Journal: PLoS ONE

Article Title: Importance of Glycosylation on Function of a Potassium Channel in Neuroblastoma Cells

doi: 10.1371/journal.pone.0019317

Figure Lengend Snippet: Western blots of wild type (Wt), N220Q, N229Q, and N220Q/N229Q Kv3.1 proteins. Kv3.1 proteins were detected when heterologously expressed in B35 cells (A). Arrows and lines denote the type of N -glycan attached to the Kv3.1 protein. Assignments of the various glycosylated and unglycosylated Kv3.1 proteins were based on immunoband shifts produced by glycosidase treatment. N220Q and N229Q proteins were digested (+) and undigested (−) with neuraminidase (B), PNGase F (C) and Endo H (D). A solid line on image indicates that samples were run on a different blot (B). The numbers adjacent to the Western blots represent the Kaleidoscope markers (in KDa). Similar migration patterns were observed on at least three separate Western blots.

Article Snippet: Electrophoresed proteins were transferred to Immobilon-P PVDF membranes (Millipore, Billercia, MA, USA) at 175 mAmps for 90–240 min. Blots were then incubated at room temperature for 20 min in blocking buffer (PBS, 3% BSA with 0.1% Tween 20) followed by incubation for 2 h with polyclonal rabbit anti-Kv3.1, anti-Kv3.3, or anti-Kv3.4 antibodies (Alamone Labs, Jerusalem, Israel) or overnight with mouse anti-Kv3.1 antibody (NeuroMab).

Techniques: Western Blot, Produced, Migration

Journal: PLoS ONE

Article Title: Importance of Glycosylation on Function of a Potassium Channel in Neuroblastoma Cells

doi: 10.1371/journal.pone.0019317

Figure Lengend Snippet: Whole cell currents for glycosylated (A, middle panel; B, top panel) and unglycosylated (A and B, bottom panels) Kv3.1 proteins were elicited from the indicated voltage protocol (A, top panel). Whole cell currents were scaled for inactivating (A) and non-inactivating (B) current types from B35 cells expressing glycosylated and unglycosylated Kv3.1 proteins. Right panels show traces at expanded time scales and grey lines denote currents at +40 and +60 mV. Traces were scaled to show differences in activation kinetics. Conductance-voltage (g/gmax) curves of both inactivating (C) and non-inactivating (D) current types for glycosylated and unglycosylated Kv3.1 channels. Rise times of inactivating (E) and non-inactivating (F) currents types. n represents number of cells.

Article Snippet: Electrophoresed proteins were transferred to Immobilon-P PVDF membranes (Millipore, Billercia, MA, USA) at 175 mAmps for 90–240 min. Blots were then incubated at room temperature for 20 min in blocking buffer (PBS, 3% BSA with 0.1% Tween 20) followed by incubation for 2 h with polyclonal rabbit anti-Kv3.1, anti-Kv3.3, or anti-Kv3.4 antibodies (Alamone Labs, Jerusalem, Israel) or overnight with mouse anti-Kv3.1 antibody (NeuroMab).

Techniques: Expressing, Activation Assay

Journal: PLoS ONE

Article Title: Importance of Glycosylation on Function of a Potassium Channel in Neuroblastoma Cells

doi: 10.1371/journal.pone.0019317

Figure Lengend Snippet: Electrophysiological parameters of glycosylated, unglycosylated, and partially glycosylated forms of the Kv3.1 channel in B35 cells.

Article Snippet: Electrophoresed proteins were transferred to Immobilon-P PVDF membranes (Millipore, Billercia, MA, USA) at 175 mAmps for 90–240 min. Blots were then incubated at room temperature for 20 min in blocking buffer (PBS, 3% BSA with 0.1% Tween 20) followed by incubation for 2 h with polyclonal rabbit anti-Kv3.1, anti-Kv3.3, or anti-Kv3.4 antibodies (Alamone Labs, Jerusalem, Israel) or overnight with mouse anti-Kv3.1 antibody (NeuroMab).

