The N-peptide–binding mode is critical to Munc18-1 function in synaptic exocytosis

Sec1/Munc18 (SM) proteins promote intracellular vesicle fusion by binding to N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs). A key SNARE-binding mode of SM proteins involves the N-terminal peptide (N-peptide) motif of syntaxin, a SNARE subunit localized to the target membrane. In in vitro membrane fusion assays, inhibition of N-peptide motif binding previously has been shown to abrogate the stimulatory function of Munc18-1, a SM protein involved in synaptic exocytosis in neurons. The physiological role of the N-peptide–binding mode, however, remains unclear. In this work, we addressed this key question using a “clogged” Munc18-1 protein, in which an ectopic copy of the syntaxin N-peptide motif was directly fused to Munc18-1. We found that the ectopic N-peptide motif blocks the N-peptide–binding pocket of Munc18-1, preventing the latter from binding to the native N-peptide motif on syntaxin-1. In a reconstituted system, we observed that clogged Munc18-1 is defective in promoting SNARE zippering. When introduced into induced neuronal cells (iN cells) derived from human pluripotent stem cells, clogged Munc18-1 failed to mediate synaptic exocytosis. As a result, both spontaneous and evoked synaptic transmission was abolished. These genetic findings provide direct evidence for the crucial role of the N-peptide–binding mode of Munc18-1 in synaptic exocytosis. We suggest that clogged SM proteins will also be instrumental in defining the physiological roles of the N-peptide–binding mode in other vesicle-fusion pathways.

A conserved function of SM proteins is to regulate the zippering of the trans-SNARE complex in the vesicle-fusion reaction (14, 18, 29 -34). While supplying the energy for membrane merging, SNAREs alone zipper inefficiently (18,35). By recognizing sequences on both the v-and t-SNAREs, SM proteins promote the zippering of the trans-SNARE complex and accelerate the kinetics of SNARE-dependent fusion reactions (18, 36 -39). SM proteins selectively activate their cognate SNARE pairs, thus augmenting the compartmental specificity of intracellular vesicle fusion (18,35,40).
A key SNARE-SM binding mode involves the N-terminal peptide (N-peptide) motif located at the extreme N terminus of syntaxin (14,18,36,37,41,42). Characterized by multiple charged residues and a hydrophobic leucine or phenylalanine residue (Fig. 1A), the N-peptide motif is accommodated within a peripheral pocket on the SM protein (43)(44)(45). The N-peptide motif recruits the soluble SM protein to the vicinity of membrane-anchored SNAREs, facilitating the interaction between the SM protein and the trans-SNARE complex (37). In reconstituted fusion assays, the N-peptide-binding mode is critical to the stimulatory function of Munc18-1 (18,36,37). The physiological function of this N-peptide-binding mode in neurons, however, has been debated. In genetic studies using cultured neurons or whole animals, mutations in either the syntaxin N-peptide or the N-peptide-binding pocket of Munc18-1 strongly inhibited synaptic exocytosis (46 -48). In other genetic studies, however, such mutations had little impact on synaptic exocytosis (49 -51). The discrepancy of these genetic data are likely due to the intrinsic limitations of point mutations, which often do not completely inhibit the N-peptide-binding mode and/or may cause protein misfolding. Thus, a new approach is needed to examine the physiological function of the Npeptide-binding mode of Munc18-1 in synaptic exocytosis.
In this work, we took advantage of a "clogged" Munc18-1 protein in which an ectopic copy of the syntaxin N-peptide motif is directly fused to Munc18-1 to block its N-peptidebinding pocket (37). The clogged Munc18-1 protein is unable to bind the native N-peptide motif on syntaxin and thus fails to stimulate SNARE-dependent membrane fusion in vitro (37). Using a reconstituted system, we found that clogged Munc18-1 failed to promote trans-SNARE zippering. When introduced into induced neuronal (iN) cells differentiated from human induced pluripotent stem cells (iPSCs), clogged Munc18-1 was defective in mediating synaptic exocytosis. Thus, both spontaneous and evoked synaptic transmission were abrogated. Consistent with our reconstitution data, these genetic results demonstrated that the N-peptide-binding mode is indispensable to Munc18-1 function in synaptic exocytosis.

