Journal: bioRxiv

Article Title: GABA B receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

doi: 10.1101/2020.04.16.045112

Figure Lengend Snippet: A Schematic drawing of the two MHb-IPN pathways. In red: the dorsal part of the MHb projects to the lateral subnuclei of the IPN. In blue: the ventral part of the MHb projects to the rostral/central subnuclei of the IPN. B Confocal image of Cav2.3 immunofluorescence signal indicates Cav2.3 presence in MHb axonal projections of both MHb-IPN pathways. C Pharmacological inhibition of Cav2.3 with SNX-482 in whole-cell recordings of rostral IPN neurons. Left: example traces before and after the application of SNX-482; middle: example time course of EPSC amplitude reduction by SNX-482; right: averaged time course of relative EPSC amplitude reduction by SNX-482. EPSC amplitudes were reduced by 83% on average (n=9 cells/9 mice). D In Tac1-ChR2-EYFP mice, SNX-482 reduced light-evoked glutamatergic EPSC amplitudes on average by 52% (n=8 cells/4 mice). E Confocal image of GABA B1 immunofluorescence signal indicates the presence of GABA B receptors (GBRs) in all IPN subnuclei. F In whole-cell recordings of rostral IPN neurons, activation of GBRs by baclofen (1 µM) produced a potentiation of electrically evoked EPSC amplitudes. Left: example EPSC traces before (black) and during the application of baclofen (red) and after washout of baclofen (blue); middle: example time course of EPSC amplitudes in one cell; right: averaged time course of relative EPSC amplitude change after baclofen (n=13 cells/9 mice). G Baclofen reduced the amplitude of light-evoked glutamatergic EPSCs in lateral IPN neurons (n=10 cells/5 mice). Scale bars in panels ( B ) and ( E ) are 100 µm. Averaged data is presented as mean ± SEM.

Article Snippet: For immunoblotting analysis, proteins were transferred to 0.45 μM polivinylidene fluoride membranes (Milipore, Burlington, MA, USA) for 2 h at 200 mA and probed with the primary antibodies rabbit anti-Flag (F7425, Sigma) and anti-Cav2.3 (ACC-006, Alomone Labs) in combination with peroxidase-coupled secondary donkey anti-rabbit antibodies (NA934, GE Healthcare, 1:10000).

Techniques: Immunofluorescence, Inhibition, Activation Assay, Produced

Journal: bioRxiv

Article Title: GABA B receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

doi: 10.1101/2020.04.16.045112

Figure Lengend Snippet: Transmission electron microscopy images of 70 nm−thick sections following pre-embedding immunolabeled IPN slices for Cav2.3 ( A ), GABA B1 ( B ), KCTD8 ( C ), KCTD12 ( D ) and KCTD12b ( E ) from synapses in the rostral (left images) and lateral (right image) IPN subnuclei. Scale bars: 200 nm. Graph on the right displays quantification of relative and absolute silver-enhanced gold particle densities in the active zone and at distances of 50 − 200 nm from the edge of the active zone (50 nm bins). F Absolute labeling densities are summarized for synapses in the rostral (left panel) and lateral IPN (right panel). Note absence of KCTD12 and KCTD12b particles in presynaptic terminals inside the lateral IPN subnuclei. KCTD12 was not included in panel F because of predominantly postsynaptic localization inside the rostral IPN. Data was pooled from two animals, showing no significant difference in gold particle distribution patterns with Kolmogorov-Smirnov test (see Supplementary Figure S1).

Article Snippet: For immunoblotting analysis, proteins were transferred to 0.45 μM polivinylidene fluoride membranes (Milipore, Burlington, MA, USA) for 2 h at 200 mA and probed with the primary antibodies rabbit anti-Flag (F7425, Sigma) and anti-Cav2.3 (ACC-006, Alomone Labs) in combination with peroxidase-coupled secondary donkey anti-rabbit antibodies (NA934, GE Healthcare, 1:10000).

