 2D no significant difference in the expression of Syt 1 and Syt 1* in injected oocytes was observed.


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Fig. 2.   Syt 1 and Syt 1* interact with Cav1.2 (Lc-channel). Oocytes injected with cRNA of alpha 11.2 (5 ng/oocyte), alpha 2delta 1 (5 ng/oocyte), beta 2a (10 ng/oocyte), and a day later with Syt 1 (5 ng/oocyte) or Syt 1* (5 ng/oocyte) are shown. Inward Ba2+ currents were elicited from a holding potential of -80 mV in response to a 160-ms pulse by voltage steps to potentials between -30 and +45 mV in 5-mV increments. A, leak-subtracted peak current-voltage relationship: collected data from oocytes expressing the three channel subunits (open circle ) together with Syt 1 (black-down-triangle ) or Syt 1* (down-triangle). The data points correspond to the mean ± S.E. of current amplitude (n = 8). B, the activation component of a typical current produced at each test pulse was fitted with a single exponential function between the lines marked by asterisks. C, the averaged time constants of activation (tau act, mean ± S.E., n = 6) are plotted against test pulses in the absence (open circle ) and in the presence of Syt 1 (black-down-triangle ) and Syt 1* (down-triangle). Two sample Student's t tests were applied, and p values <0.05 were obtained from the two-tailed tests. D, protein expression of Syt 1 and Syt 1* was determined at day 5 following cRNA (5 ng/oocyte) injection into Xenopus oocytes. The proteins were separated on 10% SDS-PAGE, transferred to nitrocellulose membrane, and subjected to Western analysis using anti-Syt 1 antibody and ECL detection.

                              
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Table I
Differential effect of syntaxin 1A Syt 1 wild type and Syt 1 mutants on the kinetic parameters of Cav 1.2 and Cav 2.3 
Whole cell Ba2+ currents were evoked from a holding potential of -80 mV by a single voltage step to test potentials as indicated by the superscripts. Values are mean ± S.D. for (n = 6-10). Test pulse duration for Cav1.2 was 160 ms and 80 ms for Cav2.3. peak, peak current; tau act, time constant of activation. Test potentials are depicted by superscripts a, b, c, d.

Syt 1 and Syntaxin 1A Are Functionally Coupled to Cav1.2 Activity-- We previously demonstrated that Cav1.2 as well as Cav2.2 (neuronal N-type channel) activities are inhibited with co-expression of syntaxin 1A (28, 32, 37). Since the inhibitory effect of syntaxin 1A on these channels is reversed by Syt 1 (31, 32, 33) we next examined the Syt 1* mutant for reversal of syntaxin 1A inhibitory effects on channel activity. Fig. 3 shows the results of co-expressing Cav1.2 and syntaxin 1A with Syt 1 and Syt*1 in Xenopus oocytes. Superimposed traces of macroscopic whole cell Ba2+ currents showed an 80% inhibition of current amplitude by syntaxin 1A, which was fully reversed in the presence of Syt 1 and partially by Syt 1* (Fig. 3A; Table I). Furthermore, peak current amplitudes normalized to maximum current (I/Imax) showed a large voltage shift in the half-maximal voltage (V1/2) induced by syntaxin 1A from V1/2 = -21 ± 1.2 mV to V1/2 = -7.5 ± 1.8 mV. This voltage shift was reverted to V1/2 = -18.6 ± 2 mV by Syt 1 and -19 ± 2.2 mV by Syt 1* (Fig. 3B). Similarly, a complete reversal of the syntaxin 1A effect on Cav1.2 activation was observed with co-expression of Syt 1 (Fig. 3C), and only partial reversal by Syt 1* (Fig. 3D). The mutation at the polylysine motif impaired Syt 1* capacity to reverse the inhibitory effects of syntaxin 1A on Cav1.2 current amplitude and activation kinetics.