Techniques:

Journal: PLoS ONE

Article Title: Importance of Glycosylation on Function of a Potassium Channel in Neuroblastoma Cells

doi: 10.1371/journal.pone.0019317

Figure Lengend Snippet: A deactivation voltage protocol (A, left panel) was utilized to obtain deactivation currents for B35 cells expressing the glycosylated (A, right panel) and unglycosylated. Scaled deactivation currents from transfected B35 cells expressing either inactivating (B) or non-inactivating (D) current types. Grey lines denote currents at −30 and −50 mV. Traces were scaled to show differences in deactivation kinetics. Deactivation time constant vs. voltage plot of B35 cells expressing glycosylated and unglycosylated Kv3.1 channels for inactivating (C) and non-inactivating (E) currents types.

Article Snippet: Electrophoresed proteins were transferred to Immobilon-P PVDF membranes (Millipore, Billercia, MA, USA) at 175 mAmps for 90–240 min. Blots were then incubated at room temperature for 20 min in blocking buffer (PBS, 3% BSA with 0.1% Tween 20) followed by incubation for 2 h with polyclonal rabbit anti-Kv3.1, anti-Kv3.3, or anti-Kv3.4 antibodies (Alamone Labs, Jerusalem, Israel) or overnight with mouse anti-Kv3.1 antibody (NeuroMab).

Techniques: Expressing, Transfection

Journal: PLoS ONE

Article Title: Importance of Glycosylation on Function of a Potassium Channel in Neuroblastoma Cells

doi: 10.1371/journal.pone.0019317

Figure Lengend Snippet: Whole cell currents were elicited from the shown voltage protocol (A, left panel) for B35 cells expressing glycosylated (A, right panel) and unglycosylated (B) Kv3.1 proteins. Traces were scaled to show differences in inactivation kinetics. Grey lines denote currents at +40 mV.

Article Snippet: Electrophoresed proteins were transferred to Immobilon-P PVDF membranes (Millipore, Billercia, MA, USA) at 175 mAmps for 90–240 min. Blots were then incubated at room temperature for 20 min in blocking buffer (PBS, 3% BSA with 0.1% Tween 20) followed by incubation for 2 h with polyclonal rabbit anti-Kv3.1, anti-Kv3.3, or anti-Kv3.4 antibodies (Alamone Labs, Jerusalem, Israel) or overnight with mouse anti-Kv3.1 antibody (NeuroMab).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Importance of Glycosylation on Function of a Potassium Channel in Neuroblastoma Cells

doi: 10.1371/journal.pone.0019317

Figure Lengend Snippet: Currents were elicited by a train of five depolarizing voltage steps to +40 mV once every 525 ms, from a holding potential of −50 mV (A, top panel) for B35 cells expressing glycosylated (A, bottom-left panel) and unglycosylated (A, bottom-right panel) Kv3.1 channels. A bar graph representing the percent of peak current amplitude remaining after the fifth pulse relative to peak current amplitude of initial pulse for the various Kv3.1 channels (B). Asterisks indicate significant differences in mean values at a probability of P <0.01 from that of glycosylated Kv3.1.

Article Snippet: Electrophoresed proteins were transferred to Immobilon-P PVDF membranes (Millipore, Billercia, MA, USA) at 175 mAmps for 90–240 min. Blots were then incubated at room temperature for 20 min in blocking buffer (PBS, 3% BSA with 0.1% Tween 20) followed by incubation for 2 h with polyclonal rabbit anti-Kv3.1, anti-Kv3.3, or anti-Kv3.4 antibodies (Alamone Labs, Jerusalem, Israel) or overnight with mouse anti-Kv3.1 antibody (NeuroMab).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Importance of Glycosylation on Function of a Potassium Channel in Neuroblastoma Cells

doi: 10.1371/journal.pone.0019317

Figure Lengend Snippet: Wound width was determined and then normalized at 0, 6, 11 and 23 h for glycosylated and unglycosylated Kv3.1 transfected and non-transfected B35 cells (A). Similar experiments were also performed for glycosylated, and partially glycosylated Kv3.1 glycoproteins (N220Q, and N229Q) transfected B35 cells (B). Data were expressed as the mean +/− SEM. Asterisks indicate significant differences in mean values at a probability of P <0.01 from that of glycosylated Kv3.1. Images were obtained at 0 h, 6 h, 11 h and 23 h of the generated wound for group I: wild type Kv3.1 (row I), N220Q/N229Q (row II) transfected B35 cells and non-transfected B35 cells (row III); and group II: wild type Kv3.1 (row IV), N220Q (row V) and N229Q (row VI) (C). The distance between the two white dashed lines represents wound width for each image. This distance becomes smaller as time increases, representing the rate of cell migration. n represents number of cell wounds. The experiments were conducted on three separate occasions. The solid black line represents a 25 µM scale bar.