Techniques: Transmission Assay, Electron Microscopy, Immunolabeling, Labeling

Journal: bioRxiv

Article Title: GABA B receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

doi: 10.1101/2020.04.16.045112

Figure Lengend Snippet: A Example image of a grid-glued replica containing the whole IPN. White line indicates demarcation of rostral/central and lateral subnuclei. Scale bar: 20 µm B Example image of a presynaptic P face and a postsynaptic E face of a habenular synapse in the rostral IPN that was double labeled with antibodies against AMPA receptors (10 nm gold) and Cav2.3 (5 nm gold). Scale bar: 100 nm. C Example image of a similar synaptic profile double labeled with antibodies against AMPA receptors (10 nm gold) and Cav2.3 (5 nm gold) in the rostral IPN of a Cav2.3 KO mouse. Scale bar: 100 nm. D Left panel: double labeling of a WT carbon-only replica with antibodies against Cav2.3 (5 nm gold) and a mixture of active zone proteins (2 nm gold), including RIM1/2, CAST and neurexin. Right panel: the same image with additional coloring of 2 nm (red) and 5 nm (blue) particles and demarcation of the active zone area based on active zone marker labeling. Scale bars: 100 nm. F Left panel: quantification of Cav2.3 labeling densities in the presynaptic P face in WT and Cav2.3 KO mice. *** indicates P < 0.001, unpaired t-test. Right panel: areas of demarcated active zones, including incomplete profiles, were not significantly different between replicas from WT and Cav2.3 KO mice. Data were obtained from 4 replicas from 4 mice of each genotype. G Comparison of active zone area demarcated with or without active zone markers and the size of the glutamatergic postsynaptic IMP clusters. P value indicates result of Kolmogorov-Smirnov test.

Article Snippet: For immunoblotting analysis, proteins were transferred to 0.45 μM polivinylidene fluoride membranes (Milipore, Burlington, MA, USA) for 2 h at 200 mA and probed with the primary antibodies rabbit anti-Flag (F7425, Sigma) and anti-Cav2.3 (ACC-006, Alomone Labs) in combination with peroxidase-coupled secondary donkey anti-rabbit antibodies (NA934, GE Healthcare, 1:10000).

Techniques: Labeling, Marker

Journal: bioRxiv

Article Title: GABA B receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

doi: 10.1101/2020.04.16.045112

Figure Lengend Snippet: A Active zones double-labeled for Cav2.3 and either GABA B1 (left), KCTD8 (middle) or KCTD12b (right) in IPN replicas. Top row images are from presynaptic terminals in the rostral IPN, bottom row images are from presynaptic terminals in the lateral IPN. Scale bar: 100 nm. B Quantification of active zone immunolabeling in the rostral and lateral IPN. With the exception of the absence of KCTD12b in lateral IPN terminals, absolute particle numbers per active zone (left graph) and particle densities (middle graph) are comparable between MHb terminals in the rostral and lateral IPN. Right graph: Over 97% of active zones positive for Cav2.3 labeling also show labeling for one of the other molecules (GABA B1 , KCTD8 or KCTD12b), suggesting co-localization of all presynaptic molecules inside the same active zone. Numbers inside the bars indicate the number of replicas used for each quantification. C Nearest neighbor distance (NND) for all presynaptic molecules in MHb terminals inside the rostral and lateral IPN based on the real (black line) and simulated random distribution (blue line). Smaller NND values in real distributions compared to simulation suggest clustering of all presynaptic molecules. P values calculated via Kolmogorov-Smirnov test.

Article Snippet: For immunoblotting analysis, proteins were transferred to 0.45 μM polivinylidene fluoride membranes (Milipore, Burlington, MA, USA) for 2 h at 200 mA and probed with the primary antibodies rabbit anti-Flag (F7425, Sigma) and anti-Cav2.3 (ACC-006, Alomone Labs) in combination with peroxidase-coupled secondary donkey anti-rabbit antibodies (NA934, GE Healthcare, 1:10000).

Techniques: Labeling, Immunolabeling

Journal: bioRxiv

Article Title: GABA B receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

doi: 10.1101/2020.04.16.045112

Figure Lengend Snippet: A Example images of active zones containing Cav2.3 and either GABA B1 (left panels) or KCTD8 (right panels) in replicas of WT (upper row) and KCTD12b KO IPN tissue (lower row). Scale bars: 100 nm B Quantification of relative densities for Cav2.3, KCTD8 and GABA B1 in active zones located in the rostral IPN of WT and KCTD12b KO mice. Densities were normalized to the average density in MHb terminals inside the lateral IPN of the same replica. The number inside the bars indicate the number of replicas used for quantification. ** indicate P < 0.01 in a Mann-Whitney test.

