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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-KD increases VGCC reactivation on a very short time scale.(A, B, E, F) Example traces of Ca2+ responses to one AP (black) and two APs separated by 2 ms for wild-type (WT) boutons (A), TeNT-LC expressing boutons (B), Munc13-KD boutons (E), and WT soma (F). (C) Average membrane AP waveform measured using ARCH at WT boutons for single and double AP (2 ms ISI) at 30°C (left) and 37°C (right) indicating AP failure for the second AP at the lower temperature but successful firing and propagation of both APs at physiological temperature. Arch recordings from Munc13-KD boutons also show successful firing of the second AP. (D) ARCH amplitude ratios of second AP peak with respect to first AP peak for WT at 30°C (left), WT at 37°C (middle) and Munc13-KD at 37°C (right). Open symbols show the mean ± SEM value for N = 4, WT 30°C; N = 7, WT 37°C, N = 7 Munc13 KD 37°C. (G) Average Ca2+ signal peak height in 2 ms paired pulses ISI normalized to the respective Ca2+ signal peak in response to 1AP. (H) Average Ca2+ signal peak calculated for the second AP during paired pulses of various ISI for WT (black) and Munc13-KD (red) cells normalized to Ca2+ signal peak in response to 1 AP. 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.07728.007
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fig4: Munc13-KD increases VGCC reactivation on a very short time scale.(A, B, E, F) Example traces of Ca2+ responses to one AP (black) and two APs separated by 2 ms for wild-type (WT) boutons (A), TeNT-LC expressing boutons (B), Munc13-KD boutons (E), and WT soma (F). (C) Average membrane AP waveform measured using ARCH at WT boutons for single and double AP (2 ms ISI) at 30°C (left) and 37°C (right) indicating AP failure for the second AP at the lower temperature but successful firing and propagation of both APs at physiological temperature. Arch recordings from Munc13-KD boutons also show successful firing of the second AP. (D) ARCH amplitude ratios of second AP peak with respect to first AP peak for WT at 30°C (left), WT at 37°C (middle) and Munc13-KD at 37°C (right). Open symbols show the mean ± SEM value for N = 4, WT 30°C; N = 7, WT 37°C, N = 7 Munc13 KD 37°C. (G) Average Ca2+ signal peak height in 2 ms paired pulses ISI normalized to the respective Ca2+ signal peak in response to 1AP. (H) Average Ca2+ signal peak calculated for the second AP during paired pulses of various ISI for WT (black) and Munc13-KD (red) cells normalized to Ca2+ signal peak in response to 1 AP. 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.07728.007

Mentions: As a second paradigm to study changes in VGCC-gating properties, we probed Ca2+ dynamics for paired stimuli on very fast time scales, at the upper limit of stimulation frequencies that could conceivably generate multiple APs (Staff et al., 2000). Since presynaptic Ca2+ clearance subsequent to AP stimulation occurs over a >100 ms time scale, and as single AP signals are well below the saturation threshold for Fluo5F, the Ca2+ influx for an inter-stimulus interval (ISI) of 2 ms should reflect the sum of the Ca2+ influx from each stimulus. These experiments showed that in WT synapses the summed response for double AP stimulation was only slightly higher than that obtained with a single AP (Figure 4A) indicating a 90% reduction in Ca2+ influx on the second AP with a 2 ms ISI (Figure 4A,G, Table 1). These results are consistent with previous reports that demonstrated profound depression of excitatory postsynaptic current (EPSC) in response to stimulus pairs at these frequencies (Stevens and Wang, 1995; Dobrunz et al., 1997; Brody and Yue, 2000). Those studies independently proposed various hypotheses for the mechanism of this depression including exocytosis-driven inhibition, failure of AP propagation, and inactivation of VGCCs. All three of these possible mechanisms would be potentially consistent with our observations of depression of Ca2+ influx for short ISIs. We reasoned that if exocytosis at the active zone leads to a brief refractory period in VGCC function then conditions that eliminate exocytosis should remove the depression in Ca2+ influx. We tested this idea in a manner that is independent of Munc13 by expressing the proteolytic light-chain of tetanus toxin (TeNT-LC) in hippocampal neurons and carried out measurements of AP-driven influx