Fig. 4.

Impact of SYT1 variants on initial exocytic rate. Hippocampal neurons transfected with SYT1-pHluorin variants or wild-type SYT1-pHluorin were stimulated with 1200 AP at 10 Hz in the presence of bafilomycin A1 and perfused with ammonia buffer to reveal total SYT1-pHluorin fluorescence. (a, c) Time course (first 5 s of stimulation) of mean ΔF/F₀ of C2B (a) or C2A (c) domain SYT1-pHluorin variants normalised to maximum fluorescence induced by ammonia buffer. SEM indicated by shading. (b, d) Initial exocytic rates for C2B (b) or C2A (d) domain variants over the first 5 s of stimulation. One-way ANOVA with Dunnett's multiple comparison test compared to WT; b: M303V p = 0.0009, S309P p = <0.0001, Y365C p = 0.020, G369D p = 0.032, I368T p = 0.0001. d: L159R p = 0.40, T196K p = 0.63, E209K p = 0.44, E219Q p = 0.16, I368T p = 0.0048. Data displayed as mean ± SEM, n = 7–11 (individual field of view from an independent coverslip, from at least 3 independent cultures each comprising at least 3 embryos; WT n = 9 for a, b, n = 10 for c, d; I368T n = 8 for a, b, n = 7 for c, d; M303V n = 8; S309P = 8; Y365C = 8; G369D = 9; L159R = 9; T196K = 11; E209K = 9; E219Q = 9). In b, d ∗ indicates p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 compared to WT; also see Supplementary Table S2h and i