Article Snippet: For immunoblotting analysis, proteins were transferred to 0.45 μM polivinylidene fluoride membranes (Milipore, Burlington, MA, USA) for 2 h at 200 mA and probed with the primary antibodies rabbit anti-Flag (F7425, Sigma) and anti-Cav2.3 (ACC-006, Alomone Labs) in combination with peroxidase-coupled secondary donkey anti-rabbit antibodies (NA934, GE Healthcare, 1:10000).

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: GABA B receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

doi: 10.1101/2020.04.16.045112

Figure Lengend Snippet: A Co-immunoprecipitation from total cell lysates of HEK293 cells transfected with Flag-tagged KCTDs and Cav2.3. Immunoprecipitation of Cav2.3 co-precipitated KCTD8 and KCTD12b, but not KCTD12. Input lanes (bottom) indicate expression of the tagged proteins in the cell lysates. B Whole-cell recordings from HEK293 cells stably expressing Cav2.3. Ba 2+ current densities measured in response to a single depolarizing voltage step from −80 to 10 mV were significantly increased in KCTD8 co-transfected cells. * P < 0.05, ** P < 0.01 one-way ANOVA with Tukey post hoc test. C Current density-to-voltage relationship demonstrating higher current densities in KCTD8-transfected cells compared with Control- and KCTD12b-transfected cells. **** P < 0.0001 two-way ANOVA with Tukey post hoc test. D Activation and inactivation curves in Control-, KCTD8- and KCTD12b-transfected cells. ** P < 0.01, two-way ANOVA with Tukey post hoc test. E Schematic representation of the distribution and function of Cav2.3, GBRs and KCTDs in ventral MHb terminals of WT and KCTD12b KO mice. Top image: In WT terminals, the active zone contains Cav2.3 and hetero-pentameric rings comprising KCTD12b in excess over KCTD8, whereas KCTD8 and GBRs are located peri-synaptically. Bottom image: In absence of KCTD12b, KCTD8 invades the active zone and compensates for the loss of KCTD12b, resulting in increased release probability, potentially via increased Ca 2+ influx through Cav2.3.

Article Snippet: For immunoblotting analysis, proteins were transferred to 0.45 μM polivinylidene fluoride membranes (Milipore, Burlington, MA, USA) for 2 h at 200 mA and probed with the primary antibodies rabbit anti-Flag (F7425, Sigma) and anti-Cav2.3 (ACC-006, Alomone Labs) in combination with peroxidase-coupled secondary donkey anti-rabbit antibodies (NA934, GE Healthcare, 1:10000).

Techniques: Immunoprecipitation, Transfection, Expressing, Stable Transfection, Activation Assay

Journal: The Journal of Neuroscience

Article Title: A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels

doi: 10.1523/JNEUROSCI.2844-07.2008

Figure Lengend Snippet: Inhibition of Cav2.1 Ca2+ currents by the R1279X EA2-truncated form of Cav2.1 protein. A, Schematic cartoon representing the main Cav2.1 constructs used in this study. B, The Ca2+ current density in HEK293 cells expressing the Cav2.1 channel protein. The cells were cotransfected with plasmids encoding the human Cav2.1 subunit, the β1b subunit, and the α2/δ1 subunit. Mean ± SEM values of the current density for cells expressing the Cav2.1 subunit alone (white bar; n = 18) or in the presence of the R1279X mutant (+R1279X) (black bar; n = 22) are presented (***p < 0.001; Student's t test). The inset shows representative current traces at TP −40 and 0 mV [holding potential (HP), −80 mV]. C, Same experiments as in B performed in the neuroblastoma cell line NG108-15. The HP was −50 mV (***p < 0.001; Student's t test). Cav2.1 subunit alone (white bar; n = 15) or in the presence of the R1279X mutant (+R1279X) (black bar; n = 22). D, Measurements of native T-type and HVA Ca2+ current densities in R1279X-transfected NG108-15 cells. The T-current density (left) was measured in proliferative NG108-15 cells (Prolif.; 2–3 d after transfection; n = 9) and in differentiated NG108-15 cells (Diff.; 6 d after transfection; 3–4 d after switching to differentiation medium; n = 11) using HP −100 mV and TP −30 mV. The HVA current density, mainly corresponding to L- and N-type channel activities, was measured using HP −50 mV and TP 0 mV (right).