for single and AP pairs at 2 ms ISI. We previously showed that expression of TeNT-LC in these cells eliminates exocytosis (Rangaraju et al., 2014). Simply eliminating exocytosis, however, did not change the reactivation behavior of presynaptic VGCCs (Figure 4B,G) probed with 2 AP stimulation with a 2 ms ISI, which were indistinguishable from that observed in WT synapses. Thus, the observed fast paired-pulse depression in presynaptic Ca2+ influx is not exocytosis driven. In order to determine if the loss of Ca2+ influx could result from AP failure, we used Arch-GFP to examine the presynaptic AP waveform for single and AP pairs at 2 ms ISI (Figure 4C) measured at individual boutons. Our measurements have all been carried out at 37°C while most of the earlier work examined synaptic depression at colder temperatures (∼22–24°C). Arch-GFP-based recordings of AP pairs showed that even at 30°C there is virtually complete failure in AP propagation for the second AP when delivered 2 ms after the first, consistent with the predictions of Brody and Yue (2000) (Figure 4C). The ratio of the ARCH signal in response to the second stimulus normalized to that of the response to the first stimulus is close to zero, once corrected for the residual signal resulting from the first AP (Figure 4D). In contrast at 37°C, similar measurements revealed that the amplitude of the second AP is similar to the first at these short ISIs in both WT and Munc13-KD (Figure 4C,D) in all cells examined (Figure 4D) (wild-type AP2/AP1 = 1.04 ± 0.05 sem, n = 7, Munc13-KD AP2/AP1 = 1.04 ± 0.16 sem, n = 7). The width of the second AP was also similar for both wild type (FWHM AP2 = 1.2 ± 0.24 ms sem, n = 7) and Munc13-KD (FWHM AP2 = 1.36 ± 0.50 ms sem, n = 7). Therefore, under these conditions both APs successfully propagate to presynaptic boutons and should be equally capable of fully driving VGCCs to the open state in both WT and Munc13-KD boutons. Thus, at physiological temperature, AP failure is not the mechanism of fast synaptic depression of Ca2+ influx (or by extension the mechanisms of the depression of exocytosis).10.7554/eLife.07728.007Figure 4.Munc13-KD increases VGCC reactivation on a very short time scale.


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-KD increases VGCC reactivation on a very short time scale.(A, B, E, F) Example traces of Ca2+ responses to one AP (black) and two APs separated by 2 ms for wild-type (WT) boutons (A), TeNT-LC expressing boutons (B), Munc13-KD boutons (E), and WT soma (F). (C) Average membrane AP waveform measured using ARCH at WT boutons for single and double AP (2 ms ISI) at 30°C (left) and 37°C (right) indicating AP failure for the second AP at the lower temperature but successful firing and propagation of both APs at physiological temperature. Arch recordings from Munc13-KD boutons also show successful firing of the second AP. (D) ARCH amplitude ratios of second AP peak with respect to first AP peak for WT at 30°C (left), WT at 37°C (middle) and Munc13-KD at 37°C (right). Open symbols show the mean ± SEM value for N = 4, WT 30°C; N = 7, WT 37°C, N = 7 Munc13 KD 37°C. (G) Average Ca2+ signal peak height in 2 ms paired pulses ISI normalized to the respective Ca2+ signal peak in response to 1AP. (H) Average Ca2+ signal peak calculated for the second AP during paired pulses of various ISI for WT (black) and Munc13-KD (red) cells normalized to Ca2+ signal peak in response to 1 AP. 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.07728.007
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4525472&req=5

fig4: Munc13-KD increases VGCC reactivation on a very short time scale.(A, B, E, F) Example traces of Ca2+ responses to one AP (black) and two APs separated by 2 ms for wild-type (WT) boutons (A), TeNT-LC expressing boutons (B), Munc13-KD boutons (E), and WT soma (F). (C) Average membrane AP waveform measured using ARCH at WT boutons for single and double AP (2 ms ISI) at 30°C (left) and 37°C (right) indicating AP failure for the second AP at the lower temperature but successful firing and propagation of both APs at physiological temperature. Arch recordings from Munc13-KD boutons also show successful firing of the second AP. (D) ARCH amplitude ratios of second AP peak with respect to first AP peak for WT at 30°C (left), WT at 37°C (middle) and Munc13-KD at 37°C (right). Open symbols show the mean ± SEM value for N = 4, WT 30°C; N = 7, WT 37°C, N = 7 Munc13 KD 37°C. (G) Average Ca2+ signal peak height in 2 ms paired pulses ISI normalized to the respective Ca2+ signal peak in response to 1AP. (H) Average Ca2+ signal peak calculated for the second AP during paired pulses of various ISI for WT (black) and Munc13-KD (red) cells normalized to Ca2+ signal peak in response to 1 AP. 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.07728.007