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Fig. 3.   Syt 1 and Syt 1* interact with Cav1.2 in the presence of syntaxin 1A. A, superimposition of macroscopic alpha 11.2, alpha 2delta 1, and beta 2a currents evoked from a holding potential -80 mV by a single voltage step (160 ms) to +20 mV in oocytes expressing the three-channel subunits in various combinations as indicated. B, peak-current amplitudes (data not shown) normalized to maximum current (I/Imax) plotted against test potentials were fitted according to Boltzmann; channel subunits (open circle ) with syntaxin 1A (), syntaxin 1A and Syt 1 (black-down-triangle ), or syntaxin 1A and Syt 1* (down-triangle). The mid-point of activation (V1/2) and Boltzmann slope (k) of alpha 11.2/alpha 2delta 1/beta 2a were V1/2 = -21.6 ± 1.2 mV, k = 2.9 ± 1.9; with syntaxin 1A, V1/2 = -7.5 ± 1.75 mV, k = 7.2 ± 1.44; with syntaxin 1A and Syt 1 V1/2 = -18.6 ± 2.1 mV, k = 2.4 ± 0.8; and with syntaxin 1A and Syt 1*, V1/2 = -19.0 ± 2.2 mV and k = 3.9 ± 2.1. C, the activation time constants (tau act, mean ± S.E., n = 8) are plotted against test pulses between -10 and +30 mV; the channel alone (open circle ), with syntaxin 1A (), with syntaxin 1A and Syt 1 (black-down-triangle ) and D, with syntaxin 1A and Syt 1* (down-triangle). Two sample Student's t tests were applied, and p values <0.05 were obtained from the two-tailed tests. See Fig. 2 for cRNA/oocyte of channel subunits, syntaxin 1A (2 ng/oocyte).

Cav1.2 Interacts with Syntaxin 1A and the Truncated Syt 11-264 and Syt 1*1-264 Mutants-- The partial reversion of the syntaxin 1A effect on Cav1.2 activation by Syt 1* compared with Syt 1 suggests that the polylysine C2A motif couples Syt 1 to channel activation (Fig. 3, C and D). To isolate the contribution of C2A domain we co-expressed truncated Syt 1 lacking the C2B domain (Syt 11-264) with Cav1.2 and syntaxin 1A. Cav1.2 whole cell currents were activated from a holding potential of -80 to 0 mV test pulse (Fig. 4A). Both the superimposed traces as well as the current-voltage relationships (Fig. 4, A-C) showed diminished current amplitudes by syntaxin 1A that were only partially reversed by Syt 11-264 and Syt 1*1-264. Syt 1*1-264 was significantly less effective than Syt 11-264. Furthermore the large shift in the half-maximal voltage induced by syntaxin 1A, (see above) was shifted back to V1/2 = -20.6 ± 2.3 mV by Syt 11-264 and only to -12.9 ± 3 mV by Syt 1*1-264 (Fig. 4D).


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Fig. 4.   Syt 11-264 and Syt 1*1-264 interact with Cav1.2 in the presence of syntaxin 1A. Inward Ba2+ currents were elicited in oocytes co-expressing alpha 11.2, alpha 2delta 1, beta 2a, syntaxin 1A, and syntaxin 1A with Syt 11-264 and syntaxin 1A with Syt 1*1-264 from a holding potential of -80 mV by voltage steps of 160 ms applied in 5-mV increments at potentials between -30 and +45 mV. A, superposition of macroscopic alpha 11.2, alpha 2delta 1, and beta 2a currents activated from a holding potential -80 by a single voltage step of 160 ms to a test potential of 0 mV in various combinations as indicated. B, leak-subtracted peak current-voltage relationship: collected data from oocytes expressing the three-channel subunits (open circle ) with syntaxin 1A (), syntaxin 1A and Syt 11-264 (black-square), or C, syntaxin 1A and Syt 1*1-264 (). The data points correspond to the mean ± S.E. of current amplitude (n = 7). D, peak current amplitudes normalized to maximum current (I/Imax) are plotted against test potentials (data from B and C) and were fitted according to Boltzmann equation. The mid-point of activation (V1/2) and the Boltzmann slope (k) of alpha 11.2/alpha 2delta 1/beta 2a were V1/2 = -21.6 ± 1.2 mV, k = 2.9 ± 1.9, with Syt 11-264 V1/2 = -20.6 ± 2.3 mV, k = 5.1 ± 2.89, and with Syt 1*1-264 V1/2 = -12.9 ± 3.1 mV, k = 5.4 ± 2.1. E, activation time constants of the channel (tau act, mean ± S.E., n = 6) are plotted against test potentials (open circle ) with syntaxin 1A (), syntaxin 1A and Syt 11-264 (black-square) and F, syntaxin 1A and Syt 1*1-264 (). Two sample Student's t tests were applied, and p values <0.05 were obtained from the two-tailed tests. (See legend to Fig. 2 for cRNA injected per oocyte,)

