A novel region in the CaV2.1 α1 subunit C-terminus regulates fast synaptic vesicle fusion and vesicle docking at the mammalian presynaptic active zone
- Max Planck Florida Institute for Neuroscience, United States
- University of Iowa, United States
- Leiden University Medical Center, Netherlands
Figures
Figure 1
CaV2.1 can be selectively ablated and functionally rescued at the calyx of Held.
(A) Cartoon depicting CaV2.1 α1 subunit distal C-terminal interaction partners. (B) Amino acid sequence of the distal Cav2.1 C-terminus indicating interaction sites and truncation mutants. (C) left: …
Figure 2 with 1 supplement
C-terminal deletions in CaV2.1 do not affect CaV2 abundance at the presynaptic terminal.
(A) Cartoons depicting CaV2.1 full transcript or mutants (top) with corresponding exemplary Ca2+ currents (bottom) triggered by 10 ms voltage steps from d -50 mV to 50 mV in 5mV steps. (B–C) …
Figure 2—figure supplement 1
Cav2.1 rescue after CKO does not affect biophysical properties of the Ca2+ current at the calyx of Held.
(A) top: Raw peak Ca2+ current amplitudes and, bottom: normalized peak currents (I/Imasx) as a function of voltage. Continuous curves represent fits according to Equation 1. (B) top: Averaged raw …
Figure 3 with 1 supplement
A novel role for a C-terminal region between amino acids 2042 and 2061 that regulates fast release independent of CaV2.1 abundance.
(A) Cartoon depicting truncated regions in our CaV2.1 α1 deletion mutants including the binding sites for CaVβ4, CASK, RBP, PXXP, RIM1/2 and Mint-1 along with the effects of C-terminal truncations …
Figure 3—figure supplement 1
Cav2.1 Full transcript rescue does not affect synaptic transmission at the calyx of Held/MNTB synapse.
(A–B) Averaged traces of SV pool measurements in noninjected control mice (black; a) and mice expressing Cre + Full length rescue construct (grey; B). Top: stimulation protocol driving SV release by …
Figure 4
The novel C-terminal region between amino acids 2042 and 2061 regulates size of the fast and total releasable pool and synaptic vesicle release kinetics.
(A–B) Average release rate trace after 3 ms (A) or 30 ms stimulation (B) from calyces expressing either Cre + full transcript rescue (grey), Δ2042–2368 (green) or Δ2016–2368 (blue); n = 10 for each …
Figure 5
The novel C-terminal region between amino acids 2042 and 2061 regulates the number of docked synaptic vesicles at the active zones.
(A–C) Representative EM images showing AZs from nontransduced calyces (A), and calyces transduced with Δ2042–2368 (B) or Δ2016–2368 (C). Transduced cells were identified by pre-embedded nanogold …
Figure 6
A novel C-terminal region in CaV2.1 α1 subunit regulates SV docking at the active zone and RRP size independent of Ca2+ signaling.
Cartoon depicting truncated regions in our CaV2.1 α1 deletion mutants including the binding sites for CaVβ, CASK, RBP, RBP, RIM1/2, Mint-1 as well as PXXP motifs and the effects of truncations on ICa…
Tables
Table 1
Electrophysiological parameters of IV relations of Ca2+ currents.
| Parameter | Mean ± SEM (n) | OW-ANOVA Dunnett’s Test |
|---|---|---|