Mentions: As a second paradigm to study changes in VGCC-gating properties, we probed Ca2+ dynamics for paired stimuli on very fast time scales, at the upper limit of stimulation frequencies that could conceivably generate multiple APs (Staff et al., 2000). Since presynaptic Ca2+ clearance subsequent to AP stimulation occurs over a >100 ms time scale, and as single AP signals are well below the saturation threshold for Fluo5F, the Ca2+ influx for an inter-stimulus interval (ISI) of 2 ms should reflect the sum of the Ca2+ influx from each stimulus. These experiments showed that in WT synapses the summed response for double AP stimulation was only slightly higher than that obtained with a single AP (Figure 4A) indicating a 90% reduction in Ca2+ influx on the second AP with a 2 ms ISI (Figure 4A,G, Table 1). These results are consistent with previous reports that demonstrated profound depression of excitatory postsynaptic current (EPSC) in response to stimulus pairs at these frequencies (Stevens and Wang, 1995; Dobrunz et al., 1997; Brody and Yue, 2000). Those studies independently proposed various hypotheses for the mechanism of this depression including exocytosis-driven inhibition, failure of AP propagation, and inactivation of VGCCs. All three of these possible mechanisms would be potentially consistent with our observations of depression of Ca2+ influx for short ISIs. We reasoned that if exocytosis at the active zone leads to a brief refractory period in VGCC function then conditions that eliminate exocytosis should remove the depression in Ca2+ influx. We tested this idea in a manner that is independent of Munc13 by expressing the proteolytic light-chain of tetanus toxin (TeNT-LC) in hippocampal neurons and carried out measurements of AP-driven influx for single and AP pairs at 2 ms ISI. We previously showed that expression of TeNT-LC in these cells eliminates exocytosis (Rangaraju et al., 2014). Simply eliminating exocytosis, however, did not change the reactivation behavior of presynaptic VGCCs (Figure 4B,G) probed with 2 AP stimulation with a 2 ms ISI, which were indistinguishable from that observed in WT synapses. Thus, the observed fast paired-pulse depression in presynaptic Ca2+ influx is not exocytosis driven. In order to determine if the loss of Ca2+ influx could result from AP failure, we used Arch-GFP to examine the presynaptic AP waveform for single and AP pairs at 2 ms ISI (Figure 4C) measured at individual boutons. Our measurements have all been carried out at 37°C while most of the earlier work examined synaptic depression at colder temperatures (∼22–24°C). Arch-GFP-based recordings of AP pairs showed that even at 30°C there is virtually complete failure in AP propagation for the second AP when delivered 2 ms after the first, consistent with the predictions of Brody and Yue (2000) (Figure 4C). The ratio of the ARCH signal in response to the second stimulus normalized to that of the response to the first stimulus is close to zero, once corrected for the residual signal resulting from the first AP (Figure 4D). In contrast at 37°C, similar measurements revealed that the amplitude of the second AP is similar to the first at these short ISIs in both WT and Munc13-KD (Figure 4C,D) in all cells examined (Figure 4D) (wild-type AP2/AP1 = 1.04 ± 0.05 sem, n = 7, Munc13-KD AP2/AP1 = 1.04 ± 0.16 sem, n = 7). The width of the second AP was also similar for both wild type (FWHM AP2 = 1.2 ± 0.24 ms sem, n = 7) and Munc13-KD (FWHM AP2 = 1.36 ± 0.50 ms sem, n = 7). Therefore, under these conditions both APs successfully propagate to presynaptic boutons and should be equally capable of fully driving VGCCs to the open state in both WT and Munc13-KD boutons. Thus, at physiological temperature, AP failure is not the mechanism of fast synaptic depression of Ca2+ influx (or by extension the mechanisms of the depression of exocytosis).10.7554/eLife.07728.007Figure 4.Munc13-KD increases VGCC reactivation on a very short time scale.

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