Journal: Frontiers in Molecular Neuroscience

Article Title: SUMOylation of the Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 2 Increases Surface Expression and the Maximal Conductance of the Hyperpolarization-Activated Current

doi: 10.3389/fnmol.2016.00168

Figure Lengend Snippet: Establishing a culture system to investigate HCN2 channel SUMOylation. Cell lysates from a Hek cell line stably expressing GFP-HCN2 channels (A) and the parental Hek cell line (B) were used in IP experiments with an antibody against GFP. WBs containing the lysate (L) and IP products (IP) were then probed with anti-HCN2 and anti-GFP antibodies. A doublet was recognized by both antibodies in the stably transfected but not parental Hek cell line, suggesting that the doublet represents GFP-HCN2 channels. The band present at 50 kDa in the IP lanes corresponds to the heavy chain of the anti-rabbit antibody used in the IP experiment. The anti-rabbit secondary antibody used in the GFP WB produced a strong signal. The anti-goat secondary antibody used in the HCN2 WB produced a much weaker signal.

Article Snippet: 1.5 mg of the mouse membrane preparation with 5 μg of anti-HCN2 antibody ( Table , Santa Cruz Biotechnology; sc-19708) were used in combination with the Classic Magnetic Co-IP Kit (Pierce), following the manufacturer’s instructions.

Techniques: Stable Transfection, Expressing, Transfection, Produced

Journal: Frontiers in Molecular Neuroscience

Article Title: SUMOylation of the Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 2 Increases Surface Expression and the Maximal Conductance of the Hyperpolarization-Activated Current

doi: 10.3389/fnmol.2016.00168

Figure Lengend Snippet: Primary antibodies.

Article Snippet: 1.5 mg of the mouse membrane preparation with 5 μg of anti-HCN2 antibody ( Table , Santa Cruz Biotechnology; sc-19708) were used in combination with the Classic Magnetic Co-IP Kit (Pierce), following the manufacturer’s instructions.

Techniques: Concentration Assay, Western Blot, Transfection, Expressing

Journal: Frontiers in Molecular Neuroscience

Article Title: SUMOylation of the Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 2 Increases Surface Expression and the Maximal Conductance of the Hyperpolarization-Activated Current

doi: 10.3389/fnmol.2016.00168

Figure Lengend Snippet: Mouse HCN2 is SUMOylated in vivo. Mouse forebrain non-denatured (A) and denatured (B) membrane fractions were used in immunoprecipitation (IP) experiments with an antibody against HCN2 or IgG (negative control). Western blots (WBs) containing the IP products were probed for HCN2, SUMO1, and SUMO2/3. The experiment was repeated three times using the brains from three different mice. Representative WBs are shown from a single experiment. The red dots indicate non-specific products pulled down in the IP. The single band at ∼120 kDa represents HCN2 channels. The asterisk indicates a non-covalently bound, SUMOylated protein. The band at ∼50 kDa represents the IP antibody.

Article Snippet: 1.5 mg of the mouse membrane preparation with 5 μg of anti-HCN2 antibody ( Table , Santa Cruz Biotechnology; sc-19708) were used in combination with the Classic Magnetic Co-IP Kit (Pierce), following the manufacturer’s instructions.

Techniques: In Vivo, Immunoprecipitation, Negative Control, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: SUMOylation of the Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 2 Increases Surface Expression and the Maximal Conductance of the Hyperpolarization-Activated Current

doi: 10.3389/fnmol.2016.00168

Figure Lengend Snippet: GFP-HCN2 channels are SUMOylated in Hek-HCN2 cells. Cell lysates from the Hek-HCN2 cell line were used in IP experiments with an antibody against GFP. WBs containing the IP products were then probed with an anti-GFP antibody or an anti-SUMO2/3 antibody. Parental Hek cell lysates were also probed with the anti-SUMO2/3 antibody. The anti-SUMO2/3 antibody recognized the GFP-HCN2 doublet in the Hek-HCN2 cell line, but not in the parental cell line, suggesting that GFP-HCN2 channels are SUMOylated.

Article Snippet: 1.5 mg of the mouse membrane preparation with 5 μg of anti-HCN2 antibody ( Table , Santa Cruz Biotechnology; sc-19708) were used in combination with the Classic Magnetic Co-IP Kit (Pierce), following the manufacturer’s instructions.

