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

  1. Matthias Lübbert
  2. R Oliver Goral
  3. Rachel Satterfield
  4. Travis Putzke
  5. Arn MJM van den Maagdenberg
  6. Naomi Kamasawa
  7. Samuel M Young Jr  Is a corresponding author
  1. Max Planck Florida Institute for Neuroscience, United States
  2. University of Iowa, United States
  3. Leiden University Medical Center, Netherlands
6 figures and 3 tables

Figures

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: …

https://doi.org/10.7554/eLife.28412.003
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) …

https://doi.org/10.7554/eLife.28412.004
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 …

https://doi.org/10.7554/eLife.28412.005
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 …

https://doi.org/10.7554/eLife.28412.006
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 …

https://doi.org/10.7554/eLife.28412.007
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 …

https://doi.org/10.7554/eLife.28412.011
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 …

https://doi.org/10.7554/eLife.28412.012
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

https://doi.org/10.7554/eLife.28412.013

Tables

Table 1

Electrophysiological parameters of IV relations of Ca2+ currents.

https://doi.org/10.7554/eLife.28412.008
ParameterMean ± SEM (n)OW-ANOVA
Dunnett’s Test
Max. Ca2+ current amplitude Imax (pA)
Wild type911 ± 63 (12)p=0.9998 (n.s.)
Full transcript925 ± 99 (10)control group
CKO464 ± 59 (11)p<0.0001 (****)
△2365–2368863 ± 53 (10)p=0.9513 (n.s.)
△2213–2368741 ± 56 (10)p=0.2261 (n.s.)
△2016–2368744 ± 67 (10)p=0.2383 (n.s.)
Membrane capacitance Cslow (pF)
Wild type20.9 ± 1.6 (12)p=0.5071 (n.s.)
Full transcript18.2 ± 1.2 (10)control group
CKO18.1 ± 1.8 (11)p>0.9999 (n.s.)
△2365–236816.9 ± 1.1 (10)p=0.9593 (n.s.)
△2213–236819.8 ± 1.4 (10)p=0.9933 (n.s.)
△2016–236817.2 ± 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 type8.0 ± 0.5 (12)p=0.9524 (n.s.)
Full transcript7.4 ± 0.5 (10)control group
CKO12.6 ± 1.3 (11)p<0.0001 (****)
△2365–23687.6 ± 0.3 (10)p=0.9997 (n.s.)
△2213–23688.4 ± 0.4 (10)p=0.7154 (n.s.)
△2016–23688.2 ± 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 type8.3 ± 0.5 (12)p=0.9111 (n.s.)
Full transcript8.9 ± 0.7 (10)control group
CKO10.3 ± 0.6 (11)p=0.3117 (n.s.)
△2365–23687.7 ± 0.7 (10)p=0.4951 (n.s.)
△2213–23688.9 ± 0.4 (10)p=0.9947 (n.s.)
△2016–23687.2 ± 0.3 (10)p=0.1578 (n.s.)
  1. *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.

