DP-2 alone does not bind to VAPA but effectively disrupts Kv2.1-VAPA association

Kv2.1 and Kv2.2 channels are recruited to ER-PM junctions via CT PRC-mediated interaction with VAPA/B (17–19). As indicated earlier, this process occurs at noncanonical FFAT binding motifs within the PRC domains of Kv2.1 and Kv2.2 (fig. S1B), which are recruited to the FFAT binding domain on VAPA/B (17–19). We used a peptide spot array assay (23) to assess the ability of DP-2, along with many mutant variants of the peptide (see below; fig. S5A), to bind VAPA in a cell-free assay. Unexpectedly, despite the fact that noncanonical FFAT motifs SFISCAT and SFTSCAT are required for Kv2.1 and Kv2.2 interaction with VAP proteins, we observed no binding between these peptides and VAPA (fig. S5A). Moreover, we observed no binding between VAPA and DP-2. These data indicate that both the DP-2 sequence and the noncanonical FFAT motifs located on Kv2.1 and Kv2.2 may not be sufficient for VAPA binding alone, at least in a cell-free environment. This assay was validated by observing strong VAPA binding to the positive control peptide (EFFDAPE), a canonical FFAT motif.

Given these results, we were thus interested in testing whether a TAT-linked derivative of the canonical FFAT motif (TAT-EFFDAPE) could disrupt Kv2.1 surface cluster formation. GFP-Kv2.1–transfected neurons were treated with TAT-EFFDAPE (TAT-FFAT; 10 μM) or a scramble control peptide TAT-FDEEFAP (TAT-FFATSc; 10 μM) for either 2 or 24 hours and analyzed for Kv2.1 cluster density. As TAT-FFAT treatment did not induce Kv2.1 cluster dispersal (fig. S5, B and C) when compared with a scrambled sequence peptide, our results suggest that a more complex interaction between VAPA and Kv2.1 may be at play.

Selective serine phosphorylation of Kv2.1 within its PRC domain appears critical for its recruitment by VAP proteins, most notably S590 (S605 on Kv2.2) (17–19). CD4-linked chimeras of Kv2.1 PRC domain peptides localize to VAPA proteins at the ER, an effect abolished by selective serine-to-alanine mutations within the PRC domain (19). Conversely, mutation of these same residues to aspartic acid restores CD4-linked PRC-VAPA interaction (19). Although these phosphorylation events had been proposed to render the PRC domain as containing noncanonical FFAT motifs (17–19), we were also unable to observe demonstrable binding to VAPA with these amino acids sequences in the peptide array. The pseudo-phosphorylated mutant DP-2 peptide sequences lacked VAPA binding capacity in our peptide spot array assay (fig. S5A). Regardless of the observed lack of VAPA binding, a TAT-linked peptide containing serine-to-aspartate mutations (TAT-DP-2D:TAT-DIDDFTD) effectively declustered Kv2.1-GFP aggregates in neurons, while the serine-to-alanine mutant (TAT-DP-2A:TAT-AIDAFTA) did not (fig. S6, A and B). Together, these results strongly indicate that TAT-DP-2–mediated Kv2.1 declustering relies on phosphorylation of serine residues, although this sequence alone is not sufficient to bind to VAPA (fig. S5A), implicating a multifaceted architecture underlying the interaction between Kv2.1 and its associated ER binding partner.

Despite the lack of demonstrable direct binding between TAT-DP-2 and VAPA, we evaluated whether TAT-DP-2 could still disrupt the Kv2.1-VAPA interaction in vitro, given its strong declustering effect. We used a proximity ligation assay (PLA; Fig. 3A), which allows for analysis of protein-protein association at distances less than 40 nm from each other (24). We treated cortical neurons with vehicle, TAT-Sc-2 (10 μM), or TAT-DP-2 (10 μM) for 2 hours. Following treatment, we used a PLA staining protocol that allowed for fluorescent labeling of Kv2.1-VAPA association based on published methods (24). We observed robust fluorescent PLA puncta formation in both vehicle and TAT-Sc-2–treated neurons (Fig. 3, B and C), indicating a high level of Kv2.1-VAPA association. In contrast, TAT-DP-2 exposure induced a significant reduction in PLA reactions (Fig. 3, B and C), demonstrating that this peptide alone could effectively disrupt Kv2.1-VAPA association, seemingly without directly binding to VAPA.

