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The active-zone protein Munc13 controls the use-dependence of presynaptic voltage-gated calcium channels.

Calloway N, Gouzer G, Xue M, Ryan TA - Elife (2015)

Bottom Line: Presynaptic calcium channel function is critical for converting electrical information into chemical communication but the molecules in the active zone that sculpt this function are poorly understood.We show that Munc13, an active-zone protein essential for exocytosis, also controls presynaptic voltage-gated calcium channel (VGCC) function dictating their behavior during various forms of activity.We demonstrate that in vitro Munc13 interacts with voltage-VGCCs via a pair of basic residues in Munc13's C2B domain.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Weill Cornell Medical College, New York, United States.

ABSTRACT
Presynaptic calcium channel function is critical for converting electrical information into chemical communication but the molecules in the active zone that sculpt this function are poorly understood. We show that Munc13, an active-zone protein essential for exocytosis, also controls presynaptic voltage-gated calcium channel (VGCC) function dictating their behavior during various forms of activity. We demonstrate that in vitro Munc13 interacts with voltage-VGCCs via a pair of basic residues in Munc13's C2B domain. We show that elimination of this interaction by either removal of Munc13 or replacement of Munc13 with a Munc13 C2B mutant alters synaptic VGCC's response to and recovery from high-frequency action potential bursts and alters calcium influx from single action potential stimuli. These studies illustrate a novel form of synaptic modulation and show that Munc13 is poised to profoundly impact information transfer at nerve terminals by controlling both vesicle priming and the trigger for exocytosis.

No MeSH data available.


Related in: MedlinePlus

Munc13 C2B domain interacts with CaV2.2 synprint.(A) Alignment of C2 domains from RIM1, synaptotagmin-1, Munc13 isoforms 1, 2, and 3, and Munc13-KR/AA showing the synprint binding polybasic sequence (green), Ca2+-interacting residues (pink), and the two mutated residues in Munc13-KR/AA to prevent synprint binding (bold). (B) Top, representative pull-down of T7-tagged CaV2.2 Synprint region by Glutathione–sepharose bound GST or GST-Munc13-1-C2B fusions in the presence of 1 mM EDTA or 1 mM Ca2+. Bottom, quantification of N = 4 independent pull-down experiments normalized to average blot intensity. Results are mean ± SEM. p < 10−6 for grouping by genotype, p = n.s. for the presence of Ca2+ or genotype–Ca2+ interaction. (C) Cartoon illustrating the relative position and orientation of the synprint binding site and the residues mutated in Munc13-KR/AA with respect to the plasma membrane and Ca2+ binding site. PDB structure 3KWU.DOI:http://dx.doi.org/10.7554/eLife.07728.003
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fig1: Munc13 C2B domain interacts with CaV2.2 synprint.(A) Alignment of C2 domains from RIM1, synaptotagmin-1, Munc13 isoforms 1, 2, and 3, and Munc13-KR/AA showing the synprint binding polybasic sequence (green), Ca2+-interacting residues (pink), and the two mutated residues in Munc13-KR/AA to prevent synprint binding (bold). (B) Top, representative pull-down of T7-tagged CaV2.2 Synprint region by Glutathione–sepharose bound GST or GST-Munc13-1-C2B fusions in the presence of 1 mM EDTA or 1 mM Ca2+. Bottom, quantification of N = 4 independent pull-down experiments normalized to average blot intensity. Results are mean ± SEM. p < 10−6 for grouping by genotype, p = n.s. for the presence of Ca2+ or genotype–Ca2+ interaction. (C) Cartoon illustrating the relative position and orientation of the synprint binding site and the residues mutated in Munc13-KR/AA with respect to the plasma membrane and Ca2+ binding site. PDB structure 3KWU.DOI:http://dx.doi.org/10.7554/eLife.07728.003

Mentions: Munc13 isoforms contain numerous protein–protein and protein–ligand interaction domains. These include a Ca2+ and lipid-interacting C2B domain homologous to the C2B domains of synaptotagmin-1 and RIM1, which are known to interact with the synaptic protein interaction (synprint) region on CaV2.2 (Sheng et al., 1997; Chapman et al., 1998; Coppola et al., 2001) (Figure 1A). Since Munc13 is known to be targeted to regions of the presynaptic membrane rich in VGCCs (Weimer et al., 2006), we wondered if Munc13 might also interact with VGCCs via this conserved C2B domain. We carried out in vitro co-precipitation assays using glutathione S-transferase (GST) Munc13-1 C2B domain fusions and epitope-tagged derivative of the synprint region of CaV2.2. We showed that the C2B domain of Munc13, like that in synaptotagmin and RIM, also interacts with the synprint region of CaV2.2 in a Ca2+-independent manner (Figure 1B). Munc13 isoforms carry a short polybasic sequence in their C2B domain similar to the synprint interaction motif on synaptotagmin that is orthogonal to the lipid-interaction face of the protein (Figure 1C). Mutating this polybasic sequence (Munc13-KR/AA) disrupted the in vitro interaction between Munc13-1 and the synprint region of CaV2.2 (Figure 1B) similar to what has been reported for both the synaptotagmin C2B-synprint and RIM C2B-synprint interactions. These findings indicate that the polybasic region in the C2B domains of these proteins is a shared motif that potentially mediates interaction with VGCCs in situ. Previous reports of C2B-synprint interactions have not included functional assays of the effect of the interaction on presynaptic Ca2+ influx. We therefore took advantage of the ability to monitor Ca2+ influx at active zones under conditions that perturb the potential Munc13-VGCC interaction.10.7554/eLife.07728.003Figure 1.Munc13 C2B domain interacts with CaV2.2 synprint.


