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. 2008 Mar 11;105(10):3986-91.
doi: 10.1073/pnas.0712372105. Epub 2008 Feb 28.

Genetic analysis of synaptotagmin-7 function in synaptic vesicle exocytosis

Anton Maximov  1 , Ye Lao, Hongmei Li, Xiaocheng Chen, Josep Rizo, Jakob B Sørensen, Thomas C Südhof
Affiliations

Genetic analysis of synaptotagmin-7 function in synaptic vesicle exocytosis

Anton Maximov et al. Proc Natl Acad Sci U S A. .

Abstract

Synaptotagmin-7 is a candidate Ca(2+) sensor for exocytosis that is at least partly localized to synapses. Similar to synaptotagmin-1, which functions as a Ca(2+) sensor for fast synaptic vesicle (SV) exocytosis, synaptotagmin-7 contains C(2)A and C(2)B domains that exhibit Ca(2+)-dependent phospholipid binding. However, synaptotagmin-7 cannot replace synaptotagmin-1 as a Ca(2+) sensor for fast SV exocytosis, raising questions about the physiological significance of its Ca(2+)-binding properties. Here, we examine how synaptotagmin-7 binds Ca(2+) and test whether this Ca(2+) binding regulates Ca(2+)-triggered SV exocytosis. We show that the synaptotagmin-7 C(2)A domain exhibits a Ca(2+)-binding mode similar to that of the synaptotagmin-1 C(2)A domain, suggesting that the synaptotagmin-1 and -7 C(2) domains generally employ comparable Ca(2+)-binding mechanisms. We then generated mutant mice that lack synaptotagmin-7 or contain point mutations inactivating Ca(2+) binding either to both C(2) domains of synaptotagmin-7 or only to its C(2)B domain. Synaptotagmin-7-mutant mice were viable and fertile. Inactivation of Ca(2+) binding to both C(2) domains caused an approximately 70% reduction in synaptotagmin-7 levels, whereas inactivation of Ca(2+) binding to only the C(2)B domain did not alter synaptotagmin-7 levels. The synaptotagmin-7 deletion did not change fast synchronous release, slow asynchronous release, or short-term synaptic plasticity of release of neurotransmitters. Thus, our results show that Ca(2+) binding to the synaptotagmin-7 C(2) domains is physiologically important for stabilizing synaptotagmin-7, but that Ca(2+) binding by synaptotagmin-7 likely does not regulate SV exocytosis, consistent with a role for synaptotagmin-7 in other forms of Ca(2+)-dependent synaptic exocytosis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Analysis of intrinsic Ca2+ binding to the synaptotagmin-7 C2A domain by NMR spectroscopy. (A) Superposition of 1H-15N HSQC spectra of the 15N-labeled synaptotagmin-7 C2A domain acquired at Ca2+ concentrations of 0–5 mM. The spectrum obtained in the absence of Ca2+ is shown in black; rainbow coloring was used for the remaining spectra (red, lowest Ca2+ concentrations; blue, highest Ca2+ concentrations). Selected cross-peak assignments are indicated (red arrows, Ca2+-dependent movement of some cross-peaks). (B–D) Superpositions of expansions of analogous 1H-15N HSQC spectra acquired in the presence of 0–40 mM Ca2+, illustrating the Ca2+-dependent movement of the cross-peaks from D227, L224, and D225 (other cross-peaks in the expansions that exhibit little or no Ca2+-dependent changes are also labeled). The Ca2+ concentrations used are indicated next to some of the cross-peaks and in the box below the spectra.
Fig. 2.
Fig. 2.
Strategy for generation of synaptotagmin-7-mutant mouse lines. (A) Diagram of the synaptotagmin-7 domain structure (Upper: TMR, transmembrane region; arrowhead, position of the alternatively spliced linker region) and sequences of the Ca2+-binding sites in the two C2 domains (C2A and C2B) that were mutated in the knockin (KI) mice (Lower: red boxes, aspartate residues essential for Ca2+ binding; asterisks, aspartate residues replaced with alanine residues in synaptotagmin-7 knockin mice). (B) Homologous recombination strategy for synaptotagmin-7 C2B- and C2AB-domain mutant mice. A NEO-resistance gene cassette flanked by LoxP sites (blue triangles) was introduced into an EcoRI site in intron 12, and a diphtheria toxin gene (DT) was inserted downstream of the short vector arm. The coding sequences of exons 11 and 13 (red boxes) were mutated to replace six aspartate residues that bind Ca2+ in the C2A and C2B domains with alanines (D225, D227, and D233 in exon 11; D303, D357, and D359 in exon 13) (see asterisks in A). Diagrams on the bottom depict the mutant synaptotagmin-7 alleles containing alanine substitutions in both exon 11 and 13 (C2AB KI/KO) or only in exon 13 (C2B KI/KO) and the subsequent Cre-recombined alleles in which the NEO cassettes were excised.
Fig. 3.
Fig. 3.
Expression of synaptotagmin-7 in mutant mice. (A) Representative immunoblots of synaptotagmin-7 (Syt 7), Rab 3, synaptotagmin-1 (Syt 1), and syntaxin-1A (Syx 1) in brain proteins (≈40 μg per lane) from synaptotagmin-7 KO and Cre-recombined C2B and C2AB knockin mice and their WT littermate controls. Symbols on the left indicate the four major brain synaptotagmin-7 splice variants (S, short form; L, long form). (B–D) Expression levels of individual synaptotagmin-7 splice variants in the brains of adult (>P30) KO (B) and Cre-recombined C2B- and C2AB-domain KI mice (C and D, respectively) determined by quantitative immunoblotting with 125I-labeled secondary antibodies and normalized to expression in their WT littermates (means ± SDs; n ≥ 3).