| Max. Ca2+ current amplitude Imax (pA) | ||
| Wild type | 911 ± 63 (12) | p=0.9998 (n.s.) |
| Full transcript | 925 ± 99 (10) | control group |
| CKO | 464 ± 59 (11) | p<0.0001 (****) |
| △2365–2368 | 863 ± 53 (10) | p=0.9513 (n.s.) |
| △2213–2368 | 741 ± 56 (10) | p=0.2261 (n.s.) |
| △2016–2368 | 744 ± 67 (10) | p=0.2383 (n.s.) |
| Membrane capacitance Cslow (pF) | ||
| Wild type | 20.9 ± 1.6 (12) | p=0.5071 (n.s.) |
| Full transcript | 18.2 ± 1.2 (10) | control group |
| CKO | 18.1 ± 1.8 (11) | p>0.9999 (n.s.) |
| △2365–2368 | 16.9 ± 1.1 (10) | p=0.9593 (n.s.) |
| △2213–2368 | 19.8 ± 1.4 (10) | p=0.9933 (n.s.) |
| △2016–2368 | 17.2 ± 1.8 (10) | p=0.8986 (n.s.) |
| IV fit: Half-maximal activation voltage Vm (mV) | ||
| Wild type | −24.6 ± 1.3 (12) | p=0.9986 (n.s.) |
| Full transcript | −25.1 ± 1.3 (10) | control group |
| CKO | −22.3 ± 1.3 (11) | p=0.3604 (n.s.) |
| △2365–2368 | −23.1 ± 1.1 (10) | p=0.6831 (n.s.) |
| △2213–2368 | −23.8 ± 1.5 (10) | p=0.9298 (n.s.) |
| △2016–2368 | −23.3 ± 0.9 (10) | p=0.7924 (n.s.) |
| IV fit: Voltage-dependence of activation km (mV) | ||
| Wild type | 8.0 ± 0.5 (12) | p=0.9524 (n.s.) |
| Full transcript | 7.4 ± 0.5 (10) | control group |
| CKO | 12.6 ± 1.3 (11) | p<0.0001 (****) |
| △2365–2368 | 7.6 ± 0.3 (10) | p=0.9997 (n.s.) |
| △2213–2368 | 8.4 ± 0.4 (10) | p=0.7154 (n.s.) |
| △2016–2368 | 8.2 ± 0.3 (10) | p=0.8481 (n.s.) |
| Boltzmann fit: Half-maximal activation voltage V0.5 (mV) | ||
| Wild type | −10.6 ± 1.4 (12) | p=0.9997 (n.s.) |
| Full transcript | −11 ± 0.1 (10) | control group |
| CKO | −1.7 ± 1.1 (11) | p<0.0001 (****) |
| △2365–2368 | −8.7 ± 1.1 (10) | p=0.6311 (n.s.) |
| △2213–2368 | −8.2 ± 1.8 (10) | p=0.4343 (n.s.) |
| △2016–2368 | −8.9 ± 1.1 (10) | p=0.7130 (n.s.) |
| Boltzmann fit: Voltage-dependence k (mV) | ||
| Wild type | 8.3 ± 0.5 (12) | p=0.9111 (n.s.) |
| Full transcript | 8.9 ± 0.7 (10) | control group |
| CKO | 10.3 ± 0.6 (11) | p=0.3117 (n.s.) |
| △2365–2368 | 7.7 ± 0.7 (10) | p=0.4951 (n.s.) |
| △2213–2368 | 8.9 ± 0.4 (10) | p=0.9947 (n.s.) |
| △2016–2368 | 7.2 ± 0.3 (10) | p=0.1578 (n.s.) |
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*One-Way ANOVA with a Dunnett’s Test with condition knockout as reference group was performed to calculate statistical significance.
Table 2
Summary of currents from synaptic vesicle pool measurements.
| Parameter | 3 ms (mean ± SEM (n) | OW-ANOVA Dunnett’s Test | 30 ms (mean ± SEM (n) | OW-ANOVA Dunnett’s Test |
|---|---|---|---|---|
| Ca2+ current amplitude (nA) | ||||
| Wild type | 0.99 ± 0.07 (10) | p=0.2671 (n.s.) | 0.93 ± 0.07 (10) | p=0.3557 (n.s.) |
| Full transcript | 1.24 ± 0.12 (10) | control group | 1.18 ± 0.12 (10) | control group |
| △2365–2368 | 1.14 ± 0.84 (10) | p=0.9320 (n.s.) | 1.01 ± 0.12 (10) | p=0.9451 (n.s.) |
| △2213–2368 | 1.04 ± 0.09 (8) | p=0.5366 (n.s.) | 0.9 ± 0.1 (8) | p=0.2766 (n.s.) |
| △2061–2368 | 1.23 ± 0.08 (10) | p=0.9999 (n.s.) | 1.13 ± 0.08 (10) | p=0.9993 (n.s.) |
| △2042–2368 | 1.13 ± 0.15 (10) | p=0.9121 (n.s.) | 1.03 ± 0.13 (10) | p=0.8410 (n.s.) |
| △2016–2368 | 0.93 ± 0.38 (10) | p=0.1091 (n.s.) | 0.86 ± 0.46 (10) | p=0.1175 (n.s.) |
| Ca2+ influx charge (pC) | ||||