Article Snippet: Anti-Ca v 2.1 polyclonal antibodies were purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Inhibition, Construct, Expressing, Mutagenesis, Transfection

Journal: The Journal of Neuroscience

Article Title: A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels

doi: 10.1523/JNEUROSCI.2844-07.2008

Figure Lengend Snippet: Effect of R1279X mutant on surface expression of Cav2.1 channel. A, HEK293 cells were cotransfected with the HA-tagged wild-type Cav2.1 subunit alone (with free GFP) or together with the R1279X mutant (fused to GFP). Left column, GFP fluorescence. Middle column, Staining of cells with monoclonal rat anti-HA antibody (primary antibody) and Alexa594 (secondary antibody). NP, Nonpermeabilized; P, permeabilized. Images were acquired using a Leica SP2 confocal microscope, with a 63× oil immersion objective. B, Luminometric ELISA assays to quantify surface expression of the HA-tagged Cav2.1 in the presence of the R1279X mutant and auxiliary subunits (**p < 0.01, ***p < 0.001; Student's t test). C, Western blots performed on HEK293 cells expressing Cav2.1-HA alone (−) or in the presence of R1279X (+) without (left) or with (right) auxiliary subunits. β1b-HA was used in these experiments (right).

Article Snippet: Anti-Ca v 2.1 polyclonal antibodies were purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Mutagenesis, Expressing, Fluorescence, Staining, Microscopy, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: The Journal of Neuroscience

Article Title: A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels

doi: 10.1523/JNEUROSCI.2844-07.2008

Figure Lengend Snippet: Channel misfolding and instability induced by the R1279X mutant. A, B, Pulse-chase experiments were performed on HEK293 cells 48 h after transfection (see Materials and Methods). Cells were labeled with 35S-methionine-cysteine for 20 min. The chase was performed for 0, 1, 2, or 4 h as indicated. After lysis, the Cav2.1 subunit was immunoprecipitated with an anti-HA antibody. The arrow indicates the band corresponding to the EA2 mutant that coimmunoprecipitates with the wild-type Cav2.1 subunit. In B (left), the β1b is detected (arrow). C, Quantification of three independent experiments using GE Healthcare software. D, Pulse-chase analyses performed on HEK293 cells transfected with CD4-AAXX alone or in the presence of R1279X. Quantification was performed as described above. E, Immunoprecipitations performed on cells coexpressing various subunit arrangements (as indicated) in the absence or presence of an untagged R1279X mutant (right) using standard SDS-PAGE (6%). Western blots (WBs) were performed with the indicated antibodies. F, Immunoprecipitations were performed between β1b-HA and GFP-R1279X as described in E. MW, Molecular weight.

Article Snippet: Anti-Ca v 2.1 polyclonal antibodies were purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Mutagenesis, Pulse Chase, Transfection, Labeling, Lysis, Immunoprecipitation, Software, SDS Page, Western Blot, Molecular Weight