A more striking difference between Syt 11-264 and Syt 1*1-264 was observed on channel activation (Fig. 4, E and F). The marked slowing effect of activation kinetics by syntaxin 1A was fully reversed by Syt 11-264, (Fig. 4C; Table I). In contrast, Syt 1*1-264 was completely ineffective (Fig. 4E). Together, these results suggest the involvement of the C2A polylysine motif in the interaction with the channel.

K189-192A Mutations Abolish Lc753-893 Binding to the C2A Domain of Syt 1-- The polylysine motif (189-192) at the C2A domain is exposed on the surface of the beta -sandwich of Syt 1 where they are accessible for interacting with potential effector molecules (7). To determine whether the loss of functional interaction with the channel is related to impaired binding to Lc743-893, the II-III linker of the Cav1.2 alpha 1 subunit (32) black-square. Two types of binding studies of C2A and mutant C2A* domains were preformed. (i) Recombinant GST-C2A, GST-C2A*, GST-Syt 1, and GST proteins were immobilized to GSH-agarose beads and incubated with equimolar concentrations of recombinant His6Lc753-893 (0.5 µM; 2.5 µg). As shown by Western analysis using anti-Lc753-893 antibody (32), C2A, C2B, and Syt 1 bind Lc753-893, but no binding of C2A* was observed (Fig. 5A). (ii) The affinity of C2A and C2A* to Lc753-893 was tested using the Biacore technology (Biacore; see "Experimental Procedures"). His6Lc753-893 was immobilized on a sensor chip surface. Recombinant samples of GST-C2A, GST-C2A* at the indicated concentrations were injected into the flow cell of the system (Biacore), and changes in resonance units were recorded as a function of time to yield sensorgrams as shown in Fig. 5B. The C2A binding to Lc753-893 is manifested as large amplitude of the surface plasmon resonance signal while no resonance signal was obtained by C2A*. GST alone showed no binding (data not shown). The calculated affinity of C2A was O.213 µM (chi 2 = 4.06; Fig. 5B). Hence the C2A mutant does not bind to the intracellular domain of the channel that comprises the Syt 1 interaction (31, 32, 35, 40, 47).


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Fig. 5.   Direct interaction of C2A and C2A* with Cav1.2 cytosolic domain Lc753-893. Lc753-893 binding to Syt 1 and C2 domains. A, GST fusion proteins (100 pmol) were immobilized on GSH-agarose beads and incubated for 60 min with His6Lc753-893 (250 pmol). After extensive washing, the remaining bound proteins were eluted with 15 mM GSH, separated on 10% SDS-PAGE, transferred to nitrocellulose, and subjected to Western analysis by using anti-Lc753-893 antibody and detected by ECL. His6Lc753-893 (30 ng) was used as a marker. B, sensorgram of C2A and C2A* interaction with Lc753-893 obtained by surface plasmon resonance spectroscopy. The solution of recombinant GST C2A, GST C2A*, and GST at concentrations as indicated was flowed over immobilized His6Lc753-893, and the interaction at the surface was recorded. The apparent equilibrium dissociation constant for C2A (Kd = 0.213 µM; chi 2 = 4.06) was calculated from the ratio of the dissociation and association rate constants (koff/kon). RU, resonance units.

The C2A Domain of Syt 1 Is Required for Functional Interactions with Cav2.3 (R-channel)-- Cav2.3 currents were elicited in oocytes co-expressing alpha 12.3/beta 2a/alpha 2delta 1 subunits (41) and Syt 1 or Syt 1* (Fig. 6). Syt 1 or Syt 1* modified neither Cav2.3 current-voltage relationship nor peak-current amplitude (Fig. 6A). Conversely, Syt 1 strongly accelerated Cav2.3 activation in the range of -20 to +5 mV, converging at more depolarized values (>5 mV) (Fig. 6B; Table I; Ref. 30). Interestingly, Syt 1 was previously shown to accelerate the activation kinetic of Cav2.2 (N-type channel; Ref. 28). The effect of Syt 1* on Cav2.3 was more complex, showing mixed effects of this mutant on channel activation (Fig. 6C). At negative potentials between -20 and -10 mV, the rate was accelerated by Syt 1* similar to Syt 1, but between -5 and 0 mV an abrupt decrease in the rate was observed, which was slower than the channel (Fig. 6D). At more positive potentials, in the range of +5 to +30 mV, tau act approached control values (Fig. 6, C and D).


