Concurrent HK and DN mutations eliminate the enhancement of SV release.

(A) Sequence alignment of the C1 and C2B domains between the worm UNC-13L, the rat Munc13-1, and the fly UNC-13A. The highly conserved residues in all three species are marked in blue, while shared only by two species are marked in light blue. The HK mutation in C1 that affects DAG binding and the DN mutations in C2B that abolish Ca2+ binding are marked bold and indicated by inverted red triangles. (B) Example traces of stimulus-evoked EPSCs from UNC-13L, LHK, LD1-5N, and LHK+D1-5N rescued animals. (C) Quantification of the evoked EPSC amplitude and charge transfer from the same genotypes as in B. (D) Representative mEPSC traces (recorded at 0mM and 1mM Ca2+) from the indicated genotypes. (E and F) Averaged mEPSC frequency and amplitude from the same genotypes as in D. (G) Representative mIPSC traces (recorded at 0mM and 1mM Ca2+) from the indicated genotypes. (H and I) Quantification of the frequency and amplitude of the mIPSCs from the same genotypes as in G. (J) Representative confocal z stack images for mApple-tagged UNC-13L, UNC-13LHK, UNC-13LDN, and UNC-13LHK+DN (all driven by the unc-129 promoter). Scale bar, 5 μm. (K) Quantification of the fluorescence intensity from the lines in J. Data are mean ± SEM (** p < 0.01, *** p < 0.001 when compared to UNC-13L rescue; n.s., non-significant when compared to UNC-13L rescue; one-way ANOVA).

The N terminus in UNC-13L regulates the functions of C1 and C2B.

(A) Cartoon depicting the HK and DN mutations in UNC-13L and UNC-13R.

(B) Example traces of stimulus-evoked EPSCs from UNC-13R, RHK, RD1-5N, and RHK+D1-5N rescued animals. (C) Quantification of the evoked EPSC amplitude and charge transfer from the same genotypes as in B. (D) Representative mEPSC traces (recorded at 0mM and 1mM Ca2+) from the indicated genotypes. (E and F) Quantification of the frequency and amplitude of the mEPSCs from the same genotypes as in D. (G) Representative mIPSC traces (recorded at 0mM and 1mM Ca2+) from the indicated genotypes. (H and I) Quantification of the frequency and amplitude of the mIPSCs from the same genotypes as in G. Data are mean ± SEM (** p < 0.01, *** p < 0.001 when compared to UNC-13L rescue; n.s., non-significant when compared to UNC-13L rescue; one-way ANOVA).

The C2A domain regulates the functions of C1 and C2B.

(A) Domain structure of UNC-13L and UNC-13LΔC2A. (B) Example traces of stimulus-evoked EPSCs from UNC-13ΔC2A, ΔC2AHK, ΔC2AD1-5N, and ΔC2AHK+D1-5N rescued animals. (C) Quantification of the evoked EPSC amplitude and charge transfer from the same genotypes as in B. (D) Representative mEPSC traces (recorded at 0mM and 1mM Ca2+) from the indicated genotypes. (E and F) Quantification of the frequency and amplitude of the mEPSCs from the same genotypes as in (D). (G) Representative mIPSC traces (recorded at 0-mM and 1-mM Ca2+) from the indicated genotypes. (H and I) Quantification of the frequency and amplitude of the mIPSCs from the same genotypes as in G. Data are mean ± SEM (** p < 0.01, *** p < 0.001 when compared to UNC-13ΔC2A rescue; n.s., non-significant; one-way ANOVA).

The X domain regulates the functions of C1 and C2B.

(A) Domain structure of UNC-13L and UNC-13ΔX. (B) Example traces of stimulus-evoked EPSCs from UNC-13ΔX, ΔXHK, ΔXD1-5N, and ΔXHK+D1-5N rescued animals. (C) Quantification of the evoked EPSC amplitude and charge transfer from the same genotypes as in B. Data are mean ± SEM (n.s., non-significant; one-way ANOVA).