Journal: The Journal of Neuroscience

Article Title: A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels

doi: 10.1523/JNEUROSCI.2844-07.2008

Figure Lengend Snippet: EA2 missense mutants act in a dominant-negative manner by misfolding and instability induction. A, Histograms of the mean Ca2+ current density (± SEM) obtained in a representative batch of NG108-15 cells expressing wild-type subunit alone (Cav2.1-HA, n = 18); the EA2 mutants alone (Cav2.1-HA-G293R, n = 33, 5 with detectable current; Cav2.1-HA-AY1593/94D, n = 6); and the wild-type and EA2 mutants together (Cav2.1-HA+Cav2.1-HA-G293R, n = 23, 3 with detectable current; Cav2.1-HA+Cav2.1-HA-AY1593/94D, n = 10). These experiments were conducted in the presence of the auxiliary β1b and α2/δ1 subunits. B, Normalized native T-type Ca2+ current densities (mean ± SEM) in the NG108-15 cells analyzed in A. The differences are not statistically significant between the various conditions tested (Student's t test). C, Western blots with the indicated antibodies (WB) were performed on HEK293 cells coexpressing Cav2.1-HA alone or in the presence of G293R and AY1593/94D mutants together with auxiliary subunits. The bottom panel shows the Western blot from cells expressing missense mutants alone. D, Pulse-chase analyses were performed on HEK293 cells transfected with wild-type Cav2.1 alone or in the presence of G293R. Quantification of three independents experiment was performed as described above. MW, Molecular weight.

Article Snippet: Anti-Ca v 2.1 polyclonal antibodies were purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Dominant Negative Mutation, Expressing, Western Blot, Pulse Chase, Transfection, Molecular Weight

Journal: The Journal of Neuroscience

Article Title: A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels

doi: 10.1523/JNEUROSCI.2844-07.2008

Figure Lengend Snippet: Endoplasmic reticulum retention and proteasomal degradation of the dominant-negative Cav mutants. A, Confocal images of nonpermeabilized NG108-15 cells expressing EA2 mutants and truncated Cav3.2 subunits. Alexa 594-coupled CT was used as plasma membrane marker (0.5 μg/ml). B, Confocal images of immunofluorescence staining performed on permeabilized NG108-15 cells expressing indicated EA2 mutants and truncated Cav3.2. Polyclonal anti-protein disulfide isomerase (Assay Designs, Ann Arbor, MI) was used as ER marker. C, Pulse-chase experiments were performed as in Figure 3 on cells transfected with EA2 mutants (R1279X and G293R) and Cav3.2 truncated forms. Chase was done for the indicated time (hours) with or without MG-132 proteasome inhibitor (50 μm). After lysis, the truncated Cav channels were immunoprecipitated with anti-GFP antibody. D, Representative pulse chase performed on HEK293 cells transfected with Cav2.1 and the R1279X mutant and the corresponding quantification (n = 3). During the chase, cells were treated with MG-132 (50 μm), leupeptin (20 μm), and NH4Cl (10 mm).

Article Snippet: Anti-Ca v 2.1 polyclonal antibodies were purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Dominant Negative Mutation, Expressing, Marker, Immunofluorescence, Staining, Pulse Chase, Transfection, Lysis, Immunoprecipitation, Mutagenesis

Journal: BMC Neuroscience

Article Title: The punctate localization of rat Eag1 K + channels is conferred by the proximal post-CNBHD region

doi: 10.1186/1471-2202-15-23

Figure Lengend Snippet: Subcellular localization of native rEag1 and rEag2 channels in young and mature hippocampal neurons in culture. Dissociated hippocampal neurons at DIV3, DIV7, and DIV12 were immunostained with the anti-rEag1 (A, B) or the anti-rEag2 (C, D) antibodies ( shown in green; left panels ), followed by counterstaining with the antibody for the dendritic marker MAP2 or the axonal marker tau ( shown in red; middle panels ). Merged images are shown in the right panels. (A) rEag1 immunoreactivities were localized in cell bodies, as well as in MAP2-positive and MAP2-negative ( arrows ) processes. (B) rEag1 immunoreactivities were present in the axonal compartment that was clearly defined by the immunofluorescence signal of tau ( arrows ). For DIV12 neurons in both (A) and (B), note the presence of punctate rEag1 staining patterns throughout proximal and distal neurites. (C, D) rEag2 immunoreactivities were present in both MAP2-positive and tau-positive processes. No significant rEag2 puncta were observed. Scale bar, 25 μm.

Article Snippet: The antibodies used in this study include rabbit anti-rEag1, rabbit anti-rEag2 (Alomone), mouse anti-β-actin (Sigma), mouse anti-MAP2 (Sigma), mouse anti-tau (Chemicon), mouse anti-PSD-95 (Cell Signaling), and mouse anti-synaptophysin (a kind gift from Dr. Erik Schweitzer, UCLA).

