Regulation of exocytosis and fusion pores by synaptotagmin-effector interactions

Abstract

Synaptotagmin (syt) serves as a Ca(2+) sensor in the release of neurotransmitters and hormones. This function depends on the ability of syt to interact with other molecules. Syt binds to phosphatidylserine (PS)-containing lipid bilayers as well as to soluble N-ethylmaleimide sensitive factor receptors (SNAREs) and promotes SNARE assembly. All these interactions are regulated by Ca(2+), but their specific roles in distinct kinetic steps of exocytosis are not well understood. To explore these questions we used amperometry recording from PC12 cells to investigate the kinetics of exocytosis. Syt isoforms and syt I mutants were overexpressed to perturb syt-PS and syt-SNARE interactions to varying degrees and evaluate the effects on fusion event frequency and the rates of fusion pore transitions. Syt I produced more rapid dilation of fusion pores than syt VII or syt IX, consistent with its role in synchronous synaptic release. Stronger syt-PS interactions were accompanied by a higher frequency of fusion events and more stable fusion pores. By contrast, syt-SNARE interactions and syt-induced SNARE assembly were uncorrelated with rates of exocytosis. This associates the syt-PS interaction with two distinct kinetic steps in Ca(2+) triggered exocytosis and supports a role for the syt-PS interaction in stabilizing open fusion pores.

Figure 1.

Protein backbone structure of the cytoplasmic domain of syt I C2AB in ribbon format, with the membrane sketched below. The solution structures of C2A and C2B were modified from (Shao et al., 1998) and (Fernandez et al., 2001). Red spheres depict bound Ca²⁺. The residues mutated for this study, R399 and T328, are indicated and their side chains are rendered in blue.

Figure 2.

Syt I mutations and isoforms affect secretion rate. (A) Sample amperometry traces for wild-type syt I and two syt I mutants. The thick horizontal line below indicates the time of depolarization by a puff of high KCl. (B) Cumulative spike counts were plotted versus time to illustrate the time course of fusion triggered by depolarization (starting at t = 0). (C) Secretion rates for the different mutants were computed as number of spikes in the first 20 s. Amperometry traces for different syt isoforms (D), cumulative spike plots (E), and secretion rates (F). Data were from 42 to 125 cells. *, p < 0.05; **, p < 0.01. Error bars represent SEM.

Figure 3.

Syt I mutations and isoforms affect fusion pore stability. (A) Sample trace with an expanded view of a single vesicle release event showing the prespike foot (PSF). (B) Fusion pore lifetime distributions for wild-type syt I and two mutants, and (C) their mean fusion pore lifetimes. (D) Fusion pore lifetime distributions for control, syt VII, and syt IX (for syt I see B), and (E) their mean fusion pore lifetimes. 315-1021 prespike feet from 42 to 125 cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars represent SEM.

Figure 4.

The effect of syt I mutations and isoforms on fusion pore dynamics. (A) A sample amperometry recording of a kiss-and-run event. (B) The model of fusion pore kinetics used to determine the rate constants k_c and k_d from mean fusion pore lifetime and fraction of kiss-and-run events (Wang et al., 2006). (C) Fraction of kiss-and-run events. (D and E) Computed values of k_c, and k_d for wild-type syt I and two mutants. (F) Fraction of kiss-and-run events. (G and H) Computed values of k_c, and k_d for syt isoforms. Same data sets as Figure 3.

Figure 5.

PS and t-SNARE binding properties of syt I mutations. (A) A sample cosedimentation gel for syt I mutants bound to the indicated concentrations of liposomes (containing 25% PS). [Ca²⁺] = 1 mM. (B) Percentage of protein bound to liposomes plotted versus liposome concentration. (C) Kd-cs values for syt mutations derived from plots in B. (D) A sample cofloatation gel for syt-t-SNARE binding. (E) Syt-t-SNARE binding in the presence of Ca²⁺ or EGTA. (F) Fold-increase in t-SNARE binding (binding in Ca²⁺/binding in EGTA) computed as the ratio from the values in E. Data from at least three independent experiments. *, p < 0.05; ***, p < 0.001. Error bars represent SEM.

Figure 6.

PS binding and t-SNARE binding by syt isoforms. (A) A sample cosedimentation gel for syt isoforms bound to the indicated concentrations of liposomes (containing 15% PS). [Ca²⁺] = 1 mM. (B) Percentage of protein bound to liposomes plotted versus liposome concentration. (C) Kd-cs values for syt mutants derived from plots in B. (D) A sample cofloatation gel for syt-t-SNARE binding. (E) Syt-t-SNARE binding in the presence of Ca²⁺ or EGTA. (F) Fold-increase in t-SNARE binding (binding in Ca²⁺/binding in EGTA) computed as the ratio from the values in E. Data from at least 3 independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars represent SEM.

Figure 7.

Syt mutants and isoforms regulate t-SNARE assembly. (A) Samples were incubated in 1 mM Ca²⁺ or 0.2 mM EGTA for 1 h as indicated by + and −. ± represents the addition of 2 mM EGTA for another 30 min following incubation in 1 mM Ca²⁺ before the coflotation experiment. The intensity ratios for SNAP-25 and syntaxin were averaged for mutants (B) and isoforms (C). Data were pooled from at least 3 independent experiments. *, p < 0.05, **, p < 0.01; ***, p < 0.001. Error bars represent SEM.

Figure 8.

Test for correlation between in vitro interactions and exocytosis in syt I mutants. Plots of secretion rate versus PS-liposome binding Kd-cs (A), fold-increase in t-SNARE binding (Ca²⁺ binding/EGTA binding) (B), and syt-induced t-SNARE assembly (C). Plots of PSF lifetime versus Kd-cs (D), fold-increase in t-SNARE binding (binding in Ca²⁺/binding in EGTA) (E), and syt-induced t-SNARE assembly (F). A statistically significant correlation was found only for PSF duration versus Kd-cs (D).

Figure 9.

Test for correlation between in vitro interactions and exocytosis for syt I isoforms. As in Figure 8 secretion rate and PSF lifetime are plotted versus PS-liposome binding, t-SNARE binding, and SNARE assembly, and again as in Figure 8, a statistically significant correlation was found only for PSF duration versus Kd-cs (D).

Figure 10.

Plots of fusion pore kinetic parameters versus PS-liposome binding (Kd-cs) for mutations (A–C) and isoforms (D–F). The fraction of kiss-and-run event and PSF duration were used to calculate the fusion pore kinetic rate constants k_c and k_d. Only the plots of k_d versus Kd-cs yielded statistically significant correlations (F). The plots of k_d (C and F) had the steepest slopes.