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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-KR/AA rescues synaptic vesicle exocytosis and reveals the effect of fast reactivation on exocytosis.(A–C) Average traces synaptic vesicle exocytosis detected by vGlut1-pHluorin in response to single APs (black) and two APs separated by 2 ms (red) for wild-type (WT) in 2 mM external Ca2+ (A), 1.2 mM external Ca2+ (B), Munc13-KD + Munc13-1 (C), and Munc13-KD + Munc13-KR/AA. Dashed line shows the response amplitude of the single AP for comparison (D). Increased exocytosis with two APs in Munc13-KD + Munc13-KR/AA cells indicates that the trends in Ca2+ influx have a demonstrable effect on exocytosis, and that the Munc13 interaction with VGCC affects neural processing on this time scale. Exocytosis for a single AP is depressed in Munc13-KD + Munc13-KR/AA terminal by 38% compared to WT rescue, consistent with reduced Ca2+ influx in these cells. (E) Average values for fast reactivation at 2 ms ISI for the cell types in A–C. Results are mean ± SEM. **p < 0.01, ***p < 0.001, all other comparisons n.s.DOI:http://dx.doi.org/10.7554/eLife.07728.010
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fig7: Munc13-KR/AA rescues synaptic vesicle exocytosis and reveals the effect of fast reactivation on exocytosis.(A–C) Average traces synaptic vesicle exocytosis detected by vGlut1-pHluorin in response to single APs (black) and two APs separated by 2 ms (red) for wild-type (WT) in 2 mM external Ca2+ (A), 1.2 mM external Ca2+ (B), Munc13-KD + Munc13-1 (C), and Munc13-KD + Munc13-KR/AA. Dashed line shows the response amplitude of the single AP for comparison (D). Increased exocytosis with two APs in Munc13-KD + Munc13-KR/AA cells indicates that the trends in Ca2+ influx have a demonstrable effect on exocytosis, and that the Munc13 interaction with VGCC affects neural processing on this time scale. Exocytosis for a single AP is depressed in Munc13-KD + Munc13-KR/AA terminal by 38% compared to WT rescue, consistent with reduced Ca2+ influx in these cells. (E) Average values for fast reactivation at 2 ms ISI for the cell types in A–C. Results are mean ± SEM. **p < 0.01, ***p < 0.001, all other comparisons n.s.DOI:http://dx.doi.org/10.7554/eLife.07728.010

Mentions: Because we observed a Munc13-mediated reduction in VGCC reactivation with 2 ms ISI-paired pulses, we tested the physiological consequences of such stimuli at the level of presynaptic exocytosis. Bursts of stimulation have been identified at this frequency throughout the nervous system (Kandel and Spencer, 1961; Llinas and Jahnsen, 1982; Gray and McCormick, 1996), and the fidelity of synaptic transmission in these bursts is strongly influenced by the ISI time scale (Harris et al., 2001; Roberts et al., 2008). Previous studies have shown that when hippocampal synapses are challenged by pairs of APs separated by only a few milliseconds, the second response is generally profoundly depressed with respect to the first (Stevens and Wang, 1995; Dobrunz et al., 1997; Brody and Yue, 2000). We measured synaptic vesicle exocytosis using vGlut1-pHluorin as previously described (Voglmaier et al., 2006; Ariel and Ryan, 2010; Hoppa et al., 2012; Ariel et al., 2013) and compared single AP responses with 2 ms ISI paired pulses in WT cells using the optical reporter. Since endocytosis and re-acidification of vGlut1-pHluorin occurs on a relatively slow time scale (s), the signals from 2 AP with only 2 ms ISI should summate. We found that in WT cells stimulation with two APs at 2 ms ISI resulted in a small (20%) increase in exocytosis over a single AP (Figure 7A,E, Table 1), corresponding to 80% inhibition of exocytosis from the second AP. This depression in the second AP response was insensitive to decreasing magnitude of influx during the first AP (Figure 7B,E), consistent with our observations that reductions in extracellular Ca2+ do not affect levels of channel reactivation (Figure 5). The complete lack of synaptic vesicle exocytosis precludes measuring fast depression in Munc13-KD cells. However, reintroduction of shRNA-resistant Munc13-1 into the KD background gave very similar fast depression to WT cells (Figure 7C,E, Table 1). In contrast, expression of Munc13-KR/AA in the Munc13-KD rescued exocytosis from a single AP to levels expected based on Ca2+ influx (see below) but failed to restore the profound depression of exocytosis for 2 ms ISI indicating that this mutant effectively decoupled the vesicle priming activity of Munc13-1 from its effects on VGCCs (Figure 7D,E, Table 1). Synaptic vesicle exocytosis was smaller for Munc13-KR/AA rescue than WT cells as one would predict from the reduced Ca2+ influx in Munc13-KR/AA rescues and the steep relationship between Ca2+ influx and vesicle exocytosis (Ariel and Ryan, 2010). In fact, exocytosis in Munc13-KR/AA rescue in response to single AP was similar to that measured in WT cells in the presence of 1.2 mM extracellular Ca2+ (not shown). The fact that VGCC failed to reactivate in 1.2 mM extracellular Ca2+ WT cells but not in Munc13-KR/AA rescue suggests that the increase in exocytosis during 2 ms ISI paired pulses in Munc13-KR/AA is a direct effect of the lack of Munc13-VGCC interaction and not a secondary effect of reduced exocytosis. We therefore conclude that this interaction site in Munc13's C2B domain mediates VGCC channel reactivation in nerve terminals.10.7554/eLife.07728.010Figure 7.Munc13-KR/AA rescues synaptic vesicle exocytosis and reveals the effect of fast reactivation on exocytosis.