(D) Representative mEPSC traces (recorded at 0mM and 1mM Ca2+) from the indicated genotypes. (E and F) Quantification of the frequency and amplitude of the mEPSCs from the same genotypes as in D. Data are mean ± SEM (** p < 0.01 when compared to UNC-13ΔX rescue; n.s., non-significant when compared to UNC-13ΔX rescue; one-way ANOVA).

(G) Representative mIPSC traces (recorded at 0mM and 1mM Ca2+) from the indicated genotypes. (H and I) Quantification of the frequency and amplitude of the mIPSCs from the same genotypes as in G. Data are mean ± SEM (* p < 0.05, ** p < 0.01, *** p < 0.001 when compared to UNC-13ΔX rescue; one-way ANOVA).

Individual C1 and C2B promotes SV release in a gain-of-function manner.

(A) Cartoons depicting UNC-13L, MUNC2C, C1MUNC2C, C2BMUNC2C, and C2Blinker3MUNC2C. (B) Example traces of stimulus-evoked EPSCs from the indicated genotypes. (C) Quantification of the evoked EPSC amplitude and charge transfer from the same genotypes as in B. (D and E) Averaged mEPSC frequency and amplitude (recorded at 0mM and 1mM Ca2+) from the same genotypes as in B. (F and G) Quantification of the mIPSC frequency and amplitude (recorded at 0mM and 1mM Ca2+) from the same genotypes as in B. Data are mean ± SEM (* p < 0.05, *** p < 0.001 when compared to MUNC2C rescue; ## p < 0.01, ### p < 0.001; n.s., non-significant; one-way ANOVA).

The polybasic motif is critical for the C2B function.

(A) Left, crystal structures of UNC-13 C2B (EBI AlphaFold database, UniProt ID: P27715) and Munc13-1 C2B (from PDB: 7T7V). The polybasic residues in each C2B are labelled in purple, and the five Ca2+-binding sites (aspartates) are labelled in blue. Right, Sequence comparison of UNC-13 C2B and Munc13-1 C2B. The polybasic residues are indicated by purple arrowheads, and the five aspartates are indicated by blue arrowheads. (B) Example traces of stimulus-evoked EPSCs from MUNC2C, C2BMUNC2C, C2BKQMUNC2C, C2Blinker3MUNC2C, and C2BKQlinker3MUNC2C rescued animals. (C) Quantification of the evoked EPSC amplitude and charge transfer from the same genotypes as in B. (D) Representative mEPSC traces (recorded at 0mM and 1mM Ca2+) from the indicated genotypes. (E and F) Quantification of the frequency and amplitude of the mEPSCs. (G) Representative mIPSC traces (recorded at 0mM and 1mM Ca2+) from the indicated genotypes in B. (H and I) Quantification of the frequency and amplitude of the mIPSCs. Data are mean ± SEM (*** p < 0.001; n.s., non-significant when compared to MUNC2C; one-way ANOVA).

Disrupting C1 and C2B membrane interaction simultaneously results in the inactivation of UNC-13S.

(A) Cartoon depicting the HK and DN mutations in UNC-13L, UNC-13R, and UNC-13S. (B) Example traces of stimulus-evoked EPSCs from UNC-13S, SHK, SD1-5N, SHK+D1-5N rescued animals. (C) Averaged evoked EPSC amplitude and charge transfer. (D) Representative mEPSC traces (recorded at 0mM and 1mM Ca2+) from the indicated genotypes in B. (E and F) Quantification of the frequency and amplitude of the mEPSCs. (G) Representative mIPSC traces (recorded at 0mM and 1mM Ca2+) from the indicated genotypes in B.(H and I) Quantification of the frequency and amplitude of the mIPSCs. (J) Representative confocal z stack images for mApple-tagged UNC-13S, SHK, SDN, and SHK+DN (all driven by the unc-129 promoter). Scale bar, 5 μm. (K) Quantification of the fluorescence intensity. Data are mean ± SEM (** p < 0.01, *** p < 0.001 when compared to UNC-13S rescue; n.s., non-significant; one-way ANOVA).

The concurrent mutations of HK and PL in UNC-13L and UNC-13S do not decrease SV release.