| Wild type | 2.95 ± 0.3 (10) | p=0.5713 (n.s.) | 25.93 ± 2.22 (10) | p=0.4932 (n.s.) |
| Full transcript | 3.62 ± 0.36 (10) | control group | 31.71 ± 2.87 (10) | control group |
| △2365–2368 | 3.89 ± 0.31 (10) | p=0.9838 (n.s.) | 33.52 ± 2.65 (10) | p=0.9943 (n.s.) |
| △2213–2368 | 3.48 ± 0.35 (8) | p=0.9996 (n.s.) | 28.09 ± 2.7 (8) | p=0.8924 (n.s.) |
| △2061–2368 | 3.87 ± 0.33 (10) | p=0.9893 (n.s.) | 34.94 ± 2.93 (10) | p=0.9132 (n.s.) |
| △2042–2368 | 3.8 ± 0.54 (10) | p=0.9977 (n.s.) | 33.71 ± 4.22 (10) | p=0.9910 (n.s.) |
| △2016–2368 | 3.12 ± 0.14 (10) | p=0.8063 (n.s.) | 27.81 ± 1.14 (10) | p=0.8255 (n.s.) |
| EPSC amplitude (nA) | ||||
| Wild type | 8.99 ± 1.12 (10) | p=0.9995 (n.s.) | 9.77 ± 0.87 (10) | p=0.8167 (n.s.) |
| Full transcript | 8.54 ± 0.9 (10) | control group | 8.31 ± 0.79 (10) | control group |
| △2365–2368 | 6.88 ± 0.78 (10) | p=0.7024 (n.s.) | 7.45 ± 0.73 (10) | p=0.9789 (n.s.) |
| △2213–2368 | 6.05 ± 1.2 (8) | p=0.3672 (n.s.) | 6.7 ± 0.94 (8) | p=0.7880 (n.s.) |
| △2061–2368 | 7.05 ± 1.25 (10) | p=0.7836 (n.s.) | 7.72 ± 1.44 (10) | p=0.9963 (n.s.) |
| △2042–2368 | 3.38 ± 0.89 (10) | p=0.0028 (**) | 5.28 ± 1.31 (10) | p=0.1689 (n.s.) |
| △2016–2368 | 2.49 ± 0.89 (10) | p=0.0004 (***) | 3.61 ± 0.91 (10) | p=0.0098 (**) |
| EPSC 10–90% rise time (ms) | ||||
| Wild type | 1.46 ± 0.14 (10) | p=0.9908 (n.s.) | 1.71 ± 0.31 (10) | p=0.9995 (n.s.) |
| Full transcript | 1.25 ± 0.13 (10) | control group | 1.41 ± 0.18 (10) | control group |
| △2365–2368 | 1.79 ± 0.09 (10) | p=0.5934 (n.s.) | 2.18 ± 0.27 (10) | p=0.9097 (n.s.) |
| △2213–2368 | 1.88 ± 0.06 (8) | p=0.4956 (n.s.) | 2.43 ± 0.3 (8) | p=0.7964 (n.s.) |
| △2061–2368 | 1.8 ± 0.12 (10) | p=0.5792 (n.s.) | 2.07 ± 0.21 (10) | p=0.9529 (n.s.) |
| △2042–2368 | 2.46 ± 0.42 (10) | p=0.0200 (*) | 4.67 ± 0.72 (10) | p=0.0046 (**) |
| △2016–2368 | 3.2 ± 0.57 (10) | p<0.0001 (****) | 6.99 ± 1.45 (10) | p<0.0001 (****) |
| Synaptic delay (ms) | ||||
| Wild type | 1.82 ± 0.13 (10) | p=0.9569 (n.s.) | 1.92 ± 0.2 (10) | p=0.9977 (n.s.) |
| Full transcript | 1.7 ± 0.12 (10) | control group | 1.73 ± 0.11 (10) | control group |
| △2365–2368 | 2.11 ± 0.1 (10) | p=0.1170 (n.s.) | 2.22 ± 0.16 (10) | p=0.8464 (n.s.) |
| △2213–2368 | 2.19 ± 0.1 (8) | p=0.0615 (n.s.) | 2.33 ± 0.19 (8) | p=0.7507 (n.s.) |
| △2061–2368 | 2.14 ± 0.14 (10) | p=0.0748 (n.s.) | 2.28 ± 0.15 (10) | p=0.7714 (n.s.) |
| △2042–2368 | 2.8 ± 0.07 (10) | p<0.0001 (****) | 3.68 ± 0.3 (10) | p=0.0019 (**) |
| △2016–2368 | 2.55 ± 0.2 (10) | p<0.0001 (****) | 4.78 ± 0.83 (10) | p<0.0001 (****) |
-
*One-Way ANOVA with a Dunnett’s Test with full transcript as a control group was performed to calculate statistical significance.
Table 3
Summary of 3ms /30ms EPSC ratios.
| EPSC ratio (3 ms/30 ms) | ||
| Wild type | 0.89 ± 0.05 (10) | p=0.5456 (n.s.) |
| Full transcript | 1.03 ± 0.03 (10) | control group |
| △2365–2368 | 0.92 ± 0.04 (10) | p=0.6887 (n.s.) |
| △2213–2368 | 0.87 ± 0.05 (8) | p=0.4631 (n.s.) |
| △2061–2368 | 0.93 ± 0.04 (10) | p=0.7858 (n.s.) |
| △2042–2368 | 0.61 ± 0.09 (10) | p=0.0004 (***) |
| △2016–2368 | 0.50 ± 0.12 (10) | p<0.0001 (****) |
