Figure 4. Syt-7 increases forward priming and decreases depriming.

(A) Simple model (Model I) featuring a single primed vesicle pool, a reversible priming reaction (forward rate: k₁; reverse rate: k_-1), and a fusion rate k_f. (B) Recovery in WT cells (persian green) and Syt-7 KO cells (vermilion) of secretion at 60 ms after an uncaging stimulus, approximately corresponding to the RRP. Stim1, Stim2 = amplitude of secretion after first, or second, stimulus. Shown are mean ± SEM, plus a fit of a mono-exponential recovery curve (lines). The fit to the Syt-7 KO is also shown after scaling to the same amplitude as the WT curve (vermilion broken line), to show the faster kinetics. The fitted parameters: Plateau and the rate constant of recovery, which is the rate constant for depriming, k_-1, under simplified assumptions (see text). Both Plateau and k_-1 are significantly different from each other (Extra sum-of-squares F test for comparison of models, p<0.0001). (C) Same as B, but secretion at 600 ms after uncaging was used, approximately corresponding to the fusion of both RRP and SRP. (D) Model (Model II) featuring a single primed vesicle pool, limited by a fixed number of release sites, PP_max, a reversible priming reaction (forward rate: k₁; reverse rate: k_-1), and a fusion rate k_f. (E) Recovery (at 60 ms) in the WT (green, Model II) with parameters recalculated from the fit of Model I to the data (panel B, see Materials and methods), and Syt-7 KO curve (vermilion), with the same change in priming and depriming rate as observed experimentally, now translated to a release site model, and after scaling to the WT amplitude (vermilion broken line). Under these circumstances, recovery in the Syt-7 KO trails the WT. (F) Recovery (at 600 ms) in the WT (Model II) with parameters recalculated from panel C, and in the KO (vermilion), when introducing the same changes as observed experimentally, translated to a release site model, and after scaling to the WT amplitude (vermilion broken lines). Under these circumstances, the Syt-7 KO leads the WT trace, but the differences are small. Data information: Data are presented as mean ± SEM. **: p<0.01; ***: p<0.001. In (B, C) Student’s t-test: test between genotypes (WT vs. KO) at the same inter stimulus intervals; Mann Whitney test: (600 ms: WT22s vs. KO22s).

Figure 4—figure supplement 1. Differences in unpriming rate can be distinguished during pool recovery, if the Primed Pool is not limited by release sites.

(A) In Model I, we assume that vesicles from a very large Depot Pool prime reversibly (priming rate: k₁; unpriming rate: k_-1) into the Primed Pool, which is free to change is size. Fusion happens from the primed pool with rate k_f. The bottom left panel shows the recovery of the primed pool (black line) with parameters from a fit to WT data (Figure 4B), k1⋅ DP = 3.21 fF/s, k_-1 = 0.043 s⁻¹, and after increasing (red line, k_-1 = 0.091 s⁻¹) or decreasing (blue line, k_-1 = 0.020 s⁻¹) the k_-1 by a factor of 2.12. The red line corresponds closely to the fit of the Syt-7 KO condition. Right-hand panel: after normalization, it is appreciated that the high-k_-1 condition recovers with faster kinetics than the WT condition, which recovers faster than the low-k₁ condition. (B) In Model II, we assume that the Primed Pool is limited by a fixed number of release sites, which sets an upper limit (PP_max) to the Primed Pool size. The bottom panel shows the recovery of the primed pool (black line) with parameters fitting the Syt-7 WT condition (black curve, see Materials and methods), and after increasing (red curve) or decreasing (blue curve) the unpriming rate (k_-1) by a factor of 2.1. Right-hand panel: normalized traces. In the presence of release sites, recovery becomes less sensitive to k_-1.

Figure 4—figure supplement 2. Double stimulation experiments in Syt-7 WT and KO cells.

(A) Mean [Ca²⁺]_i and capacitance traces of Syt-7 WT cells during double uncaging experiments. Time intervals are indicated in panel B. For time intervals 4 and 8 s, the two sequential stimulations are shown on a continuous time axis, for longer intervals they are shown overlaid. (B) Quantification of capacitance at 60 and 600 ms after flash uncaging, for the first and second stimulation. Number of cells (N) are shown on bar diagrams. (C) Mean [Ca²⁺]_i and capacitance traces of Syt-7 KO cells during double uncaging experiments. Arranged as in A. *: p<0.05; **: p<0.01; ***: p<0.001. Mann-Whitney test.