Results
In our previous biochemical studies, we engineered a clogged Munc18-1 protein in which an ectopic copy of the N-peptide motif from syntaxin-1 was directly fused to the N terminus of Munc18-1 through a flexible linker (Fig. 1, B and C) (37). The ectopic N-peptide motif occupies the N-peptide-binding pocket of Munc18-1 and prevents the latter from binding to the native N-peptide motif on syntaxin-1. As a result, the clogged Munc18-1 protein is unable to stimulate SNARE-dependent membrane fusion in vitro (Fig. 1, B and C) (37). To gain further mechanistic insights into the clogged Munc18-1 protein, we first examined its regulatory activity in trans-SNARE zippering in vitro. Synaptic exocytic SNAREs alone zippered poorly in a trans-SNARE assembly assay ( Fig. 2 (A and B) and Fig. S1). The addition of WT Munc18-1 strongly accelerated the zippering kinetics ( Fig. 2 (A and B) and Fig. S1). The clogged Munc18-1 protein, by contrast, was completely inactive in regulating the zippering process, resulting in near-background levels of trans-SNARE zippering (Fig. 2 (A and B) and Fig. S1). Next, we examined how clogged Munc18-1 regulates the upstream docking step of SNAREs. We observed that, like WT Munc18-1, clogged Munc18-1 did not influence the initial docking of SNAREs (Fig.  2, C and D). The CD spectra of WT and clogged Munc18-1 proteins were very similar (Fig. 2E), suggesting that clogged Munc18-1 was properly folded. These results demonstrated that clogged Munc18-1 is defective in promoting trans-SNARE zippering, providing a molecular explanation for its inability to accelerate the kinetics of SNARE-dependent membrane fusion.
The clogged Munc18-1 protein offered an ideal opportunity to examine the physiological role of the N-peptide-binding mode in synaptic exocytosis. First of all, the ectopic N-peptide motif completely blocks the N-peptide-binding pocket of Munc18-1 in vitro (Fig. 2, A and B) (37), in contrast to point mutations that may incompletely inhibit N-peptide binding to  (37). Yellow, Munc18-1; green, the N-terminal sequence of syntaxin-1 (amino acids  containing the N-peptide motif; purple, an engineered linker (23 residues). The model is based on the crystal structure of Munc18-1 complexed with the N-peptide motif of syntaxin-4 (Protein Data Bank code 3PUJ). Key residues of the N-peptide motif are conserved in syntaxin-1 and syntaxin-4. The sequence of the clogged Munc18-1 protein is included under "Experimental procedures."

Crucial role of the N-peptide-binding mode in exocytosis
Munc18-1. Moreover, clogged Munc18-1 does not harbor any mutation in the Munc18-1 sequence. This is important because the folding of Munc18-1 protein is highly sensitive to perturbations, including single-residue substitutions (18). Thus, mutations in the N-peptide-binding pocket of Munc18-1 may cause local misfolding of the protein, complicating functional analyses.
To determine the activity of clogged Munc18-1 in neurons, endogenous Munc18-1 needs to be eliminated and replaced with clogged Munc18-1. Our initial plan was to silence Munc18-1 expression in primary mouse cortical neurons using short hairpin RNA (shRNA), similar to a strategy used in our genetic dissection of the synaptic v-SNARE VAMP2 (52). However, we found that this shRNA method did not fully abrogate Munc18-1 expression in neurons. In this work, we characterized the clogged Munc18-1 protein in iN cells derived from human iPSCs (Fig. 3A) (53). Lentiviral expression of the transcription factor Ngn2 rapidly and efficiently (nearly 100%) programmed human iPSCs into iN cells (Fig. 3, B-D) (53). As demonstrated in this work and previous studies (53, 54), iN cells  (52). Reconstituted t-and v-SNARE liposomes were incubated at 4°C for the indicated durations before a 10-fold excess amount of inhibitory CDV2 was added to block unpaired t-SNAREs. The liposomes were subsequently solubilized, and the t-SNAREs were precipitated using nickel-Sepharose beads. Levels of full-length VAMP2 in the precipitates were measured by immunoblotting and used as an indicator for trans-SNARE assembly between liposomes. The reactions were performed in the presence of 100 mg/ml Ficoll 70. B, immunoblots showing syntaxin-1 and VAMP2 protein levels in the precipitates from the trans-SNARE assembly assays. Additional analyses of the trans-SNARE complexes are included in Fig. S1. C, the liposome docking assay was performed as described previously (35). The biotin-labeled t-SNARE liposomes containing syntaxin-1 and SNAP-25 were anchored to avidin beads and used to pull down rhodamine-labeled v-SNARE liposomes. A VAMP2NTD-TolA chimera was used as the v-SNARE in the docking assay to selectively assess the NTD-mediated docking step (66). The binding reactions were performed at 4°C for 1 h in the absence or presence of 5 M Munc18-1 (WT or clogged). D, effects of WT and clogged Munc18-1 proteins on liposome docking. Data are presented as percentage of rhodamine fluorescence intensity. Binding reactions containing biotin-labeled, protein-free liposomes were included to show the background fluorescence signals (SNARE-independent liposome docking). Error bars, S.D. E, CD spectroscopic analysis of WT and clogged Munc18-1 proteins. The CD spectra were measured using a Jasco J-815 spectropolarimeter equipped with a 1-mm quartz cell. The readings were made at 0.5-nm intervals, and each data point represents the average of six scans at a speed of 50 nm/min over the wavelength range of 200 -250 nm. The data were converted into mean residue weighted molar ellipticity using the equation, [] ϭ (100 ϫ )/Cnl, where C is the protein concentration (mM), is the measured ellipticity (millidegrees), n is the number of residues, and l is the path length (cm).