Article Snippet: Electrophoresed proteins were transferred to Immobilon-P PVDF membranes (Millipore, Billercia, MA, USA) at 175 mAmps for 90–240 min. Blots were then incubated at room temperature for 20 min in blocking buffer (PBS, 3% BSA with 0.1% Tween 20) followed by incubation for 2 h with polyclonal rabbit anti-Kv3.1, anti-Kv3.3, or anti-Kv3.4 antibodies (Alamone Labs, Jerusalem, Israel) or overnight with mouse anti-Kv3.1 antibody (NeuroMab).

Techniques: Transfection, Generated, Migration

Journal: eLife

Article Title: Kv3.3 subunits control presynaptic action potential waveform and neurotransmitter release at a central excitatory synapse

doi: 10.7554/eLife.75219

Figure Lengend Snippet: ( A ) Kv3.1 in the cochlear nucleus (outlined) of WT mouse at 1 month of age. ( B ) Kv3.3 in the same section of cochlear nucleus (outlined). ( C ) Overlay of Kv3.1 (green) and Kv3.3 (magenta) with DAPI (blue). ( D–F ) magnified section of cochlear nucleus showing Kv3.1 ( D ) and Kv3.3 ( E ) immunoreactivity. Images are overlayed in F. All scale bars = 100 µm. Antibodies and conditions have been independently validated previously using Kv3.1KO and Kv3.3KO tissues .

Article Snippet: Afterwards primary antibodies for Kv3.1b (Rabbit, Alomone APC-014, 1:1000) and Kv3.3 (Mouse, Neuromab 75–354, 1:3000) were diluted in blocking solution and incubated overnight at 4 °C.

Techniques:

Journal: eLife

Article Title: Kv3.3 subunits control presynaptic action potential waveform and neurotransmitter release at a central excitatory synapse

doi: 10.7554/eLife.75219

Figure Lengend Snippet: ( A–B ) Kv3.1 immunoreactivity spiral ganglion neurons in the WT mouse at 1 month of age. In addition to the spiral ganglion cell membranes, Kv3.1 was also present at the nodes of Ranvier (B, arrows). ( C ) No Kv3.1 staining was observed in the spiral ganglion cell bodies of Kv3.1KO mice. Non-specific signal was associated with the bony tissues of the modiolus, and assumed to be autofluorescence from the collagen fibers. ( D–E ) Kv3.3 immunoreactivity spiral ganglion neurons in the WT mouse at 1 month of age. Strong immunoreactivity was observed in the cell bodies. ( F ) Faint non-specific signal was observed in Kv3.3KO tissues, but did not appear to be associated with the spiral ganglion neurons. All scale bars = 100 µm.

Article Snippet: Afterwards primary antibodies for Kv3.1b (Rabbit, Alomone APC-014, 1:1000) and Kv3.3 (Mouse, Neuromab 75–354, 1:3000) were diluted in blocking solution and incubated overnight at 4 °C.

Techniques: Staining

Journal: eLife

Article Title: Kv3.3 subunits control presynaptic action potential waveform and neurotransmitter release at a central excitatory synapse

doi: 10.7554/eLife.75219

Figure Lengend Snippet: ( A ) Current-Voltage (I–V) relationship for potassium currents in the calyx of Held terminal of WT mice (P10–P12) in control (black; n=7 terminals, 4 mice) and TEA, 1 mM (red, n=6 terminals, 5 mice; HP = –70 mV). Inset (top): Example current traces in response to voltage command of +10 mV step (grey box in IV) in WT (black) and WT +1 mM TEA (red). Scale bars = 5 nA and 20ms. Inset (lower): Bar graph of mean currents ± SD, measured on step depolarisation to +10 mV (from HP –70 mV) ±1 mM TEA. Outward Currents are significantly reduced by TEA (student’s t-test, unpaired, P =0.0386). ( B ) WT calyx AP (black trace) evoked by 100 pA step current injection; inset - diagram of recording configuration. ( C ) WT calyx AP in the presence of TEA (1 mM, red trace); inset – overlaid WT APs ±TEA (red) as indicated by dotted box (grey) around APs in B and C. ( D ) Representative AP traces from calyx terminals of WT (black), Kv3.3KO (blue), and Kv3.1KO (orange); double arrows indicate the half-width of WT AP. AP threshold is indicated by the grey dashed line. ( E ) AP half-width measured as time difference between rise and decay phases at 50% maximal amplitude. Half-width is significantly increased in TEA and in Kv3.3KO; N is individual terminals: WT N=9 from 6 animals; TEA = 7 from 5 mice; Kv3.3KO = 6 from 3 mice and Kv3.1KO = 5 from 3 mice. ( F ) AP amplitude, ( G ) AP Decay slope, ( H ) AP rise slope (10–90%) and ( I ) membrane resistance for calyceal recordings. Average data presented as mean ± SD. Statistical test (parts E-I) were one-way ANOVAs and Tukey’s post hoc for multiple comparisons, with significant Ps indicated on the graph. Figure 1—source data 1. Relates to .

