A gain-of-function mutation in synaptotagmin-1 reveals a critical role of Ca2+-dependent soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex binding in synaptic exocytosis

Abstract

Synaptotagmin-1, the Ca2+ sensor for fast neurotransmitter release, was proposed to function by Ca2+-dependent phospholipid binding and/or by Ca2+-dependent soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex binding. Extensive in vivo data support the first hypothesis, but testing the second hypothesis has been difficult because no synaptotagmin-1 mutation is known that selectively interferes with SNARE complex binding. Using knock-in mice that carry aspartate-to-asparagine substitutions in a Ca2+-binding site of synaptotagmin-1 (the D232N or D238N substitutions), we now show that the D232N mutation dramatically increases Ca2+-dependent SNARE complex binding by native synaptotagmin-1, but leaves phospholipid binding unchanged. In contrast, the adjacent D238N mutation does not significantly affect SNARE complex binding, but decreases phospholipid binding. Electrophysiological recordings revealed that the D232N mutation increased Ca2+-triggered release, whereas the D238N mutation decreased release. These data establish that fast vesicle exocytosis is driven by a dual Ca2+-dependent activity of synaptotagmin-1, namely Ca2+-dependent binding both to SNARE complexes and to phospholipids.

Figure 1.

Immunoprecipitation analysis of native wild-type and mutant synaptotagmin-1 binding to SNARE complexes using polyclonal syntaxin-1 antibodies. A, Representative immunoblots of syntaxin-1 immunoprecipitates. Brain proteins from synaptotagmin-1 D232N-mutant and wild-type mice (WT) were solubilized with 1% Triton X-100 to promote SNARE complex formation, and immunoprecipitated with polyclonal syntaxin-1 antibodies at increasing NaCl concentrations, with or without 1 mm free Ca²⁺. Immunoprecipitates were blotted with monoclonal antibodies to synaptotagmin-1 (Syt-1), SNAP-25 (Cl 71.1), and syntaxin-1 (HPC-1); bands were probed with ¹²⁵I-labeled secondary antibodies and visualized in a phosphorimager. B, Quantitations of synaptotagmin-1 and SNAP-25 coimmunoprecipitated with syntaxin-1 at increasing NaCl concentrations in the presence or absence of 1 mm Ca²⁺. Quantitations were performed with ¹²⁵I-labeled secondary antibodies; amounts of coimmunoprecipitated proteins in this and all other immunoprecipitation experiments are normalized for the amount of the immunoprecipitated protein [i.e., syntaxin-1 (Fig. 1) or synaptobrevin-2 (Fig. 2); n = 3]. In this and all following figures, gray symbols indicate WT, red symbols indicate D232N mutant, and blue symbols indicate D238N mutant. Data shown are means ± SEMs. C, Quantitations of synaptotagmin-1, Munc18-1, and complexins 1/2 (Cpx 1/2) coimmunoprecipitated with syntaxin-1 in the presence of increasing Ca²⁺ concentrations in 100 mm NaCl (n = 3). Statistical analyses in B and C were performed with a two-way ANOVA test. n.s., Nonsignificant.

Figure 2.

Immunoprecipitation analysis of synaptotagmin-1 binding to SNARE complexes using monoclonal synaptobrevin-2 antibodies. A, Representative immunoblots of immunoprecipitations performed in 100 mm NaCl as described in Figure 1, except that monoclonal synaptobrevin-2 antibodies were used (Syt-1, synaptotagmin-1; Syp 1, synaptophysin-1; Syb 2, synaptobrevin-2). Bands were visualized by enhanced chemiluminescence. B, Quantitations of synaptotagmin-1 coimmunoprecipitated with synaptobrevin-2 in the presence or absence of 1 mm free Ca²⁺. Quantitations were performed with ¹²⁵I-labeled secondary antibodies (means ± SEMs; n = 3).

Figure 3.