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Fig. 3

TAT-DP-2 displaces Kv2.1-VAPA association in cortical neurons.

(A) Depiction of PLA is shown, indicating that PLA probes detect association between two target proteins of interest within 40 nm of each other. (B) Raw confocal images (60×) of immunolabeled cortical neurons are displayed, following 2-hour treatment with either TAT-Sc-2 or TAT-DP-2. PLA puncta-TRITC (tetramethyl rhodamine isothiocyanate) fluorescence indicates areas of Kv2.1-VAPA interaction. Note robust reduction in density of PLA puncta following TAT-DP-2 treatment. Scale bar, 10 μm. (C) Histograms represent PLA puncta per square micron of neuronal soma (# PLA puncta/sq. micron of cell soma) across four separate experiments. Data are displayed as means +/− SEM [Veh, 0.13 +/− 0.01 (n = 22 neurons); TAT-Sc-2, 0.10 +/- 0.06 (n = 15 neurons); TAT-DP-2 0.06 +/− 0.01 (n = 30 neurons)]. Analyzed via Kruskal-Wallis with Dunn’s MCT [Veh versus TAT-Sc-2, ns (P = 0.48); Veh versus TAT-DP-2 (***P < 0.0001); TAT-Sc-2 versus TAT-DP-2 (*P = 0.019)]. Ab, antibody; DAPI, 4’6-diamidino-2-phenylindole.

TAT-DP-2 prevents enhancement of Kv2.1-mediated proapoptotic potassium currents and is neuroprotective in vitro

The cell death–enabling loss of intracellular potassium in neurons can be experimentally monitored as a time-dependent enhancement in Kv2.1-mediated potassium currents under whole-cell voltage clamp conditions as new channels become incorporated into the PM (3, 8, 25). To evaluate whether TAT-DP-2–induced declustering could prevent the apoptotic enhancement of potassium currents in a manner akin to Kv2.2 CT overexpression (14), we treated cortical neurons in vitro with either TAT-DP-2 (10 μM), TAT-Sc-2 (10 μM), or vehicle for 2 hours before a 2-hour exposure to dl-threo-β-benzyloxyaspartic acid (TBOA; 60 μM) or vehicle. TBOA is a glutamate transporter inhibitor that produces a slow excitotoxic injury via prolonged N-methyl-d-aspartate (NMDA) receptor stimulation (26). This induces a pronounced potassium current surge mediated by Kv2.1, culminating in delayed apoptotic cell death, mimicking the excitotoxic conditions present in the ischemic penumbra following stroke (9, 11, 14, 26).

Delayed rectifier potassium currents, which are primarily mediated by Kv2.1 in our preparation (3), were monitored 3 to 5 hours following TBOA exposure. As expected, Kv2.1 current densities were significantly increased in the vehicle/TBOA-treated cells when compared with the vehicle/vehicle-treated group (Fig. 4, A and B). An identical effect was also seen when comparing the TAT-Sc-2/vehicle–treated group with the TAT-Sc-2/TBOA–treated cells. Notably, treatment with TAT-DP-2 completely abolished TBOA-mediated enhancement of Kv2.1 current densities, as no differences were noted between TAT-DP-2/vehicle–treated neurons and TAT-DP-2/TBOA–exposed cells. These results support the notion that DP-2–mediated Kv2.1 declustering can effectively prevent the apoptotic surge of potassium currents in neurons by disrupting their membrane insertion platform. Note that baseline Kv2.1 current densities were unaffected by the peptide treatment, demonstrating that the injury-mediated insertion of Kv2.1 channels and the normal trafficking of the channel occur via distinct processes, as we have repeatedly experimentally ascertained in prior works (3, 8, 9).