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-KR/AA rescues synaptic vesicle exocytosis and reveals the effect of fast reactivation on exocytosis.(A–C) Average traces synaptic vesicle exocytosis detected by vGlut1-pHluorin in response to single APs (black) and two APs separated by 2 ms (red) for wild-type (WT) in 2 mM external Ca2+ (A), 1.2 mM external Ca2+ (B), Munc13-KD + Munc13-1 (C), and Munc13-KD + Munc13-KR/AA. Dashed line shows the response amplitude of the single AP for comparison (D). Increased exocytosis with two APs in Munc13-KD + Munc13-KR/AA cells indicates that the trends in Ca2+ influx have a demonstrable effect on exocytosis, and that the Munc13 interaction with VGCC affects neural processing on this time scale. Exocytosis for a single AP is depressed in Munc13-KD + Munc13-KR/AA terminal by 38% compared to WT rescue, consistent with reduced Ca2+ influx in these cells. (E) Average values for fast reactivation at 2 ms ISI for the cell types in A–C. Results are mean ± SEM. **p < 0.01, ***p < 0.001, all other comparisons n.s.DOI:http://dx.doi.org/10.7554/eLife.07728.010
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fig7: Munc13-KR/AA rescues synaptic vesicle exocytosis and reveals the effect of fast reactivation on exocytosis.(A–C) Average traces synaptic vesicle exocytosis detected by vGlut1-pHluorin in response to single APs (black) and two APs separated by 2 ms (red) for wild-type (WT) in 2 mM external Ca2+ (A), 1.2 mM external Ca2+ (B), Munc13-KD + Munc13-1 (C), and Munc13-KD + Munc13-KR/AA. Dashed line shows the response amplitude of the single AP for comparison (D). Increased exocytosis with two APs in Munc13-KD + Munc13-KR/AA cells indicates that the trends in Ca2+ influx have a demonstrable effect on exocytosis, and that the Munc13 interaction with VGCC affects neural processing on this time scale. Exocytosis for a single AP is depressed in Munc13-KD + Munc13-KR/AA terminal by 38% compared to WT rescue, consistent with reduced Ca2+ influx in these cells. (E) Average values for fast reactivation at 2 ms ISI for the cell types in A–C. Results are mean ± SEM. **p < 0.01, ***p < 0.001, all other comparisons n.s.DOI:http://dx.doi.org/10.7554/eLife.07728.010
Mentions: Because we observed a Munc13-mediated reduction in VGCC reactivation with 2 ms ISI-paired pulses, we tested the physiological consequences of such stimuli at the level of presynaptic exocytosis. Bursts of stimulation have been identified at this frequency throughout the nervous system (Kandel and Spencer, 1961; Llinas and Jahnsen, 1982; Gray and McCormick, 1996), and the fidelity of synaptic transmission in these bursts is strongly influenced by the ISI time scale (Harris et al., 2001; Roberts et al., 2008). Previous studies have shown that when hippocampal synapses are challenged by pairs of APs separated by only a few milliseconds, the second response is generally profoundly depressed with respect to the first (Stevens and Wang, 1995; Dobrunz et al., 1997; Brody and Yue, 2000). We measured synaptic vesicle exocytosis using vGlut1-pHluorin as previously described (Voglmaier et al., 2006; Ariel and Ryan, 2010; Hoppa et al., 2012; Ariel et al., 2013) and compared single AP responses with 2 ms ISI paired pulses in WT cells using the optical reporter. Since endocytosis and re-acidification of vGlut1-pHluorin occurs on a relatively slow time scale (s), the signals from 2 AP with only 2 ms ISI should summate. We found that in WT cells stimulation with two APs at 2 ms ISI resulted in a small (20%) increase in exocytosis over a single AP (Figure 7A,E, Table 1), corresponding to 80% inhibition of exocytosis from the second AP. This depression in the second AP response was insensitive to decreasing magnitude of influx during the first AP (Figure 7B,E), consistent with our observations that reductions in extracellular Ca2+ do not affect levels of channel reactivation (Figure 5). The complete lack of synaptic vesicle exocytosis precludes measuring fast depression in Munc13-KD cells. However, reintroduction of shRNA-resistant Munc13-1 into the KD background gave very similar fast depression to WT cells (Figure 7C,E, Table 1). In contrast, expression of Munc13-KR/AA in the Munc13-KD rescued exocytosis from a single AP to levels expected based on Ca2+ influx (see below) but failed to restore the profound depression of exocytosis for 2 ms ISI indicating that this mutant effectively decoupled the vesicle priming activity of Munc13-1 from its effects on VGCCs (Figure 7D,E, Table 1). Synaptic vesicle exocytosis was smaller for Munc13-KR/AA rescue than WT cells as one would predict from the reduced Ca2+ influx in Munc13-KR/AA rescues and the steep relationship between Ca2+ influx and vesicle exocytosis (Ariel and Ryan, 2010). In fact, exocytosis in Munc13-KR/AA rescue in response to single AP was similar to that measured in WT cells in the presence of 1.2 mM extracellular Ca2+ (not shown). The fact that VGCC failed to reactivate in 1.2 mM extracellular Ca2+ WT cells but not in Munc13-KR/AA rescue suggests that the increase in exocytosis during 2 ms ISI paired pulses in Munc13-KR/AA is a direct effect of the lack of Munc13-VGCC interaction and not a secondary effect of reduced exocytosis. We therefore conclude that this interaction site in Munc13's C2B domain mediates VGCC channel reactivation in nerve terminals.10.7554/eLife.07728.010Figure 7.Munc13-KR/AA rescues synaptic vesicle exocytosis and reveals the effect of fast reactivation on exocytosis.

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