Techniques:

Journal: Frontiers in Molecular Neuroscience

Article Title: SUMOylation of the Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 2 Increases Surface Expression and the Maximal Conductance of the Hyperpolarization-Activated Current

doi: 10.3389/fnmol.2016.00168

Figure Lengend Snippet: Transient transfection with SUMO and Ubc9 leads to an increase in GFP-HCN2 channel SUMOylation. The stable Hek-HCN2 cell line was transiently transfected with mCherry (control), or mCherry + SUMO + Ubc9, or mCherry + SENP1. Two days after transfection, cell lysates were used in IP experiments with an anti-GFP antibody. IP products were resolved with SDS-PAGE and transferred to WBs. Blots were probed with an anti-SUMO2/3 antibody. After recording the result, blots were stripped and reprobed with an anti-GFP antibody. (A) Representative blots showing typical chemiluminescent signals for the GFP-HCN2 channel doublet after probing with the anti-SUMO2/3 antibody (upper panel) followed by stripping and re-probing with the anti-GFP antibody (bottom panel). Note that the amount of IP product varied between experiments but not across treatment groups as determined by measures of the GFP OD’s [one-way ANOVA, F (2,16) = 0.1285; p = 0.8804]. (B) The fraction of SUMOylated HCN2 channels in each treatment group (SUMO doublet OD ÷ GFP doublet OD, see text) is plotted as the mean + SEM. The treatment and the n are shown below each plot. Each n represents a single plate that was transfected and carried through the experiment to produce a single lane on a WB. Asterisk represents a statistically significant difference from control [one-way ANOVA with a Dunnett’s post hoc , F (2,16) = 4.121; p = 0.0360].

Article Snippet: 1.5 mg of the mouse membrane preparation with 5 μg of anti-HCN2 antibody ( Table , Santa Cruz Biotechnology; sc-19708) were used in combination with the Classic Magnetic Co-IP Kit (Pierce), following the manufacturer’s instructions.

Techniques: Transfection, SDS Page, Stripping Membranes

Journal: Frontiers in Molecular Neuroscience

Article Title: SUMOylation of the Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 2 Increases Surface Expression and the Maximal Conductance of the Hyperpolarization-Activated Current

doi: 10.3389/fnmol.2016.00168

Figure Lengend Snippet: Increased HCN2 channel SUMOylation augments I h G max . I h was measured in Hek-HCN2 cells transiently transfected with either mCherry (control), mCherry + SUMO + Ubc9, or mCherry + SENP1. For each treatment group, data were pooled from ≥3 transfections. (A) Representative traces for each treatment group, elicited by stepping the voltage from -50 to -120 mV in 10 mV increments. Scale bars, 500 ms and 200 pA. The kinetics of activation at -120 mV were not altered across treatment groups. Mean activation time constants: control, 400.9 ± 23.01; SUMO + Ubc9, 407.3 ± 33.67; SENP1, 497.4 ± 50.79; one-way ANOVA, F (2,32) = 2.224; p = 0.1246. (B) Plots of I h G max for each treatment group. Each data point represents a single cell. Bar represents the mean. Transfection with SUMO + Ubc9 significantly increased I h G max relative to SENP1 and control treatment groups [asterisks, p < 0.05; One-way ANOVA with Tukey’s post hoc , F (2,28) = 13.23; p < 0.0001]. (C) Plots of voltage dependence of activation. Each data point represents the mean ± SEM. There were no significant differences between treatment groups for mean V 50 [control, -93.88 ± 1.12; SUMO + Ubc9, -91.6 ± 1.29; SENP1, -87.8 ± 3.03; one-way ANOVA, F (2,32) = 3.006; p = 0.0636] or mean slope [control, -6.61 ± 0.29; SUMO + Ubc9, -7.42 ± 0.41; SENP1, -6.84 ± 0.4; one-way ANOVA, F (2,32) = 1.439; p = 0.252].

Article Snippet: 1.5 mg of the mouse membrane preparation with 5 μg of anti-HCN2 antibody ( Table , Santa Cruz Biotechnology; sc-19708) were used in combination with the Classic Magnetic Co-IP Kit (Pierce), following the manufacturer’s instructions.