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Fig. 6.   Syt 1 and Syt 1* interact with Cav2.3 (R-channel). Oocytes were injected with cRNA of alpha 12.3 (5 ng/oocyte), alpha 2delta 1 (5 ng/oocyte), beta 2a (10 ng/oocyte), and a day later, with Syt 1 (5 ng/oocyte) or Syt 1* (5 ng/oocyte). Inward Ba2+ currents were elicited from a holding potential of -80 mV in response to an 80-ms pulse to various test potentials between -30 and +45 mV in 5-mV increments. A, leak-subtracted peak current-voltage relationship: collected data from oocytes expressing the three channel subunits (open circle ) together with Syt 1 (black-down-triangle ) or Syt 1* (down-triangle). The data points correspond to the mean ± S.E. of current (n = 8). B, the activation component of a typical current produced by a test pulse was fitted with a single exponential function between the lines marked by asterisks and was applied to determine the time constant of activation (tau act). C, activation time constants (tau act, mean ± S.E., n = 6) are plotted against potentials between -20 and +30 mV in the absence (open circle ) and in the presence of Syt 1 (black-down-triangle ) or D, Syt 1* (down-triangle). Two sample Student's t test were applied, and p values <0.05 were obtained from the two tailed tests

Activation of Cav2.3 Requires the C2A Polylysine Motif-- We next examined the requirement of the Syt 1 C2A polylysine motif on channel activation using the truncated Syt 1 mutants. Cav2.3 currents were elicited in oocytes co-expressing the three channel subunits along with Syt 11-264 and Syt 1*1-264. In both mutants the C2B domain is missing, and in Syt 1*1-264 the polylysine motif was substituted with alanine residues. The effects of the truncated mutants on channel activity are shown in Fig. 7, A and B.


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Fig. 7.   Syt 11-264 and Syt 1*1-264 interact with Cav2.3. Inward Ba2+ currents were elicited in oocytes co-expressing alpha 12.3, alpha 2delta 1, and beta 2a along with Syt 11-264 and Syt 1*1-264 in response to voltage steps of 80 ms to test potentials between -30 and +40 mV in 5-mV increments. Holding potential was -80 mV. A, superimposition of alpha 12.3, alpha 2delta 1, and beta 2a current traces, either alone or in combination with Syt 11-264 and Syt 1*1-264, activated from a holding potential of -80 mV in response to a single voltage step to a 0-mV test pulse. B, leak-subtracted peak current-voltage relationship: collected data from oocytes expressing the three channel subunits alone (open circle ), with Syt 11-264 (black-square) or C, with Syt 1*1-264 (). The data points correspond to the mean ± S.E. of current (n = 8). D, peak current-amplitudes normalized to maximum current (I/Imax) are plotted against test potentials (data from B and C) were fitted according to Boltzmann. The mid-point of activation (V1/2) and the Boltzmann slope (k) of alpha 12.3/alpha 2delta 1/beta 2a were V1/2 = -8.8 ± 0.1 mV, k = 2.3 ± 0.1; with Syt 11-264 V1/2 = 0.5 ± 0.1 mV, k = 2.9 ± 0.1; and with Syt 1*1-264 V1/2 = -6.2 ± 0.1 mV and k = 2.5 ± 0.1. E, activation time constants (tau act, mean ± S.E., n = 6) are plotted against test potentials in the absence (open circle ) and in the presence of Syt 11-264 (black-square) and F, Syt 1*1-264 (). Two sample Student's t tests were applied, and p values <0.05 were obtained from the two-tailed tests. cRNA of channel subunits injected per oocyte, see Fig. 5; Syt 1*1-264 (5 ng/oocyte); Syt 11-264 (5 ng/oocyte).