Comparison of Ca²⁺-induced phospholipid binding by equivalent fragments of recombinant and native synaptotagmin-1. A, B, Analysis of the effect of incorporating phosphatidylinositides (PIP, PIP₂) into liposomes on the Ca²⁺-independent and Ca²⁺-dependent binding of native synaptotagmin-1. Panels show representative immunoblots visualized with ¹²⁵I-labeled secondary antibodies (A) and quantitations of binding (B) in the presence or absence of 0.1 mm Ca²⁺ (means ± SEMs; n = 3). Experiments were performed with the cytosolic region of synaptotagmin-1 obtained by mild trypsin digestion of total brain membranes, and with heavy liposome with a “synaptic” composition (41% phosphatidylcholine, 32% phosphatidylethanolamine, 12% phosphatidylserine, 5% phosphatidylinositol, and 10% cholesterol by weight). Binding was performed with a centrifugation assay in which synaptotagmin-1 bound to liposomes is measured by immunoblotting. C, D, Recombinant (C) and native (D) synaptotagmin-1 fragments were prepared as indicated (C₁, D₁). C₂, D₂, Representative immunoblots visualized with ¹²⁵I-labeled secondary antibodies of recombinant (C₂) and native (D₂) synaptotagmin-1. The recombinant glutathione S-transferase (GST)-rat synaptotagmin-1 (GST-rSyt1; residue 86–421) includes the trypsin-hypersensitive site that is cleaved in native synaptotagmin-1; thus, the native and recombinant trypsin-produced fragments contain identical sequences. Fragments were bound at different NaCl concentrations with and without 0.1 mm Ca²⁺ to liposomes with a phospholipid synaptic vesicle composition (phosphatidylcholine, 41%; phosphatidylethanolamine, 32%; phosphatidylserine, 12%; phosphatidylinositol, 5%; cholesterol, 10%), and with 0.1% PIP and 0.5% PIP₂. C₃, D₃, Binding was quantified for both recombinant (C₃) and native (D₃) synaptotagmin-1 using immunoblotting with ¹²⁵I-labeled secondary antibodies and phosphorimager detection, and is expressed as percentage of the maximum. Data are presented as means ± SEMs (n = 3). Statistical analyses were performed with a two-way ANOVA test.

Figure 4.

Ca²⁺-dependent binding of native wild-type or mutant synaptotagmin-1 to liposomes. All experiments were performed as in Figure 3, A and B. A, B, Analysis of the Ca²⁺ concentration dependence of the binding of native wild-type (WT) or D232N- and D238N-mutant synaptotagmin-1 to liposomes containing 0.5% PIP/0.1% PIP₂ in 100 mm NaCl. Panels show representative immunoblots (A) and quantitations of binding (B). C, NaCl concentration dependence of the binding of native WT or D232N- and D238N-mutant synaptotagmin-1 to liposomes containing 0.5% PIP/0.1% PIP₂ in the absence or presence of 1 mm free Ca²⁺. B and C show means ± SEMs (n = 3 for D232N; n = 4 for D238N). Statistical analyses were performed by two-way ANOVA test.

Figure 5.

Evoked IPSCs in synapses containing wild-type or mutant synaptotagmin-1. A, Representative traces of IPSCs evoked by focal stimulation in cortical neurons cultured from littermate wild-type (WT) and D232N- or D238N-mutant mice. Recordings were made in 2 Ca²⁺/0.8 Mg²⁺; stimulation artifacts were truncated for display purposes. B, C, Summary graphs of the amplitudes and total charge transfer during IPSCs evoked at low frequency (<1 Hz) in neurons from littermate WT and D232N- (B) or D238N-mutant mice (C). D, Representative traces of IPSCs induced by hypertonic sucrose (0.5 m). E, Total synaptic charge transfer integrated over 30 s in response to a 0.5 m sucrose application for 20 s. F, Time course of synaptic responses to isolated action potentials in cultured cortical neurons from littermate WT and D232N- or D238N-mutant neurons. Graphs show the integrated synaptic charge transfer plotted as a function of time for D232N-mutant (left panel) and D238N-mutant neurons (right panel) compared with their littermate control cultures (D232N WT, n = 18; D232N, n = 21; D238N WT, n = 23; D238N, n = 20). Data shown are means ± SEMs. Statistical significance was assessed by unpaired t test (*p < 0.05; **p < 0.01; ***p < 0.001; numbers of recorded neurons are indicated in the bars).

Figure 6.

Ca²⁺ dependency of synaptic responses in D232N- and D238N-mutant synapses. A, B, Representative traces of evoked IPSCs at the indicated concentrations of free Ca²⁺. Stimulation artifacts are removed for display purposes. C, D, Dose–response curves of evoked IPSCs in neurons from littermate wild-type (WT) and D232N- (C) or D238N-mutant mice (D). Data shown are means ± SEMs (n = 4–22 neurons depending on Ca²⁺ concentration). Statistical significance was assessed by a two-way ANOVA test. The curve shown represents the result of a fit of the data to a Hill function that is described in Table 1.