Techniques: Transfection, Activation Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: SUMOylation of the Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 2 Increases Surface Expression and the Maximal Conductance of the Hyperpolarization-Activated Current

doi: 10.3389/fnmol.2016.00168

Figure Lengend Snippet: Increased SUMOylation augments HCN2 channel surface expression. GFP-HCN2 channel cell surface expression was monitored using a biotinylation assay. Hek-HCN2 cells were transiently transfected with mCherry alone or mCherry + SUMO + Ubc9. Two days after transfection cultures were biotinylated and cell surface proteins were isolated from cell lysates using Neutravidin. Both the intracellular and cell surface fractions were run on a WB and probed with antibodies recognizing GFP, Na + /K + -ATPase, and Actin. (A) Representative WBs. (B) Plots depicting average normalized GFP-HCN2 channel surface expression (GFP doublet OD ÷ Na + /K + -ATPase OD). The treatment and the n are shown below the graph. Each n represents a single plate that was transfected, biotinylated and carried through the experiment to produce a single lane on a WB. Asterisk indicates significant difference between treatment groups (Student’s t -test, p < 0.05).

Article Snippet: 1.5 mg of the mouse membrane preparation with 5 μg of anti-HCN2 antibody ( Table , Santa Cruz Biotechnology; sc-19708) were used in combination with the Classic Magnetic Co-IP Kit (Pierce), following the manufacturer’s instructions.

Techniques: Expressing, Cell Surface Biotinylation Assay, Transfection, Isolation

Journal: Frontiers in Molecular Neuroscience

Article Title: SUMOylation of the Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 2 Increases Surface Expression and the Maximal Conductance of the Hyperpolarization-Activated Current

doi: 10.3389/fnmol.2016.00168

Figure Lengend Snippet: Identification of HCN2 channels SUMOylation sites. Amino acid sequences for the vertebrate and invertebrate HCN channels were aligned: mouse HCN1 (NP_034538), mouse HCN2 (NP_032252), mouse HCN3 (NP_032253), mouse HCN4 (NP_001074661), spiny lobster HCN (ABI94038). Putative SUMOylation sites predicted by SUMOplot freeware are indicated in blue (not mutated this study) or red (mutated in this study). The six transmembrane domains (yellow) and CNBD (blue) have been highlighted.

Article Snippet: 1.5 mg of the mouse membrane preparation with 5 μg of anti-HCN2 antibody ( Table , Santa Cruz Biotechnology; sc-19708) were used in combination with the Classic Magnetic Co-IP Kit (Pierce), following the manufacturer’s instructions.

Techniques:

Journal: Frontiers in Molecular Neuroscience

Article Title: SUMOylation of the Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 2 Increases Surface Expression and the Maximal Conductance of the Hyperpolarization-Activated Current

doi: 10.3389/fnmol.2016.00168

Figure Lengend Snippet: Overexpression of SUMO and Ubc9 enhances SUMOylation at K669. The three mutant cell lines (A–C) were transiently transfected with mCherry or mCherry + SUMO + Ubc9. Cell lysates were used in IP experiments with anti-GFP antibodies. WBs containing IP products were probed with the anti-SUMO2/3 antibody, stripped and reprobed with the anti-GFP antibody. Top Panel : Representative blots showing the GFP-HCN2 doublet that was measured. Bottom Panel: Plots of the fraction of SUMOylated GFP-HCN2 channels in each mutant cell line, mean + SEM. The treatment and the n are shown below the graph. Each n represents one plate that was transfected and carried through the experiment to produce a single lane on a WB. Asterisk significantly different (Student’s t -test, p < 0.05).

Article Snippet: 1.5 mg of the mouse membrane preparation with 5 μg of anti-HCN2 antibody ( Table , Santa Cruz Biotechnology; sc-19708) were used in combination with the Classic Magnetic Co-IP Kit (Pierce), following the manufacturer’s instructions.

Techniques: Over Expression, Mutagenesis, Transfection

Journal: Frontiers in Molecular Neuroscience

Article Title: SUMOylation of the Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 2 Increases Surface Expression and the Maximal Conductance of the Hyperpolarization-Activated Current

doi: 10.3389/fnmol.2016.00168

Figure Lengend Snippet: Enhanced SUMOylation at K669 augments I h G max . I h G max was measured following transient transfection with mCherry or mCherry + SUMO + Ubc9 in the three mutant cell lines (A–C) . Top panel : Representative traces for each treatment group for each mutant cell line. Scale bars, 500 ms, and 500 pA, Bottom Panel : Plots of normalized I h G max ( G max ÷ mean mCherry G max ) for each mutant cell line. I h G max was normalized to account for differences in overall expression of the HCN2 channels between the different mutant cell lines, most likely due to difference in the copy number of the plasmid integrated into the genome. Each data point represents a single cell. Asterisk significantly different (Student’s t -test p < 0.05, recordings were pooled from ≥3 separate transfections).

