Resolving kinetic intermediates during the regulated assembly and disassembly of fusion pores

Abstract

The opening of a fusion pore during exocytosis creates the first aqueous connection between the lumen of a vesicle and the extracellular space. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate the formation of these dynamic structures, and their kinetic transitions are tightly regulated by accessory proteins at the synapse. Here, we utilize two single molecule approaches, nanodisc-based planar bilayer electrophysiology and single-molecule FRET, to address the relationship between SNARE complex assembly and rapid (micro-millisecond) fusion pore transitions, and to define the role of accessory proteins. Synaptotagmin (syt) 1, a major Ca²⁺-sensor for synaptic vesicle exocytosis, drove the formation of an intermediate: committed trans-SNARE complexes that form large, stable pores. Once open, these pores could only be closed by the action of the ATPase, NSF. Time-resolved measurements revealed that NSF-mediated pore closure occurred via a complex 'stuttering' mechanism. This simplified system thus reveals the dynamic formation and dissolution of fusion pores.

Fig. 1. Syt1 regulation of single fusion pores measured via planar lipid bilayer electrophysiology.

a Illustration of the nanodisc-black lipid membrane (ND-BLM) system, drawn to scale, indicating the different ND preparations used in this study. The number indicates the syb2/syt1 copy number (1:1) per ND; the subscript S stands for small, 13 nm, NDs and the subscript L stands for large, 30 nm, NDs. Traces of single pores with/without syt1 are shown for ND3_S (b), ND3_L (c), and ND9_L (d). In each trace, minus (−) Ca²⁺ contains 1 mM BAPTA (in all bilayer recording experiments in this study) and plus (+) Ca²⁺ contains 500 µM [Ca²⁺]_free. Closed (C) and open (O) states are shown; the current and time scale, for all traces, is shown in the inset in b. O₁ and O₂ indicate open state currents obtained before and after addition of Ca²⁺, respectively. e Fraction of trials in which a fusion pore was detected, plotted as percentage of occurrence. ND3_S, ND3_L, and ND9_L, in the presence (+) or absence (−) of Ca²⁺ and in the presence (+) and absence (−) of syt1 were compared. Three independent sets of NDs of each type were used, and the total number of measurements obtained under each condition (n) is indicated. Pearson’s χ² analysis of pores formed by ND3_S, ND3_L, and ND9_L was performed; *p < 0.05, ***p < 0.001.

Fig. 2. Quantification of syt1-regulated fusion pore properties.

a Cumulative distribution functions (CDF) of single-channel conductances across different trials for each experimental condition. b Open dwell time histograms for ND3_S and ND9_L, plus or minus syt1 and Ca²⁺, are shown. c Pie diagrams showing the number of exponentials required to fit the open- and closed-state CDFs for individual pores under the indicated conditions. Details are described in Supplementary Fig. 4. d Conductance values (γ) (upper panel), open time (middle panel), and percentage of occurrence (lower panel) at each indicated [Ca²⁺]_free are plotted. n = 16, 10, 10, 15; three different sets of ND preparations were used. In the open time plot, mean values for the open-state dwell times of individual traces were quantified. Error bars indicate SEM. e Percentage of occurrence of pore formation, plotted for WT syt1 versus a Ca²⁺ ligand mutant (CLM), D230/232N/D363/365N, that fails to bind Ca²⁺ via either C2 domain. The number of independent trials (n) are indicated in e. Pearson’s χ² analysis was performed; ***p < 0.001.

Fig. 3. Syt1•PIP₂ interactions stabilize the open state of fusion pores.

a Traces of single pores, formed using ND3_S/syt1 plus Ca²⁺, with (+, upper panel) and without (−, lower panel) 2% PIP₂ in the BLM. Respective current histograms are shown beside the traces. Closed (C), open (O), and partially open (P) states are shown; the current and time scale applies to both traces. b Open dwell time histogram of pores (combined data from all trials), under the conditions described in a. c Fraction of time pores were open over a 30-min period, calculated from the data collected under the indicated conditions. Error bars indicate SEM from ten independent BLMs and three independent sets of each kind of ND; the Student’s T test was performed to compare the two means; ***p < 0.001.

Fig. 4. smFRET reveals the impact of syt1 on trans-SNARE complex assembly.

smFRET histograms of trans-SNARE complexes, under the conditions indicated on the far right, using FRET pairs (cy3, shown as blue dots and cy5, shown as red dots) near the N- (NN; left panels) or the C-termini (CC; right panels) of the SNARE motifs of syb2 and syntaxin-1A, respectively. For clarity, the t-SNARE heterodimer is presented by a single black line, and because NN smFRET was not affected by Ca²⁺ or syt1, only the starting condition is illustrated for the NN experiments (upper left). In the illustrations on the right (+/− Ca²⁺ and syt1), vertical lines indicate the relative distance between v- and t-SNARE NDs under each condition. NN: n = 67 (−Ca²⁺, −syt1), 61 (+Ca²⁺, −syt1), 83 (−Ca²⁺, +syt1), 64 (+Ca²⁺, +syt1). CC: n = 79 (−Ca²⁺, −syt1), 69 (+Ca²⁺, −syt1), 74 (−Ca²⁺, +syt1), 87 (+Ca²⁺, +syt1). ND1_S were used in all experiments. Data were collected using three independent sets of NDs. In all smFRET experiments, −Ca²⁺ samples contained 1 mM EGTA; +Ca²⁺ samples contained 500 µM [Ca²⁺]_free.

Fig. 5. Ca²⁺•syt1 drives trans-SNARE complexes into a functionally committed state.

a Representative recording of ND9_L lacking or containing syt1. Once pores were open, 20 µM cd-syb2 was added to each reaction. Closed (C) and open (O) states are indicated; the current/time scale for all traces is shown in the inset, left. Right panel: respective current histograms are shown. b Representative recordings of ND9_L bearing syt1 in 500 µM [Ca²⁺]_free are shown, followed by the addition of 1.5 mM BAPTA (arrow), further followed by 20 µM cd-syb2 (arrow). The current/time scale is shown on the bottom left. c Fraction of time individual pores were open over a 45-min period, plotted for each condition as indicated. Error bars indicate SEM from 12 and 8 independent BLMs for a and b, respectively; 3 independent sets of NDs were used.

Fig. 6. α-SNAP/NSF-ATP disassemble trans-SNARE complexes formed by Ca²⁺•syt1.

a Representative recording of a pore formed by ND3_S/syt1 in 500 µM [Ca²⁺]_free. After pore formation, 0.3 µM α-SNAP, 0.3 µM NSF, 1 mM ATP, and 5 mM MgCl₂ were added (red arrow). In the lower panel, ATP was replaced with 1 mM ATP-γ-S. Two different epochs of each trace are shown; the time interval between each epoch is indicated. Closed (C), open (O), and partially open (P) states are marked; the current/time scale for all traces is provided in the inset. After addition of the disassembly factors, recordings were divided arbitrarily into five 720-s epochs; the fraction of time the pores were open during each epoch was quantified for individual traces and plotted beside each panel. n = 5 BLMs using ATP, and n = 3 BLMs using ATP-γ-S; two different sets of NDs were used for each condition. b Representative current histograms for the experiments described above are shown. c Illustration (drawn to scale) shows the α-SNAP/NSF/ATP/Mg²⁺-mediated disassembly of SNAREs, leading to fusion pore closure.