Phospholipids Differentially Regulate Ca2+ Binding to Synaptotagmin-1

Abstract

Synaptotagmin-1 (Syt-1) is a calcium sensing protein that is resident in synaptic vesicles. It is well established that Syt-1 is essential for fast and synchronous neurotransmitter release. However, the role of Ca²⁺ and phospholipid binding in the function of Syt-1, and ultimately in neurotransmitter release, is unclear. Here, we investigate the binding of Ca²⁺ to Syt-1, first in the absence of lipids, using native mass spectrometry to evaluate individual binding affinities. Syt-1 binds to one Ca²⁺ with a K_D ∼ 45 μM. Each subsequent binding affinity (n ≥ 2) is successively unfavorable. Given that Syt-1 has been reported to bind anionic phospholipids to modulate the Ca²⁺ binding affinity, we explored the extent that Ca²⁺ binding was mediated by selected anionic phospholipid binding. We found that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and dioleoylphosphatidylserine (DOPS) positively modulated Ca²⁺ binding. However, the extent of Syt-1 binding to phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂) was reduced with increasing [Ca²⁺]. Overall, we find that specific lipids differentially modulate Ca²⁺ binding. Given that these lipids are enriched in different subcellular compartments and therefore may interact with Syt-1 at different stages of the synaptic vesicle cycle, we propose a regulatory mechanism involving Syt-1, Ca²⁺, and anionic phospholipids that may also control some aspects of vesicular exocytosis.

Figure 1

Syt-1 localization and structure. (a) Cartoon depiction of [Ca²⁺] gradient experienced within a presynapse immediately following Ca²⁺ influx. In this simplified model, the concentration gradient of Ca²⁺ decreases from ∼100 μM near the plasma membrane, to much lower concentrations within the center of the neuron. Syt-1 locations depend on the positions of SVs relative to this gradient. Values adapted from ref. (b) Model of Syt-1 on an SV and overall domain architecture. The construct used in this study consists of residues 96–421, encompassing both Ca²⁺ sensing domains. (c) Structure of Ca²⁺-bound Syt-1 residues 141–421 (PDB 5CCH). Green spheres depict Ca²⁺; the fifth Ca²⁺, not observed in the structure, is depicted as a red sphere labeled in gray.

Figure 2

Native mass spectrometry analysis of Syt-1. (a) Native mass spectrum of Syt-1. Measured masses are shown. (b) Detailed view of the 12⁺ charge state to demonstrate that a single binding event is present without exogenous Ca²⁺ addition (dark red circle). (c) Native mass spectrum collected following incubation of Syt-1 (12 μM) with 140 μM Ca(OAc)₂. (d) Expansion of the 12⁺ charge state demonstrates Ca²⁺ binding event from zero (apo) to five.

Figure 3

Titration of Ca²⁺ to Syt-1. (a) Stacked native mass spectra at increasing [Ca²⁺] concentrations following EDTA treatment of Syt-1 (12 μM) to remove endogenous binding. The 12⁺ charge state is shaded (pink). (b) Representative native mass spectra of the 12⁺ charge state with increasing [Ca²⁺]. (c) Plot of mole fraction for each Ca²⁺-bound state as a function of total [Ca²⁺]. Solid lines show the fit to an equilibrium binding model to determine the relative binding affinities. Individual measurements from n = 3 independent replicates are shown (colored circles). (d) Bar chart to show magnitude of K_D values for individual Ca²⁺ binding events. K_D values are reported as mean ± standard deviations, which were derived from an estimated covariance matrix.

Figure 4

Role of lipids on Ca²⁺ binding to Syt-1. (a) (Left) Representative native mass spectra of Syt-1 (12 μM) in the presence of 125 μM DOPS. (Right) magnified view of the Ca²⁺ and DOPS binding distribution. (b) (Left) Representative native mass spectra of Syt-1 (12 μM) in the presence of 125 μM PI(4,5)P₂. (Right) Magnified view of the Ca²⁺ and PI(4,5)P₂ binding distribution. (c) Representative native mass spectra of Syt-1 (12 μM) in the presence of 125 μM PI(3,4)P₂. (Inset) magnified view to show the Ca²⁺ binding distribution in the presence of 100 μM Ca²⁺. (d) (Left) Representative native mass spectra of Syt-1 (12 μM) in the presence of 125 μM PI(3,5)P₂. (Right) Magnified view to show the absence of Ca²⁺ and PI(3,5)P₂ binding. (e) (Left) representative native mass spectra of Syt-1 (12 μM) in the presence of 125 μM DPPC. (Right) Magnified view to show the absence of Ca²⁺ and DPPC binding. (f) Bar plot of the relative intensities of the Syt-1-lipid-Ca²⁺ bound state in the presence of select lipids at different [Ca²⁺]. f.c. = fold change. (g) Bar plot of the relative intensity of Syt-1-PI(3,5)P₂ at different [Ca²⁺]. Bars represent the average from n = 3 independent replicates, and error bars represent the standard deviation. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Deconvoluted mass spectra are shown in Figure S3.

Figure 5

Lipid binding regulates Ca²⁺ binding to Syt-1. Hypothetical model depicting the key roles of PI(3,5)P₂ and PI(4,5)P₂ binding to Syt-1. PI(3,5)P₂ is sequestered from membranes by Syt-1 for the assembly of V-ATPase in the absence of Ca²⁺. Upon influx, PI(3,5)P₂ is released, and PI(4,5)P₂ binds to Syt-1 for the release of neurotransmitters.