Fig. 4.
Fig. 4.
Fast synaptic transmission in neurons lacking synaptotagmin-7. (A) Representative IPSCs monitored in cortical neurons from littermate WT and synaptotagmin-7 KO mice cultured 14 days in vitro. IPSCs were recorded in 2 mM extracellular Ca2+ in the presence of AP5 and CNQX. Synaptic responses were evoked by single action potentials applied at 0.1 Hz (arrows). Scale bars apply to both traces. (Insets) Initial phases of multiple responses collected from the same neuron to illustrate the kinetics of the IPSC onset. (B) Average rise times of IPSCs recorded from WT and synaptotagmin-7-deficient neurons in 10 mM extracellular Ca2+ (means ± SDs; WT, n = 9; KO, n = 10). (C) Amplitudes and synaptic charge transfer (integrated over 1.5 s) of individual IPSCs in WT and synaptotagmin-7-deficient neurons monitored in 2 mM or 10 mM Ca2+ or 10 mM Sr2+ as indicated. Data are plotted as a fraction of the WT response (means ± SDs; 2 mM Ca2+, WT, n = 9; KO, n = 8; 10 mM Ca2+, WT, n = 14; KO, n = 14; 10 mM Sr2+, WT, n = 12; KO, n = 10; from two to four cultures from littermate WT and KO mice). (D) Time course of individual IPSCs monitored from WT (solid lines) and synaptagmin-7-deficient neurons (dashed lines). Plots exhibit the cumulative charge transfer over 1.5 s after an action potential (means ± SDs; 2 mM Ca2+, WT, n = 15; KO, n = 9; 10 mM Ca2+, WT, n = 11; KO, n = 12; 10 mM Sr2+, WT, n = 11; KO, n = 10).
Fig. 5.
Fig. 5.
Properties of inhibitory synaptic responses triggered by trains of action potentials. (A) Representative IPSCs recorded from WT and synaptotagmin-7-deficient neurons during a 5-Hz, 10-s action potential train in 2 mM extracellular Ca2+. Arrows indicate the areas defined as train release (TR, release during the stimulus train) and delayed release (DR, tail currents observed after the end of the stimulus train). Scale bars apply to both traces. (B) Average synaptic charge transfer induced by stimulus trains of 50 action potentials applied at 5–50 Hz in WT and synaptotagmin-7-deficient neurons during the train (train release), after the train (delayed release), and over the entire experiment (total charge transfer) plotted as a function of the stimulation frequency. IPSCs were triggered in 10 mM extracellular Ca2+ (means ± SDs; WT, n = 14; KO, n = 12). (C) Examples of individual IPSCs elicited by 50 action potentials applied at 20 Hz in WT and synaptotagmin-7 KO neurons. In the examples, aligned and normalized IPSCs from the beginning (solid lines, IPSCs 2–5) and end of the stimulus train (dashed lines, IPSCs 47–50) are depicted. (D) Rise times of IPSCs during a train of 50 action potentials applied at 20 Hz, plotted as a function of stimulus number. Rise times were normalized to the first response (data represent individual points from three different neurons).
Fig. 6.
Fig. 6.
Synaptotagmin-7 deletion does not suppress asynchronous release in synaptotagmin-1-deficient neurons. (A and B) Normalized amplitudes (A) and charges (B) of IPSCs triggered by isolated action potentials in WT neurons (n = 24) or neurons lacking synaptotagmin-1 (Syt 1KO, n = 20), synaptotagmin-7 (Syt 7 KO, n = 14), or both synaptotagmin-1 and -7 (Syt 1/7 DKO, n = 10). In addition, neurons lacking synaptotagmin-1 and -7 were infected with lentivirus encoding the short splice variant of synaptotagmin-7 to test rescue (Syt 1/7 DKO + Syt 7, n = 3). Note that the ns apply to all panels of this figure; data are from two independent cultures, with the rescue experiment performed for one of the two cultures. (C) Time course of single IPSCs triggered by isolated action potentials in WT neurons or neurons lacking synaptotagmin-1 (Syt 1 KO), synaptotagmin-7 (Syt 7 KO), synaptotagmin-1 and -7 (Syt 1/7 DKO), or Syt 1/7 DKO neurons infected with the lentivirus encoding the short synaptotagmin-7 splice variant (Syt 1/7 DKO + Syt 7). The time course is depicted as a cumulative plot of the synaptic charge transfer. (D and E) Representative traces (D) and mean synaptic charge transfer (normalized to that observed in synaptotagmin-7-deficient neurons) of inhibitory responses triggered by high-frequency action potential trains in synaptotagmin-7 KO and in synaptotagmin-1 and -7 double KO neurons. (F) Contribution of delayed asynchronous release to total synaptic charge transfer in WT neurons and neurons lacking synaptotagmin-1 alone or both synaptotagmin-1 and -7. IPSCs were triggered by trains of 2–50 action potentials applied at 10 Hz. The charges of delayed responses are represented as a fraction of total response and plotted as a function of the stimulus number. Data shown in all panels are means ± SDs.
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