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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

Munc13-1 but not Munc13-KR/AA rescues the Ca2+ influx phenotypes associated with Munc13-KD.(A) Example traces of Ca2+ influx detected by Fluo5F fluorescence in response to a single AP in Munc13-1 (black) and Munc13-KR/AA (red) expressing neurons in a Munc13-KD background. (B, C) Example traces of one AP (black) and two APs separated by 2 ms for Munc13-1 (B) and Munc13-KR/AA (C) expressing boutons in a Munc13-KD background. (D–F) Average values for Ca2+ influx (D), reactivation at 2 ms ISI (E), and residual inactivation (F), in Munc13-KD + Munc13-1 and Munc13-KD + Munc 13-KR/AA cells, as in Figures 3, 4. Results are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, all other comparisons n.s.DOI:http://dx.doi.org/10.7554/eLife.07728.009
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fig6: Munc13-1 but not Munc13-KR/AA rescues the Ca2+ influx phenotypes associated with Munc13-KD.(A) Example traces of Ca2+ influx detected by Fluo5F fluorescence in response to a single AP in Munc13-1 (black) and Munc13-KR/AA (red) expressing neurons in a Munc13-KD background. (B, C) Example traces of one AP (black) and two APs separated by 2 ms for Munc13-1 (B) and Munc13-KR/AA (C) expressing boutons in a Munc13-KD background. (D–F) Average values for Ca2+ influx (D), reactivation at 2 ms ISI (E), and residual inactivation (F), in Munc13-KD + Munc13-1 and Munc13-KD + Munc 13-KR/AA cells, as in Figures 3, 4. Results are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, all other comparisons n.s.DOI:http://dx.doi.org/10.7554/eLife.07728.009

Mentions: To test the significance of the observed in vitro interaction between Munc13 and the synprint region of CaV2.2, we expressed shRNA-resistant derivatives of both Munc13-1 and the mutant lacking synprint binding capacity, Munc13-KR/AA, in a Munc13-KD nerve terminals. Re-expression of Munc13-1 in Munc13-KD neurons resulted in complete restoration of Ca2+ influx from single APs (Figure 6A,D). This observation suggests that the effects seen with Munc13-KD are specific to the depletion of Munc13 expression levels as opposed to off target effects. Conversely, in Munc13-KD cells expressing Munc13-KR/AA, Ca2+ influx from single APs remained similar to Munc13-KD (Figure 6A,D) although exocytosis was largely restored (see below). This result suggests that, indeed, the synprint binding motif in the C2B domain of Munc13 is specifically responsible for the functional effects of Munc13-KD on VGCCs. Similarly, expression of WT Munc13-1 in the KD restored VGCC refractory period observed during 2 ms ISI paired pulses and eliminated the residual inactivation following 50 AP trains whereas expression of Munc13-KR/AA did not (Figure 6B,C,E,F). Thus, for these functional assays, rescue with Munc13-1 restored Ca2+ dynamics to that of WT cells, whereas Munc13-KR/AA did not and was indistinguishable from Munc13-KD. These data support the idea that the interaction we identified in vitro constitutes the primary molecular determinant of the effects on VGCCs seen in Munc13-KD neurons.10.7554/eLife.07728.009Figure 6.Munc13-1 but not Munc13-KR/AA rescues the Ca2+ influx phenotypes associated with Munc13-KD.


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)

Munc13-1 but not Munc13-KR/AA rescues the Ca2+ influx phenotypes associated with Munc13-KD.(A) Example traces of Ca2+ influx detected by Fluo5F fluorescence in response to a single AP in Munc13-1 (black) and Munc13-KR/AA (red) expressing neurons in a Munc13-KD background. (B, C) Example traces of one AP (black) and two APs separated by 2 ms for Munc13-1 (B) and Munc13-KR/AA (C) expressing boutons in a Munc13-KD background. (D–F) Average values for Ca2+ influx (D), reactivation at 2 ms ISI (E), and residual inactivation (F), in Munc13-KD + Munc13-1 and Munc13-KD + Munc 13-KR/AA cells, as in Figures 3, 4. Results are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, all other comparisons n.s.DOI:http://dx.doi.org/10.7554/eLife.07728.009
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fig6: Munc13-1 but not Munc13-KR/AA rescues the Ca2+ influx phenotypes associated with Munc13-KD.(A) Example traces of Ca2+ influx detected by Fluo5F fluorescence in response to a single AP in Munc13-1 (black) and Munc13-KR/AA (red) expressing neurons in a Munc13-KD background. (B, C) Example traces of one AP (black) and two APs separated by 2 ms for Munc13-1 (B) and Munc13-KR/AA (C) expressing boutons in a Munc13-KD background. (D–F) Average values for Ca2+ influx (D), reactivation at 2 ms ISI (E), and residual inactivation (F), in Munc13-KD + Munc13-1 and Munc13-KD + Munc 13-KR/AA cells, as in Figures 3, 4. Results are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, all other comparisons n.s.DOI:http://dx.doi.org/10.7554/eLife.07728.009
Mentions: To test the significance of the observed in vitro interaction between Munc13 and the synprint region of CaV2.2, we expressed shRNA-resistant derivatives of both Munc13-1 and the mutant lacking synprint binding capacity, Munc13-KR/AA, in a Munc13-KD nerve terminals. Re-expression of Munc13-1 in Munc13-KD neurons resulted in complete restoration of Ca2+ influx from single APs (Figure 6A,D). This observation suggests that the effects seen with Munc13-KD are specific to the depletion of Munc13 expression levels as opposed to off target effects. Conversely, in Munc13-KD cells expressing Munc13-KR/AA, Ca2+ influx from single APs remained similar to Munc13-KD (Figure 6A,D) although exocytosis was largely restored (see below). This result suggests that, indeed, the synprint binding motif in the C2B domain of Munc13 is specifically responsible for the functional effects of Munc13-KD on VGCCs. Similarly, expression of WT Munc13-1 in the KD restored VGCC refractory period observed during 2 ms ISI paired pulses and eliminated the residual inactivation following 50 AP trains whereas expression of Munc13-KR/AA did not (Figure 6B,C,E,F). Thus, for these functional assays, rescue with Munc13-1 restored Ca2+ dynamics to that of WT cells, whereas Munc13-KR/AA did not and was indistinguishable from Munc13-KD. These data support the idea that the interaction we identified in vitro constitutes the primary molecular determinant of the effects on VGCCs seen in Munc13-KD neurons.10.7554/eLife.07728.009Figure 6.Munc13-1 but not Munc13-KR/AA rescues the Ca2+ influx phenotypes associated with Munc13-KD.

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