Figure 7.

Short-term synaptic plasticity in synapses containing wild-type (WT) or mutant synaptotagmin-1. A, B, Representative IPSCs (A; interstimulus interval, 50 ms) and summary graphs (B) of paired-pulse stimulation experiments. IPSCs were examined in responses to two closely spaced action potentials elicited by focal stimulation in cultured cortical neurons. The summary graph (B) displays the ratio of the second to the first IPSC amplitude as a function of the interstimulus interval. C, Representative traces of IPSCs during a 2 s, 10 Hz stimulus train. D, Summary graphs of IPSC amplitudes during 10 Hz stimulus trains. IPSCs were normalized to the first response. Note that stimulation artifacts are removed for display purposes. In B and D, data shown are means ± SEMs (number of analyzed neurons are shown in brackets). Statistical significance was assessed by a two-way ANOVA test.

Figure 8.

Effect of D232N and D238N mutations on asynchronous release during and after a 10 Hz stimulus train. A, Representative traces of inhibitory synaptic responses in D232N- or D238N-mutant neurons and littermate wild-type (WT) control neurons when 10 stimuli at 10 Hz were elicited by focal stimulation. Delayed asynchronous release was defined as the synaptic response observed 100 ms after the last stimulus (shaded area). Recordings were from cortical neurons cultured at high density from littermate wild-type and D232N- or D238N-mutant mice. B–G, Total charge transfer during the first response (B, E), total delayed release (C, F), and total amount of release integrated over the entire stimulus train (D, G) in D232N- and D238N-mutant neurons, respectively. Note that stimulation artifacts are removed for better presentation. Data shown are means ± SEMs. *p < 0.05; **p < 0.01.

Figure 9.

Evoked EPSCs in autapses formed by D232N- and D238N-mutant neurons. A, Representative traces of EPSCs evoked in the same neuron by consecutive stimulation with an action potential (AP) (induced by somatic depolarization from −75 to 0 mV for 2 ms) and with hypertonic sucrose (0.5 m for 3 s). EPSCs were recorded in isolated hippocampal neurons cultured on glial microislands where they form hundreds of autapses. Neurons were cultured from littermate D232N- and D238N-mutant mice derived from crossings of compound heterozygous mutant mice. Capacitative and somatic currents are blanked for display purposes. B–D, Summary graphs of the synaptic charge integrals of action potential-induced EPSCs (B) and sucrose-induced EPSCs (C; corresponds to the RRP), and of the ratio of the charge integrals of action potential-induced to sucrose-induced EPSCs (D; defined as the vesicular release probability P_vr). Data shown are means ± SEMs. Statistical significance was assessed by unpaired t test (**p < 0.01; numbers of recorded neurons are indicated in the bars).

Figure 10.

Determination of the apparent Ca²⁺ affinity of release from excitatory synapses in autapses from D232N- and D238N-mutant mice. Recordings were made from autapses in hippocampal neurons cultured from littermate offspring of matings between compound heterozygous mutant mice (i.e., mice carrying one D232N and one D238N mutant allele). Ca²⁺ titrations were performed by interleafing EPSC measurements at the indicated test Ca²⁺ concentration in 1 mm Mg²⁺ with measurements under the standard conditions for autapses (with 4 mm Ca²⁺ and 4 mm Mg²⁺) to control for possible rundown of synaptic responses. A, Representative superimposed traces of action potential-evoked EPSCs recorded in D232N- (red) and D238N-mutant neurons (blue) during low-frequency stimulation (0.2 Hz for 30–60 s) at different Ca²⁺ concentrations as indicated. B, Same traces as shown in A, but normalized to the maximal amplitudes of the EPSCs (i.e., to the responses obtained in 10 mm Ca²⁺/1 mm Mg²⁺). Note that stimulus artifacts are removed for display purposes. C, D, Summary graphs of the Ca²⁺ dependence of synaptic responses in autapses. EPSC amplitudes were normalized to the EPSC obtained in standard extracellular medium containing 4 mm Ca²⁺/4 mm Mg²⁺ (C), or to the maximal EPSC amplitude (D). Data in C and D were fitted to a Hill function (Table 1). Data shown are means ± SEMs (number of analyzed neurons are shown in brackets). Statistical significance was assessed by a two-way ANOVA test.