RIM-BP2 KO reduces calcium currents and synaptic vesicle release

(A) The terminal was depolarized from −80 mV to +10 mV in WT (black) and RIM-BP2 KO (gray) hippocampal mossy fiber boutons. Ca2+ currents (ICa) and membrane capacitance (Cm) in response to a 10 ms pulse (Vm) are shown. (B) (top) The peak Ca2+ currents were plotted against the pulse duration. At 10 ms, p = 0.0499 (Wilcoxon rank sum test). (bottom) The capacitance jumps were plotted against the pulse duration. At 20 and 100 ms, p = 0.0379 and 0.0379, respectively (Wilcoxon rank sum test). Black filled circles and gray open circles represent the data from WT (n = 8) and RIM-BP2 KO (n = 8), respectively. Each data point represents mean ± SEM. (C) Experimental protocol and representative traces for presynaptic Ca2+ current measurements in WT (black) and RIM-BP2 KO (gray) boutons. Terminals were sequentially depolarized for 5 ms with 2 ms intervals from −80 mV to +70 mV by 10 mV steps. (D) Current-voltage relationships of peak Ca2+ currents in WT (black filled circle; n = 4-6) and RIM-BP2 KO (gray open circle; n = 4-5). ICas were elicited by 5 ms depolarizations. Each data point represents mean ± SEM. Numerical values of plots are provided in Figure 1-source data 1.

Source data 1. Datasets of Ca2+-current and capacitance amplitudes presented in Figure 1.

High extracellular calcium concentration rescues the decrease of synaptic vesicle release, but does not affect the RRP size

(A) (top) The ICas were plotted against the pulse duration in 4 mM [Ca2+]ext. (bottom) The Cms were plotted against the pulse duration in 4 mM [Ca2+]ext. Black filled squares and gray open squares represent the data from WT (n = 4-6) and RIM-BP2 KO (n = 3-4), respectively. For comparison, the WT data in 2mM [Ca2+]ext are superimposed (black filled circle; n = 8) (the same data set as Fig. 1B). Each data point represents mean ± SEM. (B) Average ICas (top) and Cms (bottom) in response to a 100 ms pulse in WT (black bars) and RIM-BP2 KO (gray bars) terminals. Extracellular Ca2+ concentration was 2 mM (left) or 4 mM (right). Error bars show SEM. Circles indicate individual values. Numerical values of plots are provided in Figure 2-source data 1.

Source data 1. Datasets of Ca2+-current and capacitance amplitudes presented in Figure 2.

Calcium-dependence of the release kinetics and the RRP size

(A) (top) The relationship between peak Ca2+ currents and pulse durations at different [Ca2+]exts. (bottom) The relationship between capacitance jumps and pulse durations at different [Ca2+]exts. Gray open circles, black diamonds and black triangles represent the data from RIM-BP2 KO at 2 mM [Ca2+]ext (n = 8) (the same data set as Fig. 1B), WT at 1 mM [Ca2+]ext (n = 3-5) and WT at 1.5 mM [Ca2+]ext (n = 5-7), respectively. Each data point represents mean ± SEM. (B) Capacitance jumps at various [Ca2+]exts are plotted against calcium current amplitudes. Pulses were 100 ms depolarization from −80 mV to +10 mV. Each data point represents mean ± SEM. Data points were fitted with a Hill equation with n = 3. (C) (left) Average ICas at indicated [Ca2+]exts were normalized to the response at 2 mM [Ca2+]ext in each genotype. (right) Average Cms at indicated [Ca2+]exts were normalized to the 2 mM [Ca2+]ext response in each genotype. Numerical values of plots are provided in Figure 3-source data 1.

Source data 1. Datasets of numerical values presented in Figure 3.

The effects of high EGTA on calcium currents and synaptic vesicle exocytosis

(A) Example traces in response to a 5 ms depolarizing pulse to +10 mV in WT (left) and RIM-BP2 KO (right) boutons. Ca2+ current (ICa) and membrane capacitance (Cm) recorded with 0.5 mM EGTA (black) or 5 mM EGTA (red) in the patch pipette are shown. (B) The ICas (top) and Cms (bottom) are plotted against the pulse duration. The patch pipette contained 5 mM EGTA. Black filled diamonds and gray open diamonds represent the data from WT (n = 4-5) and RIM-BP2 KO (n = 4), respectively. Each data point indicates mean ± SEM. (C, D) Average Cms (C) and ICas (D) elicited by a 20 ms pulse in WT (filled bars) and RIM-BP2 KO (hatched bars). The extracellular Ca2+ concentration was 1 mM or 2 mM. The intracellular solution contained either 0.5 mM EGTA (black) or 5 mM EGTA (red). Error bars show SEM. Circles and diamonds indicate individual values. Numerical values of plots are provided in Figure 4-source data 1.

Source data 1. Datasets of Ca2+-current and capacitance amplitudes presented in Figure 4.

RIM-BP2 deletion alters the density of Cav2.1 clusters within the AZ

(A) Confocal (top) and STED (bottom) images of Munc13-1 (magenta) and Cav2.1 (yellow) clusters at hippocampal mossy fiber terminals of WT (left) and RIM-BP2 KO (right) mice. (Scale bar: 500 nm.) (B) Confocal images of PSD95 (cyan) and VGLUT1 (white) to identify glutamatergic synapses in CA3 stratum lucidum: mossy fiber-CA3 synapses. (Scale bar: 500 nm.) The image is taken from same region shown in (A), (left). (C) Histograms (top) and cumulative distribution plots (bottom) of the total signal intensity of Cav2.1 (left), Cav2.2, and Munc13-1 (right) at AZs in WT (black) and RIM-BP2 KO (red) mice. (D) The average nearest neighbor distance between Cav2.1 and Munc13-1 clusters in WT (black; n = 4 animals) and RIM-BP2 KO (red; n = 4 animals) mice. Several hundreds of AZs per image were analyzed. Data show the average value of distance per animal and error bars represent SEM. Each data point indicates individual values. Numerical values of plots are provided in Figure 5-source data 1.

Source data 1. Datasets of numerical values presented in Figure 5.

The relationship between synaptic vesicle release and total Ca2+ charge

Capacitance jumps elicited by various pulse durations are plotted against total Ca2+ charge. The total Ca2+ charge is calculated by multiplying a peak Ca2+ current amplitude by pulse duration. Each symbol represents a data point obtained from WT (black filled symbols) and RIM-BP2 KO (gray open symbols) mice at various [Ca2+]ext. Data points were fitted by a single exponential function. Numerical values of plots are provided in Figure 3-figure supplement 1-source data 1.

Figure supplement 1-source data 1. Datasets of Ca2+ charge and ΔCm presented in Figure 3-figure supplement 1.

STED microscopy confirmed loss of RIM-BP2 proteins in KO terminals

Confocal (top) and STED (middle) images of RIM-BP2 (yellow) in WT (left) and RIM-BP2 KO (right) hippocampal mossy fiber terminals identified by PSD95 (cyan) and VGLUT1 (white) (bottom). (Scale bar: 500 nm.) The bottom images show the same region as top and middle.

STED imaging of Cav2.2 at hippocampal mossy fiber terminals

Confocal (top) and STED (middle) images of Munc13-1 (magenta) and Cav2.2 (yellow) clusters in WT (left) and RIM-BP2 KO (right) mice. Confocal images of PSD95 (cyan) and VGLUT1 (white) in the same region as top and middle are also shown (bottom). (Scale bar: 500 nm.)