Techniques: Marker, Immunofluorescence, Staining

Journal: BMC Neuroscience

Article Title: The punctate localization of rat Eag1 K + channels is conferred by the proximal post-CNBHD region

doi: 10.1186/1471-2202-15-23

Figure Lengend Snippet: Synaptic localization of rEag1 channels. (A) Hippocampal neurons were double-stained for rEag1/rEag2 ( left panels ) and the postsynaptic density marker PSD-95 ( middle panels ). Scale bar, 25 μm. (B) Quantification of the number of puncta per 100-μm neurite (puncta/100 μm) for PSD-95, rEag1, and rEag2. The number in parenthesis denotes the amount of neurites analyzed, and the asterisk indicates a significant difference ( t -test, p < 0.05) from PSD-95. Data were collected from 7-11 different neurons. (C) Quantification of the co-localization of PSD-95 with rEag1 or rEag2. The data illustrate the fraction of PSD-95 puncta that were co-localized with rEag1/2 puncta, as well as the fraction of rEag1/2 puncta that were co-localized with PSD-95 puncta. The number in parenthesis denotes the amount of neurites analyzed, and the asterisk indicates a significant difference ( t -test, p < 0.05) from rEag1. Data were collected from 7-8 different neurons. (D) Subcellular fractionation of rat brains: the homogenate (H), the soluble fraction (S1), the crude membrane fraction (P2), the synaptosomal fraction (SPM), and the two postsynaptic density (PSD) preparations (PSD I: one Triton X-100 wash; PSD II: two Triton X-100 washes). The left panel ( 25 μg ) illustrates the primary fractionation profile, whereas the right panel ( 5 μg ) exemplifies the further enrichment pattern in the three sub-fractions of synaptosomes. All fractions were subject to immunoblotting analyses with the indicated antibodies. 25 μg and 5 μg refer to the amount of total protein loaded in each lane.

Article Snippet: The antibodies used in this study include rabbit anti-rEag1, rabbit anti-rEag2 (Alomone), mouse anti-β-actin (Sigma), mouse anti-MAP2 (Sigma), mouse anti-tau (Chemicon), mouse anti-PSD-95 (Cell Signaling), and mouse anti-synaptophysin (a kind gift from Dr. Erik Schweitzer, UCLA).

Techniques: Staining, Marker, Fractionation, Western Blot

Journal: BMC Neuroscience

Article Title: The punctate localization of rat Eag1 K + channels is conferred by the proximal post-CNBHD region

doi: 10.1186/1471-2202-15-23

Figure Lengend Snippet: Expression of GFP-rEag1 and GFP-rEag2 channels in HEK293T cells and hippocampal neurons. (A) Immunofluorescence staining and functional expression of GFP-rEag1 and GFP-rEag2 K + channels in HEK293T cells. GFP fluorescence ( shown in green ) and rEag1/rEag2 immunofluorescence ( shown in red ) signals demonstrated lucid co-localization at the membrane region. Scale bar, 10 μm. Whole-cell patch clamp parameters: the holding potential for rEag1 and rEag2 was -90 and -110 mV, respectively; the pulse protocol comprised 300-ms depolarizing test pulses ranging from -70 to +50 mV (rEag1) or from -90 to +30 mV (rEag2), with 10-mV increments. (B) Over-expression of GFP-rEag1/rEag2 in DIV12 hippocampal neurons. GFP signal is shown in green, and MAP2 immunofluorescence signal in red. Note the presence of prominent GFP puncta for rEag1, but not rEag2. Scale bar, 25 μm. (C) ( Top ) Schematic representation of the structural topology of Eag K + channel. ( Bottom ) Protein sequence alignment between rEag1 and rEag2 over the post-CNBHD region. Yellow shade: identical residues. Green shade: homologous residues. Sequence alignment analysis was implemented with the Vector NTI software (InforMax).

Article Snippet: The antibodies used in this study include rabbit anti-rEag1, rabbit anti-rEag2 (Alomone), mouse anti-β-actin (Sigma), mouse anti-MAP2 (Sigma), mouse anti-tau (Chemicon), mouse anti-PSD-95 (Cell Signaling), and mouse anti-synaptophysin (a kind gift from Dr. Erik Schweitzer, UCLA).