Superimposed traces of whole cell current were activated from a holding potential of -80 mV by a single voltage step to 0-mV test pulse (Fig. 7A). Syt 11-264 appeared to inhibit current amplitude by 60% at 0 mV, while Syt 1*1-264 displayed no effect on current amplitude but significantly slowed channel inactivation (inactivation kinetics were not explored in the present study). Current-voltage relationships were significantly shifted in the presence of Syt 11-264 but not Syt 1*1-264 (Fig. 7, B and C). Peak current amplitudes normalized to maximum current (I/Imax) show that the half-maximal voltage activation (V1/2) was significantly displaced by Syt 11-264 from -8.8 ± 0.1 mV to 0.5 ± 0.1 mV and only marginally to -6.2 ± 0.1 mV by Syt 1*1-264, (Fig. 7D). This voltage shift can account for the apparent reduction in current amplitude. The slope factors were directly comparable between control conditions and those expressing Syt 11-264 or Syt 1*1-264 (Fig. 6D). Syt 11-264 was also efficient at accelerating Cav2.3 activation in the -20 to -5 mV range similar to full-length Syt 1 (Fig. 7E; Fig. 6C). In contrast, the acceleration of activation by Syt 1*1-264 was smaller and was detected only in -20 to -10 mV range (Fig. 6F). The effects of Syt 11-264 and Syt 1*1-264 on Cav2.3 kinetics were specific for this channel as co-expression of these proteins result in no effect on Cav1.2 activation kinetics (see Table I). Together, these data suggest that Cav2.3 activation involves the C2A domain of Syt 1. Moreover, mutation of the polylysine motif modifies the interaction of the C2A domain with the channel.

A Cross-interaction of Syntaxin 1A with Syt 1 Mutants and Cav2.3-- Superimposed traces of macroscopic whole cell Cav2.3 current elicited from a holding potential of -80 mV by a single voltage step to a 0-mV test pulse showed a partial reversal by Syt 1 (from 50% to 18%) of the syntaxin 1A-mediated current inhibition but not by Syt 1* (Fig. 8A). Current-voltage relationships obtained in the presence of syntaxin 1A or syntaxin 1A with either one of the Syt 1 mutants indicated a shift toward more positive potentials by Syt 1* (Fig. 8, B and C) that could account for the reduction in current amplitude at 0 mV (Fig. 7A). Peak current amplitudes normalized to maximum current (I/Imax) showed no shift in the half-maximal voltage of Cav2.3 (V1/2 = -8.8 ± 0.1 mV) by syntaxin 1A (V1/2 = -8.0 ± 2.1 mV) (Fig. 8D), unlike the large shift induced by syntaxin 1A in Cav1.2 (Fig. 3B). In the presence of syntaxin 1A, V1/2 was shifted toward more positive potentials to -4.3 ± 1.2 mV by Syt 1 and to -0.4 ± 0.7 mV by Syt 1*, (Fig. 8D). Hence, Syt 1 and Syt 1* differently modify the syntaxin-associated channel.


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Fig. 8.   Syt 1 and Syt 1* modify Cav2.3 properties in the presence of syntaxin 1A. A, superposition of macroscopic alpha 12.3, alpha 2delta 1, and beta 2a current traces evoked in response to an 80-ms pulse from a holding potential of -80 mV by a single voltage step to a 0-mV test pulse in oocytes co-expressing the three-channel subunits alone and together with either Syt 1 or Syt 1*. B, leak-subtracted peak current-voltage relationship: collected data from oocytes expressing the three-channel subunits (open circle ) with syntaxin 1A (), syntaxin 1A and Syt 1 (black-down-triangle -), and C, syntaxin 1A and Syt 1* (down-triangle). The data points correspond to the mean ± S.E. of current (n = 8). D, Peak current amplitudes normalized to maximum current (I/Imax) (data from B and C) are plotted against test potentials displayed with a Boltzmann fit. The mid-point of activation (V1/2) and the Boltzmann slope (k) of alpha 12.3/alpha 2delta 1/beta 2a were V1/2 = -8.8 ± 0.1 mV, k = 2.3 ± 0.1; with Syt 1 V1/2 = -2.8 ± 0.8 mV, k = 3.9 ± 0.25, and with Syt 1*1-264 V1/2 = -2.7 ± 1.38 mV, k = 4.2 ± 0.4. E, the activation time constants (tau act, mean ± S.E., n = 6) are plotted against test potentials between -20 and +25 mV: the channel alone (open circle ), with syntaxin 1A (), syntaxin 1A and Syt 1 (black-down-triangle ), or F, syntaxin 1A and Syt 1* (down-triangle). Two sample Student's t tests were applied, and p values <0.05 were obtained from the two-tailed tests. cRNA/oocyte, see Fig. 3; Syntaxin 1A (2 ng/oocyte).