Article Snippet: Afterwards primary antibodies for Kv3.1b (Rabbit, Alomone APC-014, 1:1000) and Kv3.3 (Mouse, Neuromab 75–354, 1:3000) were diluted in blocking solution and incubated overnight at 4 °C.

Techniques: Injection

Journal: eLife

Article Title: Kv3.3 subunits control presynaptic action potential waveform and neurotransmitter release at a central excitatory synapse

doi: 10.7554/eLife.75219

Figure Lengend Snippet: Individual images are identified in rows 1–6 and columns A-F, as indicated by the central labels. Four quadrants of 9 images are shown, each 3 × 3 matrix is from the named genotype and stained as specified in the title of each quadrant. The top row of each quadrant (rows 1 and 4) are single optical sections from 3 different MNTB neurons, in which their calyceal synaptic profiles are labelled with bassoon (purple) and co-labelled with either Kv3.1 or Kv3.3 antibodies (yellow): from Kv3.1KO and stained for Kv3.3 ( A1-C1 ); the Kv3.3KO stained for Kv3.1 ( D1-F1 ); WT stained for Kv3.3 ( A4-C4 ); WT stained for Kv3.1 ( D4-F4 ). In each MNTB neuron (rows 1 & 4) two synaptic regions of interest (ROI) containing bassoon are indicated by the red and green arrowheads. These magnified ROIs are displayed below (in rows 2+3 or 5+6) bordered by the same colour, respectively. The neuronal compartments are labelled: ‘post’ – postsynaptic; ‘pre’ – presynaptic; ‘term’ – synaptic terminal. In each image, the dark grey arrows point to presynaptic Kv3 labelling, and the white arrows point to postsynaptic Kv3 labelling. Scale bars are indicated for each row in column A (5 µm in rows 1 and 4: 1 µm in rows 2,3,5, and 6). Tissue was used from mice aged P28-P30.

Article Snippet: Afterwards primary antibodies for Kv3.1b (Rabbit, Alomone APC-014, 1:1000) and Kv3.3 (Mouse, Neuromab 75–354, 1:3000) were diluted in blocking solution and incubated overnight at 4 °C.

Techniques: Staining

Journal: eLife

Article Title: Kv3.3 subunits control presynaptic action potential waveform and neurotransmitter release at a central excitatory synapse

doi: 10.7554/eLife.75219

Figure Lengend Snippet: ( A ) Superimposed calyceal EPSCs generated from each genotype (age P21-P25): wildtype (WT; black), Kv3.3KO (blue), and Kv3.1KO mice (orange). Thin lines show traces from individual neurons (each is mean of 5 EPSCs) with thick line showing the population mean for each genotype. Grey dashed line indicates the average WT amplitude; N=WT, 22 neurons (11 mice); Kv3.3KO, 22 neurons (10 mice); Kv3.1KO, 17 neurons (8 mice). Inset shows recording and stimulation configuration. ( B ) EPSC amplitude increased in the Kv3.3KO. ( C ) EPSC rise time (10–90%) no difference was found between groups (one-way ANOVA, p=0.1576). ( D ) EPSC decay tau and ( E ) EPSC total charge were increased in the Kv3.3KO relative to WT. ( F ) EPSC traces from WT, Kv3.3KO, and Kv3.1KO mice, before and after the addition of 1 mM TEA. ( Centre): EPSC amplitudes recorded before and after perfusion of TEA (1 mM); n=WT, 9 neurons (7 mice); Kv3.3KO, 6 neurons (3 mice); Kv3.1KO, 5 neurons (3 mice). ( G ) Increase in EPSC amplitude by 1 mM TEA. ( H ). The amplitude increase induced by TEA was significantly reduced in Kv3.3KO mice compared to WT. Average data presented as mean ± SD; statistics was using one-way ANOVA with Tukey’s post hoc for multiple comparisons. Kruskal-Wallis ANOVA with Dunn’s multiple corrections was used to compare change to EPSC amplitude in TEA due to a non-gaussian distribution in WT. Figure 3—source data 1. Relates to .