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Fig. 4

TAT-DP-2 blocks proapoptotic Kv2.1 current surge and is neuroprotective in vitro.

(A) Whole-cell potassium current densities (−80- to 30-mV voltage step) 3 to 5 hours following 2-hour treatment with combinations of TAT-linked peptide (10 μM), vehicle (dH₂O), and TBOA (60 μM). Data are displayed as means ± SEM [vehicle, 86.53 ± 14.01 (n = 17); vehicle + TBOA, 162.50 ± 18.61 (n = 14); TAT-Sc-2, 71.42 ± 10.27 (n = 16); TAT-Sc-2 + TBOA, 130.80 ± 9.43 (n = 14); TAT-DP-2, 66.86 ± 4.76 (n = 14); TAT-DP-2 + TBOA, 65.68 ± 12.07 (n = 13)]. Kruskal-Wallis/Dunn: Veh versus Veh + TBOA, **P = 0.0057; TAT-Sc-2 versus TAT-Sc-2 + TBOA, **P = 0.0032; TAT-DP-2 versus TAT-DP-2 + TBOA, ns (P > 0.9999). Current density traces are depicted for each condition (50 pA/pF, 50 ms). (B) Current density–voltage curves displayed for treatment groups noted above. (C) Experimental schematic is shown. (D) Relative toxicity values ([LDH]_TBOA/[LDH]_Veh) presented as means ± SEM [Veh, 1.84 ± 0.19 (n = 7); TAT-Sc-2, 1.57 ± 0.15 (n = 7); TAT-DP-2, 1.01 ± 0.15 (n = 7)]. Analyzed via ANOVA/Holm-Sidak’s: Veh versus TAT-DP-2, **P = 0.0047; TAT-Sc-2 versus TAT-DP-2, *P = 0.0276.

With evidence that TAT-DP-2–mediated declustering prevents enhancement of proapoptotic Kv2.1 potassium currents following injury, we next tested the neuroprotective actions of TAT-DP-2 in cortical neurons in vitro. Cells were exposed to either vehicle, TAT-DP-2 (3 μM), or TAT-Sc-2 (3 μM) for 2 hours before an overnight exposure to either vehicle or TBOA (60 μM). In addition to the preincubation period, peptides were present for the first 4 hours of the TBOA exposure (Fig. 4C), a period of time when current enhancement normally begins following initial injury (3, 8). Neurotoxicity was assessed by the release of lactate dehydrogenase (LDH) into the culture medium, as described previously (27). Consistent with the observed inhibition of apoptotic potassium currents, TAT-DP-2, but not TAT-Sc-2, significantly attenuated TBOA-induced toxicity when compared to the scrambled peptide, measured as relative toxicity ratios ([LDH]_TBOA/[LDH]_Veh) (Fig. 4D). On the basis of these findings, we next evaluated the potential neuroprotective actions of TAT-DP-2 in an in vivo model of ischemia-reperfusion injury, where we have previously observed a substantial contribution of Kv2.1-enabled neuronal cell death (9).

Ethical approval

The Institutional Animal Care and Use Committee at the University of Pittsburgh School of Medicine approved all animal protocols detailed in this study.

Neuronal cell culture

In vitro experiments used primary cortical neuronal cultures prepared from embryonic day 16 to 17 Sprague-Dawley rats of either sex in a protocol described previously (10). Timed-pregnant rats (Charles River Laboratories, Wilmington, MA) were sacrificed by CO₂ inhalation. Embryonic cortices were dissociated with trypsin (0.6 mg/ml), and cells were plated in sterile six-well plates on poly-l-ornithine–coated glass coverslips at a density of 670,000 cells per well. Cytosine arabinoside (1 to 2 μM) was used to inhibit nonneuronal proliferation on day 14 in vitro (DIV); cultures were used at 21 to 25 DIV.