Cav2.3 activation was accelerated in cells expressing syntaxin 1A and was not modified further by Syt 1 (Fig. 8E). In contrast, Syt 1* slowed the activation kinetics in the presence of syntaxin 1A, in particular in potentials between 0-15 mV (Fig. 8F). Thus, mutation of the polylysine motif increased the current voltage shift of Cav2.3 and was less effective than Syt 1 at reversing the syntaxin 1A inhibition. In addition, the mutation appeared to affect the interaction of the channel with syntaxin 1A, slowing the activation kinetics. Together, these data suggest that the Syt 1 C2A polylysine motif participates in the syntaxin 1A modulation of Cav2.3 activation.

Since Syt 1 and Syt 1 mutants affect syntaxin 1A modulation of the channel, their effect on syntaxin 1A expression in oocytes was tested (Fig. 9). Oocytes were injected with syntaxin 1A cRNA (5 ng/oocyte) and cRNA encoding the various Syt 1 mutants (5 ng/oocyte) as indicated. At day five after injection, oocytes were lysed and proteins were separated on SDS-PAGE and analyzed using monoclonal anti-syntaxin 1A antibody (Fig. 9). As shown by the Western blot analysis there were no significant changes in syntaxin 1A expression in the presence of either one of the four Syt 1 mutants (Fig. 9).


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Fig. 9.   The effect of Syt 1 wild type and mutants on syntaxin 1A expression in oocytes. Xenopus oocytes were injected with cRNA of alpha 12.3 (5 ng/oocyte), alpha 2delta 1 (5 ng/oocyte), beta 2a (10 ng/oocyte), and alpha 11.2 (5 ng/oocyte) with the corresponding subunits, syntaxin 1A (5 ng/oocyte), and with Syt 1, Syt 1*, Syt 11-264, or Syt 1*1-264 (5 ng/oocyte) as indicated. Five days later, oocytes were lysed and proteins were separated on 10% SDS-PAGE and transferred to a nitrocellulose membrane. The level of syntaxin 1A expressed in the oocytes was determined in a Western analysis by using monoclonal anti-syntaxin 1A antibody and detection by ECL.

The contribution of the KKKK motif to the channel interaction with syntaxin 1A was examined by using the two truncated Syt 1 mutants lacking the C2B domain. Currents were evoked from a holding potential of -80 mV to 0 mV test pulse (Fig. 10A). As shown current amplitude was reduced by syntaxin 1A and was partially reversed by the two mutants. Current-voltage relationships showed that Syt 11-264 and Syt 1*1-264 were equally effective at reverting syntaxin 1A inhibition of Cav2.3 current amplitude (Fig. 10B). The half-maximal voltage (V1/2) of the channel was not affected by syntaxin 1A (see Fig. 8D), but a small shift toward more positive potentials was observed by Syt 11-264 to 2.7 ± 1.4 mV and to -2.8 ± 0.8 mV by Syt 1*1-264(Fig. 10C). Syntaxin 1A accelerated Cav2.3 activation (Fig. 10D), which was further increased in the presence of Syt 11-264 (Fig. 10E). In contrast, Syt 1*1-264 lost the ability to accelerate Cav2.3/syntaxin 1A activation (Fig. 10F). Together, these data show that the mutant failed to modify Cav2.3 interaction with syntaxin and suggest that the C2A polylysine motif participates in the syntaxin 1A cross-talk with Cav2.3.