Crucial role of the N-peptide-binding mode in exocytosis
were morphologically and functionally similar to primary excitatory cortical neurons. Because gene KO and rescue were carried out in iPSCs, we could generate virtually unlimited supplies of iN cells with defined genetic alterations. We used CRISPR/ Cas9 genome editing to ablate the MUNC18-1 gene in the iPSCs. Rescue genes expressing WT or clogged Munc18-1 were expressed in MUNC18-1 KO iPSCs using lentiviral expression. The iPSCs were then differentiated into iN cells (Fig. 3A).
To rule out clonal variations of iPSCs, we chose to generate iN cells using pooled KO iPSCs. CRISPR genome editing is expected to be particularly effective for MUNC18-1 KO because the stability of Munc18-1 protein is highly sensitive to perturbations (18). Thus, even in-frame indel mutations will likely create loss-of-function alleles. Indeed, MUNC18-1 expression in iN cells was abolished by CRISPR genome editing using a guide RNA (gRNA) targeting an early exon of the MUNC18-1 gene (Fig. 3B).
The suitability of this iN cell system for genetic dissection of Munc18-1 function is supported by four lines of evidence. First, similar amounts of iN cells were used for functional analysis across the experiments. Because MUNC18-1 KO reduces the viability of neurons (28,55), the numbers of seeded iPSCs were adjusted to achieve similar densities of iN cell populations at the time of functional measurements (Fig. 3, C and D). The