Article Snippet: Afterwards primary antibodies for Kv3.1b (Rabbit, Alomone APC-014, 1:1000) and Kv3.3 (Mouse, Neuromab 75–354, 1:3000) were diluted in blocking solution and incubated overnight at 4 °C.

Techniques: Generated

Journal: eLife

Article Title: Kv3.3 subunits control presynaptic action potential waveform and neurotransmitter release at a central excitatory synapse

doi: 10.7554/eLife.75219

Figure Lengend Snippet: Short-term depression was accelerated and enhanced in mice lacking Kv3.3. Values shown are for parameters measured from data presented in Figure 3 for WT, Kv3.3KO and Kv3.1KO genotypes at 100 Hz to 600 Hz range. Paired pulse depression of EPSC responses recorded in MNTB neurons (EPSC 2 /EPSC 1 ) was increased in Kv3.3 KO animals during high frequency stimulation of the calyx. The increased depression was maintained throughout the stimulation train (EPSC 80 /EPSC 1 ) across all frequencies. The rate of short term-depression in EPSC amplitudes during EPSC trains (duration 800ms), measured as short-term depression (STD) decay tau was significantly increased in Kv3.3 KOs at 100 and 200 Hz compared to WT. This STD was more severe in mice lacking Kv3.3, as shown by decreased normalized steady-state EPSC amplitudes compared to WT. STD tau and steady state amplitudes were measured using a single exponential fit to normalized EPSC amplitudes throughout the 800ms stimulation trains. n=number of neurons. Values in bold are significantly different to WT (see statistics table for more detail). Data represented as mean ± SD.

Article Snippet: Afterwards primary antibodies for Kv3.1b (Rabbit, Alomone APC-014, 1:1000) and Kv3.3 (Mouse, Neuromab 75–354, 1:3000) were diluted in blocking solution and incubated overnight at 4 °C.

Techniques:

Journal: eLife

Article Title: Kv3.3 subunits control presynaptic action potential waveform and neurotransmitter release at a central excitatory synapse

doi: 10.7554/eLife.75219

Figure Lengend Snippet:

Article Snippet: Afterwards primary antibodies for Kv3.1b (Rabbit, Alomone APC-014, 1:1000) and Kv3.3 (Mouse, Neuromab 75–354, 1:3000) were diluted in blocking solution and incubated overnight at 4 °C.

Techniques: Knock-Out, Software

Journal: eLife

Article Title: Ankyrin-R regulates fast-spiking interneuron excitability through perineuronal nets and Kv3.1b K + channels

doi: 10.7554/eLife.66491

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal antibodies against AnkR ( ) (RRID: AB_2833096 ), Ank1 (Thermo Fisher Scientific Cat# PA5-63372, RRID: AB_2638015 ), neurofilament M (Millipore Cat# AB1987, RRID: AB_91201 ), parvalbumin (Novus Cat# NB120-11427, RRID: AB_791498 ), somatostatin (Peninsula Laboratories Cat# T-4103.0050, RRID: AB_518614 ), versican (Millipore Cat# AB1032, RRID: AB_11213831 ), PlexinA4 (Abcam Cat# ab39350, RRID: AB_944890 ), neuropilin-1 (GeneTex Cat# GTX16786, RRID: AB_422398 ), Kv3.1 (LSBio (LifeSpan), Cat# LS-C322374, RRID: AB_2891125 ), Kv3.1b (Alomone Labs Cat# APC-014, RRID: AB_2040166 ), Kv3.3 (Alomone Labs Cat# APC-102, RRID: AB_2040170 ), GFP (Thermo Fisher Scientific, Cat# A-11122, RRID: AB_221569 ).

Techniques: Sequencing, Transfection, Construct, Plasmid Preparation, Immunoprecipitation, Software