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Fig. 10.   Syt 11-264 and Syt 1*1-264 modify Cav2.3 kinetics in the presence of syntaxin 1A. A, superposition of macroscopic alpha 12.3, alpha 2delta 1, and beta 2a current traces evoked in response to an 80-ms pulse from a holding potential -80 mV by a single voltage step to a 0-mV test pulse in oocytes co-expressing the three-channel subunits alone and together with Syt 11-264 or Syt 1*1-264 as indicated. B, leak-subtracted peak current-voltage relationship: collected data from oocytes expressing the three-channel subunits (open circle ), syntaxin 1A (), or with syntaxin 1A and Syt 11-264 (black-square) or syntaxin 1A and Syt 1*1-264 (). The data points correspond to the mean ± S.E. of current amplitude (n = 9). C, peak current amplitudes normalized to maximum current (I/Imax) (data from B) are plotted against test potentials and displayed with a Boltzmann fit. The mid-point of activation (V1/2) and the Boltzmann slope (k) of alpha 12.3/alpha 2delta 1/beta 2a were V1/2 = -8.8 ± 0.01 mV, k = 2.3 ± 0.1; with syntaxin 1A, V1/2 = -8 ± 2.1mV, k = 3.9 ± 0.7; with syntaxin 1A and Syt 11-264, V1/2 = -2.8 ± 0.8 mV, k = 3.9 ± 0.25; and with syntaxin 1A and Syt 1*1-264 V1/2 = -2.7 ± 1.4 mV, k = 4.2 ± 0.4. D, the activation time constants (tau act, mean ± S.E., n = 6-8) of the channel (open circle ) or with syntaxin 1A (-) were plotted against test potentials between -20 and +30 mV as indicated. E, the activation time constants were measured in oocytes expressing both syntaxin 1A and Syt 11-264 (black-square) and F, syntaxin 1A and Syt 1*1-264 (). Two sample Student's t tests were applied, and p values <0.05 were obtained from the two-tailed tests. cRNA (ng/oocyte) injected, see Figs. 6 and. 7.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A role for the C2A domain of Syt 1 in calcium-triggered neurotransmitter release has been well established in neurons and neuroendocrine cells (15-17, 42). Mutation of a polylysine motif distal to the calcium coordination site reverses the inhibitory effect of injected Syt C2A fragments on calcium-regulated secretion (17, 43). Since mutation of the polylysine motif does not affect the overall structure or Ca2+ binding properties of the C2A domain, calcium-independent properties involving the polylysine motif are important for the Syt-mediated steps leading to neurotransmitter release (17). However the nature of these interactions remained unknown.

We addressed the possibility that the voltage-gated Ca2+ channel (Cav1.2, Cav2.1, Cav2.2, and Cav2.3), an established effector for Syt 1 (30-32, 34, 40), may be functionally coupled through the polylysine C2A domain. Using the Xenopus oocytes expression system we examined the functional consequences of mutating the C2A polylysine motif on Cav1.2 (Lc-type) the channel that supports evoked secretion in PC12 cells and the neuronal Cav2.3 (R-type) channel. The changes induced in the activation kinetics and current amplitude of voltage-sensitive Ca2+ channels demonstrate that the C2A polylysine motif participates in the interaction of Syt 1 with both Cav1.2 and Cav2.3.

Modulation of Cav1.2-- Since the Syt 1/syntaxin 1A interaction occurs independently of the C2A polylysine motif (17), the observed differences in syntaxin 1A modulation of channel activity in the presence of Syt 1 may result from either a direct interaction with the channel or with a new site formed by the association of syntaxin 1A with the channel. The full-length Syt 1 reversed syntaxin 1A inhibition of Cav1.2 activity, while Syt 1* was significantly less effective. The marked slowing of activation kinetics of Cav1.2 by syntaxin 1A was reversed by Syt 11-264 but not by Syt 1*1-264. The mutation of the polylysine motif in the Syt 11-264 protein lacking a C2B domain (Syt 1*1-264) results in a complete loss of function. These results were further substantiated when no binding of His6Lc753-893 to the isolated C2A domain were observed. Lc753-893, the intracellular domain of Cav1.2 alpha 1 subunit, was previously shown to be the site of interaction of Syt 1, C2A, and C2B (31, 32). The four mutated KKKK residues abolished GST-C2A* binding to His6Lc753-893 in two methods, GSH-agarose beads and plasmon resonance spectroscopy. Therefore, these results, in part, could provide an explanation to why unlike the intact C2A, polylysine-mutated C2A peptide when injected into PC12 was unable to interfere with transmitter release (17).