TAT-linked peptides

The UniProt Consortium protein sequence database was used to align protein sequences. TAT (YGRKKRRQRRR)–linked peptides with 95.1 to 99.8% purity analyzed by high-performance liquid chromatography were generated by GenScript (Piscataway, NJ) (table S2). Notably, clustering residues within the PRC domain of Kv2.1 (S587, S590, F591, and S593; rat sequence, UniProt P15387) correspond to S583, S586, F587, and S589 in (20).

Plasmid constructs

pCMV-DP-1 and pCMV-Sc-1 plasmids were generated by standard cloning into the multiple cloning sites of pCMV-IRES2-GFP (Clontech, catalog no. 6029-1). The IRES2-GFP region of the parent plasmid was subsequently removed. Successful cloning was confirmed by sequencing. Table S3 lists all plasmids used in this study.

Transfection

Transfections were performed with Lipofectamine 2000 (L2K; Invitrogen, Carlsbad, CA). Cortical neuronal cultures on glass coverslips were placed into wells containing 500 μl of Dulbecco’s minimum essential medium with 2% HyClone bovine serum (D2C, Thermo Fisher Scientific, Waltham, MA). A mixture of 1.5 μg of total cDNA and 2 μl of L2K was prepared in 100 μl of Opti-MEM I (Life Technologies Corp., Grand Island, NY) for each well and added. Cultures were used 18 to 24 hours following transfection. cDNA amounts for each transfection experiment are described below for each figure [Fig. 1: Kv2.1-GFP plasmid (0.375 μg per well), pcDNA3 vector plasmid (0.495 μg per well), and either DP-1 or Sc-1 or Kv2.2 CT–expressing plasmid (0.63 μg per well); Fig. 2 and figs. S2 to S4 and S6: Kv2.1-GFP plasmid (0.375 μg per well), tdTomato plasmid (0.15 μg per well; from G. Ryffel; Addgene plasmid no. 30530), and pcDNA3 vector plasmid (0.975 μg per well)].

Immunohistochemistry

Eight-week-old C57BL/6J males (28 to 30 g; The Jackson Laboratory, Bar Harbor, ME) were administered intraperitoneal injections of TAT-DP-2 (6 nmol/g; n = 3) or TAT-Sc-2 peptide (6 nmol/g; n = 3). Two hours after injection, mice were anesthetized with 3% isoflurane (Henry Schein Animal Health, Dublin, OH) in a 3:1 NO/O₂ gas mixture. Fifty milliliters of ice-cold 1× sterile phosphate-buffered saline (PBS) was transcardially perfused (5 to 10 ml/min), immediately followed by 50 ml of 4% sterile paraformaldehyde (PFA; 5 to 10 ml/min). Brains were removed and placed in 4% PFA at 4°C for 24 hours and then transferred to 30% sucrose in 1× PBS at 4°C for 48 to 72 hours. Brains were embedded with Tissue-Tek OCT Compound (Sakura Finetek USA Inc., Torrance, CA) onto an SM2010R cyrosectioner (Leica Biosystems, Wetzlar, Germany), sectioned (30 μm), and stored.

Brain sections were rinsed in sterile 24-well plates in tris-buffered saline (TBS) solution. Sections were then transferred to new wells containing 0.5% Triton X-100 and 10% normal goat serum (NGS) in TBS for 30 min at room temperature. Next, sections were again transferred to wells containing primary anti-Kv2.1 antibody [mouse monoclonal immunoglobulin G (IgG), RRID AB_192761, University of California (UC) Davis/NeuroMab Facility], diluted 1:200 in 0.3% Triton X-100 and 3% NGS in TBS for 24 hours at 4°C. Sections were washed in TBS and incubated with secondary anti-mouse IgG Sigma fluorescein isothiocyanate–tagged antibody (goat monoclonal IgG; catalog F0257, Sigma-Aldrich, St. Louis, MO), diluted 1:500 in 0.3% Triton X-100 and 3% NGS in TBS for 1 hour at room temperature, and then mounted.