Crucial role of the N-peptide-binding mode in exocytosis
expression levels of ␣-tubulin were comparable across the iN cell populations (Fig. 3B), confirming our cell density estimation. Second, the overall morphologies of the WT and mutant iN cells were similar without apparent cell death (Fig. 3, C and  D). Third, the expression levels of the rescue Munc18-1 (WT or clogged) were comparable with endogenous Munc18-1 expression (Fig. 3B). Finally, the phenotype of the MUNC18-1 KO iN cells was similar to that of primary neurons isolated from Munc18-1 KO mice, and the phenotype could be fully rescued by the WT Munc18-1 gene (see below).
We then examined synaptic neurotransmitter releases in the iN cells using electrophysiological measurements. We first measured miniature excitatory postsynaptic currents (mEPSCs), which reflected spontaneous neurotransmitter releases. As expected, KO of MUNC18-1 strongly reduced the frequency of mEPSCs in iN cells (Fig. 4, A and B). The frequency of mEPSCs was fully rescued by the WT Munc18-1 gene but not by the gene encoding clogged Munc18-1 (Fig. 4, A and B). The amplitudes of mEPSCs were similar in all the iN cell populations (Fig. 4C), suggesting that the loading of neurotransmitters into synaptic vesicles was unchanged. These results demonstrated that clogged Munc18-1 failed to mediate spontaneous synaptic releases.
Next, we measured evoked excitatory postsynaptic currents (EPSCs) in the iN cells, which reflected evoked neurotransmitter releases. MUNC18-1 KO strongly decreased the amplitudes of EPSCs triggered by local electrical stimulation (Fig. 5). The EPSCs were fully restored by the expression of WT Munc18-1 but not by clogged Munc18-1 (Fig. 5). Thus, the clogged Munc18-1 protein was unable to mediate evoked synaptic neurotransmitter release.
To rule out potential off-target effects of CRISPR genome editing, we designed another two gRNAs targeting early exons of the MUNC18-1 gene. These two gRNAs were simultaneously introduced into iPSCs to mutate the MUNC18-1 gene. WT and clogged Munc18-1 were then expressed in the KO iPSCs before the cells were differentiated into iN cells. Immunoblotting of the iN cells showed that MUNC18-1 expression was eliminated in the KO cells, whereas WT and clogged Munc18-1 proteins were expressed to the endogenous level of MUNC18-1 (Fig.   S2A). Electrophysiological measurements showed that the frequency of mEPSCs was severely diminished in the KO iN cells (Fig. S2, B and C). The frequency of mEPSCs was fully rescued by the expression of WT Munc18-1 but not by clogged Munc18-1 (Fig. S2, B and C). Likewise, the amplitudes of evoked EPSCs were strongly reduced in the MUNC18-1 KO iN cells (Fig. S3). The EPSCs were fully restored by WT Munc18-1 but not by clogged Munc18-1 (Fig. S3). These findings are consistent with the results of the genetic experiments using a single gRNA (Figs. 4 and 5), indicating that the phenotype of the mutant cells was due to specific loss-of-function mutations in the MUNC18-1 gene.
Together, these genetic data demonstrated that clogged Munc18-1 is defective in mediating synaptic exocytosis in neurons, correlating with its inability to promote trans-SNARE zippering in vitro. Thus, we conclude that the N-peptide-binding mode is crucial to Munc18-1 function in synaptic exocytosis.

Discussion
A major discovery of our previous reconstitution studies was that the SM protein Munc18-1 promotes SNARE-dependent membrane fusion, and its stimulatory function requires binding to the N-peptide motif of syntaxin-1 (18,36,37). In this study, we provided direct genetic evidence for the crucial role of the N-peptide-binding mode in synaptic exocytosis in neurons. Our findings also further support the notion that the N-peptide motif promotes membrane fusion by facilitating the association between Munc18-1 and the membrane-anchored SNARE complex, rather than allosterically activating Munc18-1.
Our genetic data helped resolve a major controversy in the field regarding the physiological role of the N-peptide-binding mode. The binding of the N-peptide motif to Munc18-1 involves a number of intermolecular interactions (44). In other genetic studies, point mutations were introduced to disrupt a subset of these interactions, which was expected to reduce but not abolish the N-peptide binding. Partial inhibition of N-peptide binding may lead to variable consequences in vivo, strongly dependent on experimental conditions such as protein expression levels. Extensive mutations of the N-peptide-Munc18-1  Table S1. Statistical analysis was performed using Student's t test comparing a test data set with the control experiment of WT cells. Data shown in summary graphs are mean Ϯ S.E. (error bars). ***, p Ͻ 0.001. n.s., not significant. C, summary graph of mEPSC amplitudes. Data shown in summary graphs are mean Ϯ S.E. n.s., not significant. Scatter plots of B and C are show in Fig. S4A.