Techniques: Expressing, Immunofluorescence, Staining, Functional Assay, Fluorescence, Patch Clamp, Over Expression, Sequencing, Plasmid Preparation, Software

Journal: BMC Neuroscience

Article Title: The punctate localization of rat Eag1 K + channels is conferred by the proximal post-CNBHD region

doi: 10.1186/1471-2202-15-23

Figure Lengend Snippet: Characterization of rEag1-I and rEag2-I chimeric channels. (A) Schematic representation of the construction of rEag1-I and rEag2-I chimeras. For all schematic cartoons hereafter, rEag1 and rEag2 sequences are shown in red and black, respectively. (B) Representative K + currents recorded from Xenopus oocytes over-expressing the indicated Eag constructs. Two-electrode voltage clamp parameters: the holding potential for rEag1 and rEag2 was -90 and -110 mV, respectively; the pulse protocol comprised 300-ms depolarizing test pulses ranging from -70 to +60 mV (rEag1) or from -100 to +40 mV (rEag2), with 10-mV increments. (C) Membrane localization of GFP-rEag1-I/rEag2-I channels in HEK293T cells. Scale bar, 10 μm. (D) Expression of GFP-rEag1-I/rEag2-I channels in DIV12 hippocampal neurons. Scale bar, 25 μm. (E) Quantification of the number of GFP puncta per neuron for GFP-rEag1, GFP-rEag1-I, GFP-rEag2-I, and GFP-rEag2. The number in parenthesis denotes the amount of neurons analyzed. (*: significantly different from GFP-rEag1; t -test, p < 0.05)(#: significantly different from GFP-rEag2; t -test, p < 0.05)

Article Snippet: The antibodies used in this study include rabbit anti-rEag1, rabbit anti-rEag2 (Alomone), mouse anti-β-actin (Sigma), mouse anti-MAP2 (Sigma), mouse anti-tau (Chemicon), mouse anti-PSD-95 (Cell Signaling), and mouse anti-synaptophysin (a kind gift from Dr. Erik Schweitzer, UCLA).

Techniques: Expressing, Construct

Journal: BMC Neuroscience

Article Title: The punctate localization of rat Eag1 K + channels is conferred by the proximal post-CNBHD region

doi: 10.1186/1471-2202-15-23

Figure Lengend Snippet: Characterization of rEag1-II, III, and IV chimeric channels. (A) Schematic representation of the construction of rEag1-II, rEag1-III, and rEag1-IV chimeras. (B) Representative K + currents recorded from Xenopus oocytes over-expressing the indicated rEag1 constructs. (C) Membrane localization of the GFP-rEag1 chimeric channels in HEK293T cells. Scale bar, 10 μm. (D) Expression of the GFP-rEag1 chimeric channels in DIV12 hippocampal neurons. Scale bar, 25 μm. (E) Quantification of the number of GFP puncta per neuron for the GFP-rEag1 chimeric channels. Note the presence of rEag2-like GFP puncta density in rEag1-II only. (*: significantly different from GFP-rEag1; t -test, p < 0.05)(#: significantly different from GFP-rEag2; t -test, p < 0.05)

Article Snippet: The antibodies used in this study include rabbit anti-rEag1, rabbit anti-rEag2 (Alomone), mouse anti-β-actin (Sigma), mouse anti-MAP2 (Sigma), mouse anti-tau (Chemicon), mouse anti-PSD-95 (Cell Signaling), and mouse anti-synaptophysin (a kind gift from Dr. Erik Schweitzer, UCLA).

