The RIM^R>H mutation tightens influx-release coupling while leaving spatially averaged presynaptic Ca²⁺ transients unaffected. (A) Representative traces of spatially averaged Ca²⁺ transients in type Ib boutons of RIM^wt (grey) and RIM^R>H (red) NMJs (average of 8–12 traces per bouton). (B) Summary graphs for peak amplitude (ΔF/F), average baseline fluorescence (F baseline) and tau decay in RIM^wt (n = 28 boutons, 10 NMJs, nine animals) and RIM^R>H (n = 36 boutons, 10 NMJs, seven animals). (C) Averaged focally recorded eEPSCs in RIM^wt (grey) and RIM^R>H (red; n_eEPSC = 60 per experiment) before (continuous lines) and after application of BAPTA-AM (dashed lines). (D and E) Summary graphs for normalized eEPSC amplitude reduction and normalized eEPSC PPR increase induced by BAPTA in RIM^wt (grey, n = 22 experiments from 12 NMJs in six animals for control and BAPTA, respectively) and RIM^R>H (red, n = 21 experiments from 14 and 16 NMJs in seven and six animals for control and BAPTA, respectively). All displayed measurements made with L3 larvae expressing indicated RIM variant as UAS-RIM^X transgene under control of the motor neuron-specific ok6-GAL4 enhancer. See also Supplementary Table 5.