Crucial role of the N-peptide-binding mode in exocytosis
interactions, on the other hand, likely cause local misfolding of the protein, and the folding state may differ across experimental systems, contributing to conflicting phenotypes.
Although we cannot completely exclude the possibility that the N-peptide-binding pocket of Munc18-1 also recognizes another molecule, we consider this scenario unlikely. The N-peptide motif of syntaxin is a highly specialized sequence not known to exist in other vesicle-fusion regulators. Moreover, the peripheral N-peptide-binding pocket of Munc18-1 is physically separate from other SNARE-binding regions (e.g. domain 3a) and is not known to associate with other molecules. Nevertheless, SNAREs and Munc18-1 are known to bind other regulatory factors, including Munc13 (31,56). Further research will be needed to determine whether and how the N-peptidebinding mode is influenced by Munc13 and other regulatory factors. Likewise, additional studies will be required to examine whether the ectopic N-peptide motif binds Munc18-1 in a similar way as the native N-peptide.
Whereas the N-peptide-binding mode is widespread in vesicle fusion, it is absent in certain SM proteins, including the yeast exocytic SM protein Sec1p. For these SM proteins, the recruitment role of the N-peptide motif is likely fulfilled by another SNARE-SM binding mode or by a regulatory protein physically linking the SM protein and SNAREs.
We suggest that clogged SM protein is a powerful approach to determine the functional roles of the N-peptide-binding mode in other vesicle-fusion pathways both in vitro and in vivo. For example, the N-peptide motif of syntaxin-4 is recognized by Munc18c/Munc18-3, a SM protein involved in insulin-stimulated GLUT4 exocytosis in adipocytes and skeletal muscles (35,(57)(58)(59). It is still unclear whether N-peptide binding is required for Munc18c function in GLUT4 exocytosis. Like neurons, adipocytes and skeletal muscles are post-mitotic cells difficult to genetically manipulate in culture. Recently, human iPSCs have been successfully programmed into functional adipocytes and muscle cells (60 -62). Thus, the gene KO and rescue strategy described in this study will be instrumental in characterizing the regulatory activities of clogged Munc18c in iPSC-derived adipocytes and muscle cells.

Recombinant protein expression and purification
Recombinant proteins were expressed and purified from Escherichia coli using nickel-affinity chromatography (37,52). The synaptic exocytic t-SNARE complex was composed of untagged rat syntaxin-1 and mouse SNAP-25 with an N-terminal His 6 tag (36). The sequence of the clogged Munc18-1 protein used in this work is as follows (the syntaxin-1 sequence containing the N-peptide motif is underlined, whereas the linker sequence is shown in boldface type): MKDRTQELR-TAKDSDDDDDVTVTVDRDRFMGGGGGGGGGGSRYPY- The sequence of the VAMP2NTD-TolA chimera, in which the C-terminal domain of VAMP2 was replaced with a bacterial TolA sequence, is as follows (TolA sequence is underlined): MSATAATVPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQ-VDEVVDIMRVNVDKVLERDQKGGSSIDAVMVDSGAVVE-QYKRMQSQKRKYWWKNLKMMIILGVICAIILIIIIVYFST.  Table S1. Statistical analysis was performed using Student's t test comparing a test data set with the control experiment of WT cells. A scatter plot of C is shown in Fig. S4B.

Crucial role of the N-peptide-binding mode in exocytosis Trans-SNARE assembly assay
The v-and t-SNARE liposomes were prepared as described previously (35,63,64). The v-and t-SNARE liposomes were incubated at 4°C for the indicated durations in the presence or absence of 5 M Munc18-1 before a 10-fold excess amount of inhibitory VAMP2 cytosolic domain (CDV2) was added. Each reaction contained 5 M t-SNAREs and 1.5 M t-SNARE. The liposomes were solubilized with 1% CHAPS, and the t-SNAREs were precipitated using nickel-Sepharose beads through binding to the His 6 tag on SNAP-25. The levels of full-length VAMP2 proteins in the precipitates were measured by immunoblotting using monoclonal anti-VAMP2 antibodies (clone Cl69.1, Synaptic Systems), which was used as an indicator for trans-SNARE assembly between liposomes. Syntaxin-1 was detected using monoclonal anti-syntaxin-1 antibodies (clone HPC-1, Synaptic Systems).

Liposome docking assay
SNARE liposomes were prepared in a similar way as in the trans-SNARE assembly assay except that 2% biotin-conjugated 1-(12-biotinyl(aminododecanoyl))-2-oleoyl-sn-glycero-3-phosphoethanolamine was included in the t-SNARE liposomes, and WT VAMP2 was replaced with a VAMP2NTD-TolA chimera in the v-SNARE liposomes. Because membrane docking is mediated by the N-terminal domains (NTDs) of SNAREs, the VAMP2NTD-TolA chimera was ideally suited for monitoring the upstream docking step. The biotin-labeled t-SNARE liposomes were incubated with avidin-conjugated Sepharose beads at room temperature for 1 h. The bead-bound t-SNARE liposomes were then used to pull down rhodamine-labeled v-SNARE liposomes. The pulldown reactions were performed at 4°C in the presence or absence of 5 M Munc18-1. After washing three times with the reconstitution buffer, CHAPS was added to a final concentration of 1% to solubilize the beadbound liposomes. After removing avidin beads by centrifugation, rhodamine fluorescence in the supernatant was measured in a BioTek microplate reader. The binding reaction containing biotin-labeled, protein-free liposomes was used as a negative control to obtain the background fluorescence signal. The background rhodamine fluorescence was subtracted from other binding reactions to calculate SNARE-mediated liposome docking.