https://doi.org/10.7554/eLife.28412.009
Parameter3 ms
(mean ± SEM (n)
OW-ANOVA
Dunnett’s Test
30 ms
(mean ± SEM (n)
OW-ANOVA
Dunnett’s Test
Ca2+ current amplitude (nA)
Wild type0.99 ± 0.07 (10)p=0.2671 (n.s.)0.93 ± 0.07 (10)p=0.3557 (n.s.)
Full transcript1.24 ± 0.12 (10)control group1.18 ± 0.12 (10)control group
△2365–23681.14 ± 0.84 (10)p=0.9320 (n.s.)1.01 ± 0.12 (10)p=0.9451 (n.s.)
△2213–23681.04 ± 0.09 (8)p=0.5366 (n.s.)0.9 ± 0.1 (8)p=0.2766 (n.s.)
△2061–23681.23 ± 0.08 (10)p=0.9999 (n.s.)1.13 ± 0.08 (10)p=0.9993 (n.s.)
△2042–23681.13 ± 0.15 (10)p=0.9121 (n.s.)1.03 ± 0.13 (10)p=0.8410 (n.s.)
△2016–23680.93 ± 0.38 (10)p=0.1091 (n.s.)0.86 ± 0.46 (10)p=0.1175 (n.s.)
Ca2+ influx charge (pC)
Wild type2.95 ± 0.3 (10)p=0.5713 (n.s.)25.93 ± 2.22 (10)p=0.4932 (n.s.)
Full transcript3.62 ± 0.36 (10)control group31.71 ± 2.87 (10)control group
△2365–23683.89 ± 0.31 (10)p=0.9838 (n.s.)33.52 ± 2.65 (10)p=0.9943 (n.s.)
△2213–23683.48 ± 0.35 (8)p=0.9996 (n.s.)28.09 ± 2.7 (8)p=0.8924 (n.s.)
△2061–23683.87 ± 0.33 (10)p=0.9893 (n.s.)34.94 ± 2.93 (10)p=0.9132 (n.s.)
△2042–23683.8 ± 0.54 (10)p=0.9977 (n.s.)33.71 ± 4.22 (10)p=0.9910 (n.s.)
△2016–23683.12 ± 0.14 (10)p=0.8063 (n.s.)27.81 ± 1.14 (10)p=0.8255 (n.s.)
EPSC amplitude (nA)
Wild type8.99 ± 1.12 (10)p=0.9995 (n.s.)9.77 ± 0.87 (10)p=0.8167 (n.s.)
Full transcript8.54 ± 0.9 (10)control group8.31 ± 0.79 (10)control group
△2365–23686.88 ± 0.78 (10)p=0.7024 (n.s.)7.45 ± 0.73 (10)p=0.9789 (n.s.)
△2213–23686.05 ± 1.2 (8)p=0.3672 (n.s.)6.7 ± 0.94 (8)p=0.7880 (n.s.)
△2061–23687.05 ± 1.25 (10)p=0.7836 (n.s.)7.72 ± 1.44 (10)p=0.9963 (n.s.)
△2042–23683.38 ± 0.89 (10)p=0.0028 (**)5.28 ± 1.31 (10)p=0.1689 (n.s.)
△2016–23682.49 ± 0.89 (10)p=0.0004 (***)3.61 ± 0.91 (10)p=0.0098 (**)
EPSC 10–90% rise time (ms)
Wild type1.46 ± 0.14 (10)p=0.9908 (n.s.)1.71 ± 0.31 (10)p=0.9995 (n.s.)
Full transcript1.25 ± 0.13 (10)control group1.41 ± 0.18 (10)control group
△2365–23681.79 ± 0.09 (10)p=0.5934 (n.s.)2.18 ± 0.27 (10)p=0.9097 (n.s.)
△2213–23681.88 ± 0.06 (8)p=0.4956 (n.s.)2.43 ± 0.3 (8)p=0.7964 (n.s.)
△2061–23681.8 ± 0.12 (10)p=0.5792 (n.s.)2.07 ± 0.21 (10)p=0.9529 (n.s.)
△2042–23682.46 ± 0.42 (10)p=0.0200 (*)4.67 ± 0.72 (10)p=0.0046 (**)
△2016–23683.2 ± 0.57 (10)p<0.0001 (****)6.99 ± 1.45 (10)p<0.0001 (****)
Synaptic delay (ms)
Wild type1.82 ± 0.13 (10)p=0.9569 (n.s.)1.92 ± 0.2 (10)p=0.9977 (n.s.)
Full transcript1.7 ± 0.12 (10)control group1.73 ± 0.11 (10)control group
△2365–23682.11 ± 0.1 (10)p=0.1170 (n.s.)2.22 ± 0.16 (10)p=0.8464 (n.s.)
△2213–23682.19 ± 0.1 (8)p=0.0615 (n.s.)2.33 ± 0.19 (8)p=0.7507 (n.s.)
△2061–23682.14 ± 0.14 (10)p=0.0748 (n.s.)2.28 ± 0.15 (10)p=0.7714 (n.s.)
△2042–23682.8 ± 0.07 (10)p<0.0001 (****)3.68 ± 0.3 (10)p=0.0019 (**)
△2016–23682.55 ± 0.2 (10)p<0.0001 (****)4.78 ± 0.83 (10)p<0.0001 (****)
  1. *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.

https://doi.org/10.7554/eLife.28412.010
EPSC ratio (3 ms/30 ms)
Wild type0.89 ± 0.05 (10)p=0.5456 (n.s.)
Full transcript1.03 ± 0.03 (10)control group
△2365–23680.92 ± 0.04 (10)p=0.6887 (n.s.)
△2213–23680.87 ± 0.05 (8)p=0.4631 (n.s.)
△2061–23680.93 ± 0.04 (10)p=0.7858 (n.s.)
△2042–23680.61 ± 0.09 (10)p=0.0004 (***)
△2016–23680.50 ± 0.12 (10)p<0.0001 (****)

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