Augmenting neurotransmitter release by enhancing the apparent Ca2+ affinity of synaptotagmin 1

Abstract

Synaptotagmin 1 likely acts as a Ca2+ sensor in neurotransmitter release by Ca2+-binding to its two C2 domains. This notion was strongly supported by the observation that a mutation in the C2A domain causes parallel decreases in the apparent Ca2+ affinity of synaptotagmin 1 and in the Ca2+ sensitivity of release. However, this study was based on a single loss-of-function mutation. We now show that tryptophan substitutions in the synaptotagmin 1 C2 domains act as gain-of-function mutations to increase the apparent Ca2+ affinity of synaptotagmin 1. The same substitutions, when introduced into synaptotagmin 1 expressed in neurons, enhance the Ca2+ sensitivity of release. Mutations in the two C2 domains lead to comparable and additive effects in release. Our results thus show that the apparent Ca2+ sensitivity of release is dictated by the apparent Ca2+ affinity of synaptotagmin 1 in both directions, and that Ca2+ binding to both C2 domains contributes to Ca2+ triggering of release.

Fig. 1.

Models of the C2A domain (Upper) and C2B domain (Lower) of synaptotagmin 1 before (Left) and after (Right) mutating exposed hydrophobic residues of the Ca²⁺-binding loops to tryptophans. The mutated residues and the tryptophans are shown as orange space-filling models, and the Ca²⁺ ions are represented by cyan spheres. The models illustrate the increase in exposed hydrophobic surface area and the drastic change in shape that results from the mutations.

Fig. 2.

Ca²⁺-dependent phospholipid binding properties of SytWT, SytC2A^3W, SytC2B^3W, and SytC2A^3WC2B^3W. Wild-type or mutant forms of GST-synaptotagmin 1 C2AB domains were incubated with liposomes (41% phosphatidylcholine, 32% phosphatidylethanolamine, 10% phosphatidylserine, 5% phosphatidylinositol, 10% cholesterol) containing no PIP and PIP₂ (Upper), or 0.25% PIP + 0.05% PIP₂ (Lower) in the presence of free Ca²⁺ at the concentrations shown, clamped by Ca²⁺/Mg²⁺/EGTA buffers. Liposomes were centrifuged and washed, and bound proteins were analyzed by SDS/PAGE and Coomassie blue staining. Data shown are representative of experiments performed multiple times.

Fig. 3.

Enhanced apparent Ca²⁺ sensitivity of glutamate release in hippocampal synapses expressing SytC2^3W mutants. (A) Exemplary evoked synaptic currents from hippocampal Syt-/- neurons rescued by overexpression of SytWT (black), SytC2A^3WB^3W mutant (red), and negative control mutant SytC2A3DAC2B3DA (gray), which contains 6 C2A/B top loop aspartate-to-alanine substitutions that eliminate Ca²⁺-dependent phospholipid binding and rescue activity (28). (Inset) Bar plot shows mean EPSC amplitudes of SytWT (black), SytC2A^3W (gray), SytC2B^3W (blue), and SytC2A^3WB^3W (red) rescues. Number of cells is indicated within each bar. (B) Exemplary EPSCs from SytWT rescues (Left) and SytC2A^3WB^3W rescues (Right) at 0.5 mM (gray trace), 4 mM (black), and 8 mM (red) external Ca²⁺. (C) Mean apparent Ca²⁺ sensitivity of EPSC amplitudes in SytWT and the three SytC2^3W rescues. EPSC amplitudes measured at 0.2-12 mM external Ca²⁺ (with 1 mM Mg²⁺ held constant) were normalized to the response evoked at the highest Ca²⁺ concentration, and the data were fitted with a Hill function. SytC2A^3WB^3W, n = 12; SytWT, n = 18; SytC2A^3W, n = 10; and SytC2B^3W, n = 12.

Fig. 4.

Increased vesicular release probability in the gain-of-function synaptotagmin 1 mutants. (A) Plot of mean normalized EPSC amplitudes of SytWT and all three SytC2^3W mutants rescues during a high stimulation 10-Hz action potential train (5-s duration). (B) Bar plot of the vesicular release probability (Left) and miniature EPSC (mEPSC) frequency (Right). (C) Correlation of vesicular release rates during spontaneous release and at the peak of the evoked response in SytWT and SytC2A^3W, SytC2B^3W, and SytC2A^3WB^3W mutant rescues. The linear fit corresponds to a constant increase in release rate by the Ca²⁺-triggering event by ≈18,200-fold.

Fig. 5.

Enhanced Ca²⁺ sensitivity of triggered neurotransmitter release does not affect the time course of neurotransmitter release. (A Upper) Exemplary EPSCs from SytWT (black trace) and SytC2A^3WB^3W rescues (red trace). (A Lower) Corresponding vesicle release rate after deconvolution of EPSC with mean mEPSC time course. (B) Bar plot of mean time course (Left) and relative amplitude of the fast component of release (Right). The time courses of the decay phase were individually fitted with two exponentials to reveal the fast and slow components of vesicular release. Color code as in A. The time course of the slow component of release was not significantly changed. (SytWT, n = 22; Syt6W, n = 26.)