Generation of iN cells
Human iPSCs (clone I 50 -2) were generated from human adult fibroblasts (ATCC) using a RNA-based reprogramming method and were characterized previously (64). The iPSCs were programmed into iN cells following a procedure described previously (53). The iPSCs were cultured on Matrigel (BD Biosciences)-coated coverslips in Essential 8 medium (Life Technologies, Inc.) supplemented with 10 M Y-27632 dihydrochloride (Tocris). Lentivirus particles containing the Ngn2 expression gene were added to Essential 8 medium containing 8 g/ml Polybrene. After 24 h (day 0), the culture media were replaced with N2/DMEM/F12/NEAA (Life Technologies) containing human brain-derived neurotrophic factor (10 g/liter; PeproTech), human NT-3 (10 g/liter; PeproTech), and mouse laminin (0.2 mg/liter; Life Technologies). Meanwhile, doxycy-cline (2 g/ml; Clontech) was added to induce TetO gene expression. On day 1, 1 g/ml puromycin was added to select for transduced cells. After 24 h (day 2), the cells were fed with Neurobasal medium (Gibco), and mouse glia cells were added. The iN cells were analyzed between day 16 and day 21.

CRISPR/Cas9 genome editing
The lentiCRISPR plasmid (Addgene) was used to delete the MUNC18-1 gene in iPSCs. For gene KO using one gRNA, the following guide sequence targeting the second exon of the MUNC18-1 gene was selected: 5Ј-GATAAAGAAGGTC-AAGAAGAAGG-3Ј.
The guide sequences were individually subcloned into the BsmBI sites of the plentiCRISPR vector. Lentiviral particles prepared with the lentiCRISPR plasmids were used to infect iPSCs. On the next day, the cells were treated with 1 g/ml puromycin for 24 h to select for transduced cells. CRISPR/Cas9 is expected to induce compound heterozygous mutations that are functionally equivalent to homozygous KO (65). In gene rescue experiments, the rat Munc18-1 gene was subcloned into the BamHI and KpnI sites of the TRC2 pLKO vector. To prevent cleavage of the rescue gene by Cas9 in the single gRNA CRISPR KO, silent mutations were introduced into the rat Munc18-1 rescue gene near the PAM site using the following mutagenesis primer: 5Ј-GCATGATGTTATCAAGAAGGTG-AAAAAAAAAGGCGAGTGGAAGGTGCTGGTG-3Ј. In CRISPR KO using two gRNAs, the rescue gene was not mutated, because neither of the gRNAs targets the Munc18-1 rescue gene.

Electrophysiological recordings
EPSCs and mEPSCs were recorded in iN cells using our established procedures (52). Evoked synaptic transmission was triggered by 1-ms current injections using a concentric bipolar microelectrode (FHC; model CBAEC75) placed about 100 -150 m from the cell bodies of patched iN cells. The extracellular Crucial role of the N-peptide-binding mode in exocytosis stimuli were manipulated using an isolated pulse stimulator (World Precision Instruments). The evoked responses were measured by whole-cell recordings using a Multiclamp 700B amplifier (Molecular Devices). The whole-cell pipette solution contained 135 mM CsCl, 10 mM HEPES-CsOH (pH 7.25), 0.5 mM EGTA, 2 mM MgCl 2 , 0.4 mM NaCl-GTP, and 4 mM NaCl-ATP. The bath solution contained 140 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 0.8 MgCl 2 , 10 mM HEPES-NaOH (pH 7.4), and 10 mM glucose. The mEPSCs of the neurons were sampled at 10 kHz in the presence of 1 M tetrodotoxin (Sigma). The resistance of pipettes was 3-5 megaohms. The series resistance was adjusted to 8 -10 megaohms once the whole-cell configuration was achieved.
The detailed numbers of cultures and neurons are listed in Table S1. The electrophysiological data were processed using the pClamp 10 software (Molecular Devices). For statistical calculations, all data are shown as mean Ϯ S.E. The p values were calculated using Student's t test. In gene rescue experiments, all data are compared with the results of WT cells.