Article Snippet: 1.5 mg of the mouse membrane preparation with 5 μg of anti-HCN2 antibody ( Table , Santa Cruz Biotechnology; sc-19708) were used in combination with the Classic Magnetic Co-IP Kit (Pierce), following the manufacturer’s instructions.

Techniques: Transfection, Mutagenesis, Expressing, Plasmid Preparation

Journal: Frontiers in Molecular Neuroscience

Article Title: SUMOylation of the Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 2 Increases Surface Expression and the Maximal Conductance of the Hyperpolarization-Activated Current

doi: 10.3389/fnmol.2016.00168

Figure Lengend Snippet: Enhanced SUMOylation at K669 increases GFP-HCN2 channel surface expression. The K669R mutant cell line was transiently transfected with mCherry or mCherry + SUMO + Ubc9, followed by the biotinylation assay to measure GFP-HCN2 surface expression. (A) Representative blots probed with anti-GFP, anti-Na/K ATPase, and anti-actin antibodies. (B) Plots of normalized GFP-HCN2 channels in each treatment group. The treatment and the n are shown below the graph. Each n represents a single plate that was transfected, biotinylated, and carried through the experiment to produce a single lane on a WB. There was no significant difference between treatment groups (Student’s t -test, p = 0.5606).

Article Snippet: 1.5 mg of the mouse membrane preparation with 5 μg of anti-HCN2 antibody ( Table , Santa Cruz Biotechnology; sc-19708) were used in combination with the Classic Magnetic Co-IP Kit (Pierce), following the manufacturer’s instructions.

Techniques: Expressing, Mutagenesis, Transfection, Cell Surface Biotinylation Assay

Journal: JCI Insight

Article Title: Abolishing cAMP sensitivity in HCN2 pacemaker channels induces generalized seizures

doi: 10.1172/jci.insight.126418

Figure Lengend Snippet: (A) Structural model of the CNBD of HCN channels. The 2 key residues R591 (yellow) and T592 (pink) that are crucial for binding of cAMP are highlighted. (B) Horizontal brain slices of WT and HCN2EA mice. The position of the dLGN (red) and the VB (blue) is indicated. (C) Detection of HCN2 and HCN4 in Western blot analysis of punched dLGN and VB regions. Images are representatives of n = 3/group. (D) Western blot analysis of membrane preparations of HCN2EA and WT mice probed for HCN2 and a loading control (Na+/K+-ATPase). Images are representatives of n = 3/group. (E) Quantification of HCN2 expression level in relation to the Na+/K+-ATPase (n = 3).

Article Snippet: Co-IPs were performed with anti-HCN2 antibody (Alomone, APC-030), anti-HCN4 (Alomone, APC-052), or anti-Trip8b (N212/7, Neuromab) using Novex Protein G magnetic beads (Thermo Fisher Scientific) according to the manufacturer’s protocol. qRT-PCR.

Techniques: Binding Assay, Western Blot, Expressing

Journal: JCI Insight

Article Title: Abolishing cAMP sensitivity in HCN2 pacemaker channels induces generalized seizures