Immunocytochemistry

The in vitro Kv2.1 immunofluorescence experiment (fig. S4) was carried out in DIV 21 to 26 primary rat neuronal cultures, as described above. Each coverslip was treated with either TAT-DP-2 or TAT-Sc-2 in its individual well to reach a final concentration of 10 μM. After 3 hours, coverslips were transferred to PBS and immediately fixed with 4% PFA/4% sucrose for 15 min. Coverslips were rinsed with PBS three times and then permeabilized with 0.25% Triton X-100 for 15 min. Cells were rinsed with PBS three times and then blocked with 2% bovine serum albumin (BSA)/10% NGS for 1 hour at room temperature. Neurons were stained with mouse anti-Kv2.1 antibody (1:250; clone K89/34, Abcam, RRID: AB_2750677) using the microdrop method at 4°C overnight. Cells were rinsed in PBS three times and then stained with Alexa Fluor 568 goat anti-mouse secondary antibody (1:500; Thermo Fisher Scientific, RRID: AB_2534072) for 1 hour at room temperature. Last, cells were rinsed in PBS three times before mounting. Confocal Z projections of well-isolated neurons were obtained for evaluation of Kv2.1 cluster density (described below). The experimenter was blinded to the treatment groups during image analysis.

Confocal imaging and Kv2.1 cluster analysis

To analyze Kv2.1 distribution in live primary cortical neurons, cells were transfected with both a tdTomato construct and a GFP-tagged Kv2.1 construct (21) and imaged on a Nikon A1+ confocal microscope (Nikon, Tokyo, Japan). Cells were chosen at random by looking only at the tdTomato signal (561 nm) to avoid bias. At least three separate culture dates were used per experiment, and three coverslips were transfected per condition each time. For these experiments, n refers to the total number of cells imaged. Five to 15 optical sections (0.5 μm) capturing the cell surface of single cells were obtained at ×60 magnification (47), creating a maximum intensity projection (MAX-IP) of Kv2.1-GFP distribution. To analyze Kv2.1 distribution in the cerebral cortex of fixed brain slices, 51 to 54 image fields were obtained at ×60 magnification across 18 sections from three fixed mouse brains per treatment (total of six animals). Image fields were captured by first focusing on an area of superficial cortex closest to the cover glass. Next, 24 optical sections (0.5 μm) were obtained moving deeper into the tissue to produce a 12-μm MAX-IP for analysis of Kv2.1 distribution. Laser power, HV (high-voltage/gain), offset, pinhole size, and all camera parameters were kept identical between all samples in a given experiment. Nikon Instruments Software Advanced Research (NIS-Elements AR) was used to analyze GFP-tagged Kv2.1 channel cluster distribution. For live images of transfected neurons, object count parameters were customized to define a Kv2.1 cluster as an area of high-intensity GFP fluorescence (compared to background), measuring 0.05 μm² or larger (47). Kv2.1 cluster density was calculated by normalizing the number of Kv2.1 clusters counted to the somatic area of each neuron (number of Kv2.1 clusters per square micrometer). Because of high variability in GFP-Kv2.1 expression between individual neurons, intensity thresholding required cell-specific local background subtraction. In each neuron analyzed, two populations on Kv2.1-GFP expression were first noted: (a) highly localized signals of clustered Kv2.1 channels and (b) diffuse background staining that represents freely dispersed, conducting Kv2.1 channels on the PM. A small region of interest (ROI) within region (b) was set as the background subtraction standard, the image creating an optimal signal-to-noise ratio for clustered Kv2.1 channels. Last, automated intensity thresholding was set to identify Kv2.1 clusters as well-defined regions with a significantly higher Kv2.1-GFP signal than local background. This protocol was also adapted for analysis of cortical neurons immunostained with an anti-Kv2.1 antibody to visualize endogenous Kv2.1 distribution, except that Alexa Fluor 568 fluorescence was correlated with Kv2.1 cluster localization (fig S4). For analysis of Kv2.1 channel cluster distribution in whole-field images of fixed cortical tissue, NIS-Elements AR object count parameters were applied to the entire image field, each of which contained approximately 60 to 80 cell bodies. Kv2.1 channel clusters were designated as areas of fluorescence with pixel intensity (absolute fluorescence units; AFU) between 2200 and 4095, that measured within 3 SDs of previous empiric measurements of average Kv2.1 cluster area (0.41 to 1.37 μm²), as reported earlier (21).