(A) Positions of the PL mutation in UNC-13L and UNC-13S. (B, D) Example traces of stimulus-evoked EPSCs from unc-13 mutants rescued with wild-type UNC-13L and S, and UNC-13L or UNC-13S carrying HK, PL, and HK+PL mutations. (C, E) Quantification of the evoked EPSC amplitude and charge transfer from the same genotypes as in B and D. (F, I) Representative mEPSC traces (recorded at 0mM and 1mM Ca2+) from the same genotypes in B and D. (G-K) Quantification of the frequency and amplitude of the mEPSCs (G and J, 0mM Ca2+; H and K, 1mM Ca2+). (L, O) Representative mIPSC traces (recorded at 0mM and 1mM Ca2+) from the indicated genotypes. (M-Q) Averaged mIPSC frequency and amplitude in 0mM Ca2+ (M, P) and 0mM Ca2+ (N, Q). Data are mean ± SEM (* p < 0.05, ** p < 0.01, *** p < 0.001 when compared to UNC-13L or UNC-13S rescue; # p < 0.05, ## p < 0.01; n.s., non-significant; one-way ANOVA).

Concurrent HK and D3,4N mutations also eliminate the enhancement of SV release.

(A) Quantification of the evoked EPSC amplitude and charge transfer from UNC-13L, LHK, LD3,4N, and LHK+D3,4N rescued animals. (B and C) Quantification of the frequency and amplitude of the mEPSCs (recorded at 0mM and 1mM Ca2+) from the same genotypes in A. (D and E) Quantification of the frequency and amplitude of the mIPSCs (recorded at 0mM and 1mM Ca2+) from the same genotypes as in A. Data are mean ± SEM (* p < 0.05, ** p < 0.01, *** p < 0.001 when compared to UNC-13L; n.s., non-significant when compared to UNC-13L; one-way ANOVA).

(related to Figure 1).

The mutual inhibition between C1 and C2B requires the linkers.

(A) Averaged evoked EPSC amplitude and charge transfer from unc-13 mutants rescued with MUNC2C, C1MUNC2C, C2BMUNC2C, and C1C2BMUNC2C. (B, C) Quantification of the mEPSC frequency and amplitude in 0mM Ca2+ and 1mM Ca2+, respectively. (D, E) Summary of the mIPSC frequency and amplitude in 0mM Ca2+ and 1mM Ca2+, respectively. Data are mean ± SEM (* p < 0.05, ** p < 0.01; n.s., non-significant; one-way ANOVA).

(related to Figure 5).

The polybasic motifs in synaptotagmin-1 and rabphilin-3A.

Left, crystal structures of rat synaptotagmin-1 C2B (from PDB: 1TJM) and rat rabphilin-3A C2B (from PDB: 5LO8). The polybasic residues in each C2B are labelled in purple, and the five Ca2+-binding sites (aspartates) are labelled in blue. Right, Sequence alignment of the C2B domains in UNC-13, Munc13-1, synaptotagmin-1, and rabphilin-3A. The polybasic residues are indicated by purple arrowheads, and the five aspartates are indicated by blue arrowheads.

(related to Figure 6).

The polybasic mutations in UNC-13L do not affect SV release.

(A) Example traces of stimulus-evoked EPSCs from unc-13 mutants rescued with wild-type UNC-13L and UNC-13LKQ. (B) Quantification of the evoked EPSC amplitude and charge transfer. (C, F) Representative mEPSC and mIPSC traces (recorded at 0mM and 1mM Ca2+) from the same genotypes in A. (D,E, G, H) Quantification of the frequency and amplitude of the mEPSCs and mIPSCs in C and F (D and G, 0mM Ca2+; E and H, 1mM Ca2+). Data are mean ± SEM (one-way ANOVA).

(related to Figure 6).

The HK and DN mutations do not alter release kinetics in UNC-13S and UNC-13R.

(A, B) Although evoked EPSCs were enhanced in UNC-13S and UNC-13R by the HK and DN mutations, the release kinetics (10-90% risetime and decay) remains unchanged. Data are mean ± SEM (** p < 0.01 when compared to wild type; n.s., non-significant; one-way ANOVA).

(related to Figure 7).