Techniques: Expressing, Construct

Journal: BMC Neuroscience

Article Title: The punctate localization of rat Eag1 K + channels is conferred by the proximal post-CNBHD region

doi: 10.1186/1471-2202-15-23

Figure Lengend Snippet: Characterization of rEag2-II, III, and IV chimeric channels. (A) Schematic representation of the construction of rEag2-II, rEag2-III, and rEag2-IV chimeras. (B) Representative K + currents recorded from Xenopus oocytes over-expressing the indicated rEag2 constructs. (C) Membrane localization of the GFP-rEag2 chimeric channels in HEK293T cells. Scale bar, 10 μm. (D) Expression of the GFP-rEag2 chimeric channels in DIV12 hippocampal neurons. Scale bar, 25 μm. (E) Quantification of the number of GFP puncta per neuron for the GFP-rEag2 chimeric channels. Note the presence of rEag1-like GFP puncta density in rEag2-II only. (*: significantly different from GFP-rEag1; t -test, p < 0.05)(#: significantly different from GFP-rEag2; t -test, p < 0.05)

Article Snippet: The antibodies used in this study include rabbit anti-rEag1, rabbit anti-rEag2 (Alomone), mouse anti-β-actin (Sigma), mouse anti-MAP2 (Sigma), mouse anti-tau (Chemicon), mouse anti-PSD-95 (Cell Signaling), and mouse anti-synaptophysin (a kind gift from Dr. Erik Schweitzer, UCLA).

Techniques: Expressing, Construct

Journal: BMC Neuroscience

Article Title: The punctate localization of rat Eag1 K + channels is conferred by the proximal post-CNBHD region

doi: 10.1186/1471-2202-15-23

Figure Lengend Snippet: Expression of GFP-rEag1-K848X channels in hippocampal neurons. ( Left panel ) GFP-rEag1-K848X was over-expressed in DIV12 hippocampal neurons. Similar to GFP-rEag1 channels, the GFP signal arising from the truncation mutant also displayed the characteristic punctate pattern. Scale bar, 25 μm. ( Right panel ) Quantification of the number of GFP puncta per neuron for rEag1-K848X. (*: significantly different from GFP-rEag1; t -test, p < 0.05)(#: significantly different from GFP-rEag2; t -test, p < 0.05)

Article Snippet: The antibodies used in this study include rabbit anti-rEag1, rabbit anti-rEag2 (Alomone), mouse anti-β-actin (Sigma), mouse anti-MAP2 (Sigma), mouse anti-tau (Chemicon), mouse anti-PSD-95 (Cell Signaling), and mouse anti-synaptophysin (a kind gift from Dr. Erik Schweitzer, UCLA).

Techniques: Expressing, Mutagenesis

Journal: BMC Neuroscience

Article Title: The punctate localization of rat Eag1 K + channels is conferred by the proximal post-CNBHD region

doi: 10.1186/1471-2202-15-23

Figure Lengend Snippet: The biophysical properties of the chimeric Eag channels. Comparison of the voltage-dependent gating properties of the rEag1 (A) or rEag2 (B) chimeras with their wild-type (WT) counterparts. Steady-state voltage dependence (activation curve) is illustrated as the fraction of open channels ( P o) against the corresponding membrane potential. Activation time constants at indicated potentials were obtained from single exponential fits to the late rising phase of Eag K + currents. Deactivation time constants were derived from single exponential fits to the decay phase of Eag K + currents at the indicated tail potential in response to a +40 mV test pulse. All values are presented as mean ± SEM. Data were collected and analyzed as described previously .

Article Snippet: The antibodies used in this study include rabbit anti-rEag1, rabbit anti-rEag2 (Alomone), mouse anti-β-actin (Sigma), mouse anti-MAP2 (Sigma), mouse anti-tau (Chemicon), mouse anti-PSD-95 (Cell Signaling), and mouse anti-synaptophysin (a kind gift from Dr. Erik Schweitzer, UCLA).

Techniques: Activation Assay, Derivative Assay

Journal: BMC Neuroscience

Article Title: The punctate localization of rat Eag1 K + channels is conferred by the proximal post-CNBHD region

doi: 10.1186/1471-2202-15-23

Figure Lengend Snippet: Steady-state voltage-dependent activation parameters of the rEag1 and rEag2 chimeric channels

Article Snippet: The antibodies used in this study include rabbit anti-rEag1, rabbit anti-rEag2 (Alomone), mouse anti-β-actin (Sigma), mouse anti-MAP2 (Sigma), mouse anti-tau (Chemicon), mouse anti-PSD-95 (Cell Signaling), and mouse anti-synaptophysin (a kind gift from Dr. Erik Schweitzer, UCLA).

Techniques: Activation Assay