Proximity ligation assay

Duolink PLA technology (Sigma-Aldrich, St. Louis, MO) was used to assess in situ protein-protein interaction between Kv2.1 and VAPA. Primary cortical neurons on glass coverslips were treated in 24-well plates with vehicle (dH₂O), TAT-Sc-2 (10 μM), or TAT-DP-2 (10 μM) for 2 hours at 37°C/5% CO₂. Neurons were then rinsed with 2 ml of ice-cold sterile PBS and then transferred to 1 ml of 4% PFA in PBS for 20 min for fixation. Coverslips were transferred to 1 ml of 0.1 M glycine (in PBS) to quench fixation and rinsed three times in 1 ml of PBS. Membrane permeabilization was accomplished with 1 ml of 0.25% Triton X-100 (Sigma-Aldrich, St. Louis, MO), and nonspecific binding was blocked with 10% BSA (in PBS) for 30 min. Primary immunolabeling was accomplished by 18 to 24 hours of exposure to rabbit anti-Kv2.1 polyclonal antibody (1:500 in 3% BSA; RRID AB_2040162, Alomone Labs, Jerusalem, Israel), mouse anti-VAPA monoclonal antibody (1:500 in 3% BSA; RRID AB_2722702, UC Davis/NeuroMab Facility), and chicken anti-MAP2 polyclonal antibody (1:1000 in 3% BSA; RRID AB_5392, Abcam, Cambridge, MA) in 3% BSA (in PBS) at 4°C under gentle agitation. Secondary immunolabeling of MAP2 staining was accomplished by incubation with a Cy5-tagged donkey anti-chicken antibody (1:500 in 3% BSA) for 1 hour. Coverslips were then rinsed four times for 5 min in PBS before beginning the PLA. Duolink protocol was followed according to factory instructions with adaptation from (24). Briefly, fixed and immunocytochemically labeled neurons were first incubated with oligonucleotide-conjugated anti-mouse minus (1:5 in 3% BSA) and anti-rabbit plus (1:5 in 3% BSA) probes for 1 hour at 37°C to probe for anti-VAPA and anti-Kv2.1 primary antibodies, respectively. Coverslips were then rinsed in Duolink PLA Wash Buffer A two times for 5 min. Oligonucleotide ligation of plus and minus probes was accomplished by incubation with ligase (1:40) in Duolink PLA ligation solution (1:5 in dH2O) for 45 min at 37°C, before coverslips were again rinsed in Wash Buffer A two times for 2 min. Rolling circle amplification reaction and subsequent fluorescently labeled oligonucleotide hybridization with this concatemeric product was accomplished by incubating coverslips with Duolink amplification orange (1:5 in dH₂O) and polymerase enzyme (1:40) for 100 min at 37°C. Last, coverslips were washed in Duolink Wash Buffer B two times for 10 min, followed by a final wash in 0.01× Wash Buffer B for 1 min. Coverslips were then mounted onto glass slides with Duolink 4′,6-diamidino-2-phenylindole mounting media for confocal imaging.

