Genetic analysis of synaptotagmin-7 function in synaptic vesicle exocytosis

Maximov et al. 10.1073/pnas.0712372105.

Supporting Information

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Fig. 7. Properties of inhibitory synaptic responses triggered by trains of action potentials. Depression of synchronous IPSCs triggered in WT and synaptotagmin-7-deficient neurons by trains of action potentials applied at 5, 10, and 20 Hz as indicated. (A and B) Synaptic responses were recorded in 2 mM (A) and 10 mM (B) extracellular Ca²⁺. IPSCs amplitudes were normalized to the amplitude of first IPSC in train and are plotted as a function of stimulus number. Each panel shows averaged data from 9-11 WT or synaptotagmin-7-deficient neurons.

SI Methods

NMR Spectroscopy. The synaptotagmin-7 C₂A domain was expressed in bacteria as a GST-fusion protein, isolated by affinity chromatography, cleaved off GST with thrombin, and purified by gel filtration on a Superdex 75 column (Amersham Pharmacia Biosciences). For ¹⁵N- and ¹³C-labeling of the C2A domain, bacteria were grown in minimal media containing ¹⁵NH4Cl and ¹³C6-glucose as the sole nitrogen and carbon sources, respectively. All NMR spectra were acquired at 25°C on a Varian INOVA500 spectrometer with samples dissolved in 20 mM Tris·HCl (pH 7.3) and 100 mM NaCl. Backbone resonance assignments were obtained with a 0.7 mM uniformly ¹⁵N,¹³C-labeled synaptotagmin-7 C₂A-domain solution using HNCACB and CBCACONH experiments as described previously for the synaptotagmin-1 C₂A domain (1). Ca²⁺ titrations were performed as described previously (2) with a 100 mM uniformly ¹⁵N-labeled sample using Ca²⁺ concentrations ranging from 0-40 mM. The Ca²⁺ dependence of the chemical shifts of multiple cross-peaks was analyzed with Sigma Plot using standard protein-ligand binding equations as described previously (2).

Generation of Synaptotagmin-7-Mutant Mice. To generate the mutant synaptotagmin-7 mouse lines, several genomic clones overlapping exons 9-14 of murine synaptotagmin-7 gene were isolated and characterized. These clones were then used to construct a targeting vector that contained a NEO-resistance gene flanked by loxP sites introduced into EcoRI site in a noncoding sequence between exons 12 and 13. The aspartate residues responsible for Ca²⁺ coordination in both C₂A and C₂B domains were substituted with alanines (D225, D227, and D233 in exon 11; D303, D357, and D359 in exon 13).

After homologous recombination in ES cells, we selected the clones that included either the mutations in both the C₂A and the C₂B domains or only in the C₂B domain. Both groups of clones carrying targeted synaptotagmin-7 alleles were used to produce the original founder chimera mouse lines that were then backcrossed to C57B/6 WT females. For in vivo NEO-cassette excision, the heterozygous offspring of chimera/C57B/6 parents were crossed to a transgenic line expressing Cre recombinase. Genotyping of knockin lines containing a NEO cassette was performed by using PCR with oligonucleotide primers YL 113 and YL 97 (5'-CAGGTGCTGAGGCTTATCACAGCAGG-3' and 5'-GTGGGAGCTGGCTCCATGGTTT TTAAGTGGGTG-3', respectively) for WT allele (product size ~400 bp) and YL 113 and YL 111 (5'-GGCTGCAGGAATTCGATATCAAGCTT-3') for mutant allele (product size ~ 380 bp). Genotyping of Cre-excised knockin lines was performed with oligonucleotide primers YL 113 and YL 97 (product sizes ~400 bp for WT allele and ~500 bp for mutant allele).

Neuronal Cultures and Electrophysiology. Analyses of synaptic transmission in cultured cortical neurons were performed as described previously (3). Briefly, the cortexes were dissected from the newborn pups, dissociated by trypsin digestion, and plated on circle glass coverslips coated with Matrigel. The cultures were maintained in MEM medium (Gibco) supplemented with B-27 (Gibco), L-glutamine, 0.5% glucose, 5% FBS, and 2 mM Ara-C (Sigma-Aldrich). The cultures were used for experiments at 13-16 days in vitro. Inhibitory synaptic responses were triggered by 1-ms current injection (900 mA) through a local extracellular electrode (FHC) and recorded in a whole-cell mode using Multiclamp 700A amplifier (Axon Instruments). All experiments were performed at room temperature. The frequency, duration, and magnitude of extracellular stimulus were controlled with Model 2100 Isolated Pulse Stimulator (A-M Systems). The whole-cell pipette solution contained 135 mM CsCl2, 10 mM Hepes, 1 mM EGTA, 1 mM Na-GTP, 4 mM Mg-ATP, and 1 mM QX-314 (pH 7.4). The bath solution contained 140 mM NaCl, 5 mM KCl, 2 or 10 mM CaCl2, 0.8 mM MgCl2, 10 mM Hepes, and 10 mM glucose (pH 7.4). Excitatory AMPA and NMDA currents were suppressed by addition of 50 mM APV and 20 mM CNQX (Sigma-Aldrich) to the bath solution. The currents were sampled at 10 kHz and analyzed off-line by using pClamp9 (Axon Instruments) and Origin7 (Microcal) software. All data are shown as means ±SEM.

Miscellaneous Procedures. SDS/PAGE electrophoresis and quantitative immunoblotting were performed using standard procedures.

1. Shao X, Südhof TC, Rizo J (1998) J Biol NMR 10:307-308.

2. Ubach J, Zhang X, Shao X, Südhof TC, Rizo J (1998) EMBO J 17:3921-3930.

3. Maximov A, Pang ZP, Tervo DG, Sudhof TC (2007) J Neurosci Methods 161:75-87.