Interestingly, the effect of a mutated C2A polylysine motif in full-length Syt 1*, appeared to be partially attenuated by C2B domain, consistent with a functional relationship between the two C2 domains of Syt 1 and the calcium channel (24). Interactions through the Syt 1 C2B domain are also functionally important for neurosecretion (19, 20, 22, 24). More recent studies using a genetic rescue approach in Drosophila reveals a role for the polylysine motif of the C2B domain in evoked release (25). Moreover, the C2B domain promotes the Ca2+-dependent binding of syntaxin 1A to C2A, suggesting a level of functional synergy between the two C2 domains of Syt 1 (44). Interestingly, secretion from PC12 cells carried out using the cracked cell method showed that dense-core vesicle exocytosis does not require vesicular synaptotagmin 1 but may use instead the plasma membrane synaptotagmins 3 and 7 as Ca2+ sensors (45).

Modulation of Cav2.3-- Previously, induction of faster activation by Syt 1 was observed for the neuronal Ca2+ channels, Cav2.2 and Cav2.3, in contrast to no effect on Cav1.2 (28-30). Here we show that truncated Syt 1 (Syt 11-264) accelerated Cav2.3 activation suggesting that C2A and not C2B domain is responsible for the observed effects. Mutation of the polylysine motif in C2A abolished the stimulatory effect on Cav2.3 activation, indicating the role of this motif in the interaction of the vesicular protein with the channel as well as with Cav2.3 associated with syntaxin 1A.

The truncated mutant Syt 1*1-264 partially restored (~75%) current amplitude but did not reverse the syntaxin 1A effect on activation. Thus modulation of the syntaxin/Cav2.3 kinetics was affected by Syt 11-264 but was lost in Syt 11-264*. In contrast, Syt 1 and Syt 1* effectively reversed the syntaxin 1A inhibition of Cav2.3 current amplitude. These results propose that the C2B domain partially compensates for the mutation in the C2A polylysine motif.

Together, the data indicate that the C2A polylysine motif affects the activation of the channel and modulates the kinetics of syntaxin 1A-associated channel. In Fig. 11 we showed a schematic model of putative interactions of the channel, syntaxin, and Syt 1. 


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Fig. 11.   A schematic model illustrating putative cross-talk interfaces of the voltage-gated calcium channel, syntaxin 1A (SX), and the C2A domains of synaptotagmin (Syt 1) and synaptotagmin mutated at the C2A* polylysine motif (Syt 1*). Transmembrane II6 and III1 are the boundary of Lc753-893, the cytosolic domain of Cav1.2.

In summary, our studies provide compelling evidence that the Syt 1 C2A domain is involved in a functional coupling of the vesicle with the voltage-gated Ca2+-channels, Cav1.2 and Cav2.3. The C2A polylysine motif appears to participate in this interaction and likely functions independently of the Syt 1 Ca2+-mediated interactions with phospholipids or syntaxin 1A (17). The effects of C2A polylysine motif on transmitter release in PC 12 cells as previously reported, may result from a direct modification of the activation kinetics of the Ca2+ channel or function indirectly by competing with endogenous Syt 1 for interactions with the channel. The ability of Syt 1, syntaxin 1A, and the Ca2+ channel to interact is consistent with the formation of a functional exocytotic unit, the excitosome (32). The excitosome complex composed of the Ca2+ channel, syntaxin 1A, SNAP-25, and Syt 1 displays distinct kinetic properties required for calcium-triggered secretion (28, 29, 32, 33, 39, 46). Therefore, inhibition of neurotransmitter release by C2A domain might occur by interfering with generating the excitosome complex and the ensuing propagation of the signal from the channel to the fusion/docking machinery rather than Ca2+ binding to Syt 1 (30). The physiological relevance and the consequences of the different modulation of neuroendocrine (Cav1.2) and neuronal (Cav2.3) Ca2+ channels by Syt 1 during the steps leading to transmitter release will require further studies.

    ACKNOWLEDGEMENTS

D. Atlas thanks the H. L. Lauterbach fund.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 972-2-658-5406; Fax: 972-2-658-5413; E-mail: datlas@vms.huji.ac.il.

Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M210270200

    ABBREVIATIONS

The abbreviations used are: Syt, synaptotagmin; EDC, N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride; GST, glutathione S-transferase.

    REFERENCES
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ABSTRACT
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DISCUSSION
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