PLA puncta were imaged on a Nikon A1+ confocal microscope (Nikon, Tokyo, Japan) with a 60× objective. Random neuronal somas were identified on each coverslip using the Cy5 filter to visualize MAP2 staining. PLA puncta signals were then obtained by capturing 25 optical sections (0.5 μm) that constrained the entire Z width of PLA-TRITC (tetramethyl rhodamine isothiocyanate) fluorescence as MAX-IP files. Laser power, HV (gain), offset, pinhole size, and all camera parameters were kept identical between all samples in four separate experiments. NIS-Elements AR object count feature was then used to analyze the quantity of PLA puncta per cell. To do this, cell somas and proximal dendrites were first selected as ROIs, using MAP2-positive staining to confirm analysis of neurons. As distinct local PLA puncta-TRITC autofluorescence (markedly different in appearance from PLA puncta) was noted, local background subtraction using a small ROI within this region was performed in a protocol similar to that described earlier (48). Next, PLA puncta were counted and measured by a semi-automated object count feature within the NIS-Elements AR analysis program. Thresholding was set for each image by choosing well-defined, highly fluorescent PLA puncta compared to background fluorescence and setting inclusion thresholds based on these puncta. Object counts were verified by manual counting.

Electrophysiology

Whole-cell voltage clamp currents from rat cortical neurons were obtained with Axopatch 200B amplifier using pClamp software (Molecular Devices, Sunnyvale, CA) using 3- to 5-megohm electrodes. The extracellular solution contained the following: 2.0 mM MgCl₂, 2.5 mM KCl, 115 mM NaCl, 10 mM Hepes, 10 mM d-glucose, 1.0 mM CaCl₂, and 0.25 μM tetrodotoxin (pH 7.2). The electrode solution contained the following: 100 mM potassium gluconate, 1 mM MgCl, 10 mM KCl, 1 mM CaCl₂, 2 mM MgCl₂–adenosine triphosphate, 0.33 mM guanosine triphosphate, 11 mM EGTA, and 10 mM Hepes (pH 7.2). Series resistance was compensated (80%), and currents were digitized at 10 kHz and filtered at 2 kHz. Potassium currents were evoked with a series of 200-ms voltage steps from a holding potential of −80 to 80 mV in 10-mV increments. A 30-ms pre-pulse to +10 mV was used to inactivate A-type potassium currents. Delayed rectifier currents were measured relative to baseline at 180 ms after the initiation of each voltage step. Currents were normalized to cell capacitance. Current density analyses were limited to the +30-mV voltage step to minimize voltage errors due to large whole-cell current amplitudes at higher holding potentials, which can be significant even after compensation for series resistance.

Cell toxicity assay

Cortical neurons were pretreated with vehicle, TAT-DP-2 (3 μM), or TAT-Sc-2 (3 μM) for 2 hours in Hepes-buffered minimal essential media with 0.01% BSA (MHB). Next, cells were incubated with the same peptide concentrations along with either vehicle or 60 μM TBOA for 6 hours. Each well was washed two times with either 0.5 ml of vehicle or 60 μM TBOA, and neurons were incubated overnight. Twenty-four hours following initial TBOA treatment, medium was collected for LDH assays (Sigma-Aldrich, St. Louis, MO). Relative toxicity was quantified by calculating the ratio of LDH concentration in TBOA-treated over non–TBOA-treated wells. In cell culture, LDH release measures neuronal cell death and is not specific to any one mechanism of cell demise.