The active-zone protein Munc13 controls the use-dependence of presynaptic voltage-gated calcium channels.

Calloway N, Gouzer G, Xue M, Ryan TA - Elife (2015)

Munc13 C2B domain interacts with CaV2.2 synprint.(A) Alignment of C2 domains from RIM1, synaptotagmin-1, Munc13 isoforms 1, 2, and 3, and Munc13-KR/AA showing the synprint binding polybasic sequence (green), Ca2+-interacting residues (pink), and the two mutated residues in Munc13-KR/AA to prevent synprint binding (bold). (B) Top, representative pull-down of T7-tagged CaV2.2 Synprint region by Glutathione–sepharose bound GST or GST-Munc13-1-C2B fusions in the presence of 1 mM EDTA or 1 mM Ca2+. Bottom, quantification of N = 4 independent pull-down experiments normalized to average blot intensity. Results are mean ± SEM. p < 10−6 for grouping by genotype, p = n.s. for the presence of Ca2+ or genotype–Ca2+ interaction. (C) Cartoon illustrating the relative position and orientation of the synprint binding site and the residues mutated in Munc13-KR/AA with respect to the plasma membrane and Ca2+ binding site. PDB structure 3KWU.DOI:http://dx.doi.org/10.7554/eLife.07728.003
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Related In: Results  -  Collection

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fig1: Munc13 C2B domain interacts with CaV2.2 synprint.(A) Alignment of C2 domains from RIM1, synaptotagmin-1, Munc13 isoforms 1, 2, and 3, and Munc13-KR/AA showing the synprint binding polybasic sequence (green), Ca2+-interacting residues (pink), and the two mutated residues in Munc13-KR/AA to prevent synprint binding (bold). (B) Top, representative pull-down of T7-tagged CaV2.2 Synprint region by Glutathione–sepharose bound GST or GST-Munc13-1-C2B fusions in the presence of 1 mM EDTA or 1 mM Ca2+. Bottom, quantification of N = 4 independent pull-down experiments normalized to average blot intensity. Results are mean ± SEM. p < 10−6 for grouping by genotype, p = n.s. for the presence of Ca2+ or genotype–Ca2+ interaction. (C) Cartoon illustrating the relative position and orientation of the synprint binding site and the residues mutated in Munc13-KR/AA with respect to the plasma membrane and Ca2+ binding site. PDB structure 3KWU.DOI:http://dx.doi.org/10.7554/eLife.07728.003
Mentions: Munc13 isoforms contain numerous protein–protein and protein–ligand interaction domains. These include a Ca2+ and lipid-interacting C2B domain homologous to the C2B domains of synaptotagmin-1 and RIM1, which are known to interact with the synaptic protein interaction (synprint) region on CaV2.2 (Sheng et al., 1997; Chapman et al., 1998; Coppola et al., 2001) (Figure 1A). Since Munc13 is known to be targeted to regions of the presynaptic membrane rich in VGCCs (Weimer et al., 2006), we wondered if Munc13 might also interact with VGCCs via this conserved C2B domain. We carried out in vitro co-precipitation assays using glutathione S-transferase (GST) Munc13-1 C2B domain fusions and epitope-tagged derivative of the synprint region of CaV2.2. We showed that the C2B domain of Munc13, like that in synaptotagmin and RIM, also interacts with the synprint region of CaV2.2 in a Ca2+-independent manner (Figure 1B). Munc13 isoforms carry a short polybasic sequence in their C2B domain similar to the synprint interaction motif on synaptotagmin that is orthogonal to the lipid-interaction face of the protein (Figure 1C). Mutating this polybasic sequence (Munc13-KR/AA) disrupted the in vitro interaction between Munc13-1 and the synprint region of CaV2.2 (Figure 1B) similar to what has been reported for both the synaptotagmin C2B-synprint and RIM C2B-synprint interactions. These findings indicate that the polybasic region in the C2B domains of these proteins is a shared motif that potentially mediates interaction with VGCCs in situ. Previous reports of C2B-synprint interactions have not included functional assays of the effect of the interaction on presynaptic Ca2+ influx. We therefore took advantage of the ability to monitor Ca2+ influx at active zones under conditions that perturb the potential Munc13-VGCC interaction.10.7554/eLife.07728.003Figure 1.Munc13 C2B domain interacts with CaV2.2 synprint.

Bottom Line: Presynaptic calcium channel function is critical for converting electrical information into chemical communication but the molecules in the active zone that sculpt this function are poorly understood.We show that Munc13, an active-zone protein essential for exocytosis, also controls presynaptic voltage-gated calcium channel (VGCC) function dictating their behavior during various forms of activity.We demonstrate that in vitro Munc13 interacts with voltage-VGCCs via a pair of basic residues in Munc13's C2B domain.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Weill Cornell Medical College, New York, United States.

ABSTRACT
Presynaptic calcium channel function is critical for converting electrical information into chemical communication but the molecules in the active zone that sculpt this function are poorly understood. We show that Munc13, an active-zone protein essential for exocytosis, also controls presynaptic voltage-gated calcium channel (VGCC) function dictating their behavior during various forms of activity. We demonstrate that in vitro Munc13 interacts with voltage-VGCCs via a pair of basic residues in Munc13's C2B domain. We show that elimination of this interaction by either removal of Munc13 or replacement of Munc13 with a Munc13 C2B mutant alters synaptic VGCC's response to and recovery from high-frequency action potential bursts and alters calcium influx from single action potential stimuli. These studies illustrate a novel form of synaptic modulation and show that Munc13 is poised to profoundly impact information transfer at nerve terminals by controlling both vesicle priming and the trigger for exocytosis.

No MeSH data available.


Related in: MedlinePlus