(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: …
(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) …
(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 …
(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 …
(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 …
(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 …
(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 …
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…
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.) |
*One-Way ANOVA with a Dunnett’s Test with condition knockout as reference group was performed to calculate statistical significance.
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.
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 (****) |