MCAo microsurgery

Mice were anesthetized with isoflurane, and the common carotid artery was exposed. A single-use silicon-coated suture (MCAo suture no. 602256PK10, Doccol Corporation, Sharon, MA) was advanced 8 to 9 mm into the internal carotid artery, occluding the MCA. The suture was secured with 6-0 silk ligatures for 50 min and then removed. Twenty-four hours following cerebral reperfusion, mice were sacrificed, and four 2-mm coronal brain sections were obtained from each animal. Sections were stained with 0.05% 2,3,5-triphenyltetrazolium chloride in PBS for 30 min at room temperature. Brain sections were digitally scanned following staining, and a blinded experimenter measured infarct ratios (infarct area/total section area) using National Institutes of Health (NIH) ImageJ software. Visual representation of this protocol is in Fig. 6B.

To analyze neurological deficits following MCA stroke, testing was performed by a blinded experimenter on additional cohorts, both at baseline (all initial scores were 0) and on post-stroke days 1, 2, 3, 5, 7, 10, 14, 21, 28, 35, and 42. An objective MNS (table S1) was assigned on each day, ranging from 0 (no neurological deficit) to 8 (stroke-related death) (9).

Peptide-blot array

Standard 9-fluorenylmethoxy carbonyl chemistry was used to synthesize peptides and spot them onto CelluSpots nitrocellulose disks prederivatized with a polyethylene glycerol spacer (Intavis), using the ResPep peptide synthesizer. The peptides were spotted on 20 membranes fitted on microscope glass slides (Intavis) using an Intavis MultiPep robot. For hybridization and immunoblotting of arrays, CelluSpots slides were washed in TBST [50 mM tris-HCl (pH 7.4), 150 mM NaCl, and 0.1% Tween 20] for 10 min and 5% nonfat dry milk and then blocked for 1 hour at room temperature with gentle shaking in TBST containing 5% nonfat dry milk. Lysates from HEK293 cells transfected as before (9, 11, 49) with pEGFP-N1-VAPA, 1-242 (Addgene no. 18874) were made in 20 mM tris, 50 mM NaCl, 2 mM MgCl₂, 1% Tergitol, 0.5% sodium deoxycholate, 0.1% SDS with protease (catalog no. B14002, Bimake), phosphatase (catalog no. B15002, Bimake) inhibitor cocktails, and universal nuclease (5 U/ml; catalog no. 88702, Thermo Fisher Scientific) added extemporaneously. The concentration of lysates was measured by bicinchoninic acid (catalog no. 23227, Thermo Fisher Scientific), and peptide arrays were incubated with total protein (1 mg/ml) overnight at 4°C (9, 11, 50). Peptide arrays were washed three times for 5 min at room temperature with TBST and incubated with the primary antibody anti-VAPA (RRID AB_2722707, UC Davis/NIH NeuroMab Facility) for 2 hours at room temperature with gentle shaking in TBST and 5% BSA. After washing with TBST, the membranes were incubated in secondary antibody (IRDye 800CW Goat anti-Mouse IgG secondary antibody; RRID AB_621842, LI-COR) for 45 min, washed three times for 5 min in TBST, and visualized by infrared fluorescence (LI-COR). Four independent peptide spot arrays were used in this study. Fluorescence intensity was analyzed with Image studio (LI-COR).

We thank B. Kühn (University of Pittsburgh School of Medicine) for valuable feedback and guidance and K. Hartnett, G. Kosobucki, and S. Guar (University of Pittsburgh School of Medicine) for valuable technical support. We thank G. Begum for technical guidance on immunohistochemical studies. We thank B. Molyneaux for valuable feedback on the clinical context of this work. Funding: This work was funded by NIH grant NS043277 to E.A. A.J.S. was supported, in part, by Physician Scientist Training Program Scholarship. Author contributions: E.A. funded the work. E.A., A.J.S., J.A.J., and D.S. designed the experiments. E.A., A.J.S., J.A.J., C.-Y.Y., B.N.O., O.J.P., M.P.H., A.M., and R.K. collected and analyzed data. A.J.S. and E.A. wrote the manuscript. All authors critically edited and approved the final manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors. Contact E.A. (ude.ttip@xoder) for data files or materials.