doi: 10.1172/jci.insight.126418

Figure Lengend Snippet: (A) Distribution of HCN2 and HCN4 in the VB region of mouse thalamus. Scale bar: 200 μm. (B) Overlay of anti-HCN2 (green), anti-HCN4 (red), and Hoechst (blue). Scale bars: 200 μm. Upper: Single channels for HCN2 (green) and HCN4 (red) staining. Lower: Magnification (scale bars: 25 μm) of the dLGN (left) and VB (middle). (C) Magnified HCN2 stainings of WT (upper), HCN2EA litters (middle), and HCN2-KO mice (lower panel) in the VB region. Scale bars: 20 μm. (D) Analysis of the mean intensity of the HCN2 fluorescence in WT and HCN2EA soma and dendrites (WT: gray squares, soma [n = 20], dendrites [n = 26]; EA: blue squares, soma [n = 17], dendrites [n = 26]). (E) Magnified HCN4 stainings of WT (upper) and HCN2EA litters (middle). Staining in the absence of primary antibody is shown in the bottom panels. Scale bars: 20 μm. (F) Analysis of the mean intensity of the HCN4 fluorescence in WT and HCN2EA soma and dendrites (WT: gray circles, soma [n = 23], dendrites [n = 25]; EA: orange circles, soma [n = 25], dendrites [n = 25]) ***P < 0.001 by 1-way ANOVA with Bonferroni’s post hoc test. NS, not significant.

Article Snippet: Co-IPs were performed with anti-HCN2 antibody (Alomone, APC-030), anti-HCN4 (Alomone, APC-052), or anti-Trip8b (N212/7, Neuromab) using Novex Protein G magnetic beads (Thermo Fisher Scientific) according to the manufacturer’s protocol. qRT-PCR.

Techniques: Staining, Fluorescence

Journal: JCI Insight

Article Title: Abolishing cAMP sensitivity in HCN2 pacemaker channels induces generalized seizures

doi: 10.1172/jci.insight.126418

Figure Lengend Snippet: (A) EEG traces of WT (left), HCN2EA (middle), and global HCN2-KO mice (right panel). Spike and wave discharges (SWDs) in the EEG of HCN2EA and global HCN2-KO are marked by asterisks. SWDs concur with episodes of low activity in the EMG. Higher magnifications of SWD traces are displayed below the EEG of HCN2EA and HCN2-KO mice. Representative traces of animals analyzed in B. (B) SWD characteristics of HCN2EA (light blue, n = 5) and global HCN2-KO (dark blue, n = 6) animals compared with WT traces (gray, n = 6) regarding the time between 2 SDWs (left), mean SWD duration (middle), and the time per day in SWDs (right). NS, not significant (1-way ANOVA, Bonferroni’s post hoc test). (C) Time spent in different vigilance states (wake, NREM and REM sleep) during light and dark conditions (HCN2EA, blue; WT, gray; n = 7). (D) Power spectra of NREM sleep in HCN2EA (blue, n = 7) and WT animals (gray, n = 7). **P < 0.01 by 2-way ANOVA. (E) Mean sigma power in HCN2EA (blue, n = 7) and WT animals (gray, n = 7). *P = 0.0175 by Mann-Whitney test.

Article Snippet: Co-IPs were performed with anti-HCN2 antibody (Alomone, APC-030), anti-HCN4 (Alomone, APC-052), or anti-Trip8b (N212/7, Neuromab) using Novex Protein G magnetic beads (Thermo Fisher Scientific) according to the manufacturer’s protocol. qRT-PCR.

Techniques: Activity Assay, MANN-WHITNEY

Journal: JCI Insight

Article Title: Abolishing cAMP sensitivity in HCN2 pacemaker channels induces generalized seizures

doi: 10.1172/jci.insight.126418

Figure Lengend Snippet: (A) Strategy for specific deletion of HCN channels in the VB. Left panel: Scheme of the AAV2/8-hSyn-Cre-EGFP vector that was injected stereotactically into the VB of HCNfl/fl mice. The schematic brain shows the injected region in the thalamus. Only cells in the VB show the green EGFP signal due to viral transfection. (B) Staining with anti-HCN2 antibody (red) in the VB region shows that Cre/EGFP–positive neurons (green) lack HCN2, while nontransduced (EGFP-negative) cells express HCN2 in the plasma membrane (marked by white arrows). Scale bar: 5 μm. VM, ventromedial region. (C) VB-specific HCN2-KO mice show SWDs (marked with an asterisk) in EEG traces (representative trace, n = 3). The inset shows a magnification of the SWD. (D) Representative EEG (green) and EMG (black) traces of an HCN2fl/fl mouse that was injected with a control AAV vector that expresses only EGFP (n = 3). (D) Representative EEG (green) and EMG (black) traces of a VB-specific HCN4-KO animal that was generated by injecting the same vector as displayed in Figure 7A into the VB of an HCN4fl/fl mouse (n = 3).

Article Snippet: Co-IPs were performed with anti-HCN2 antibody (Alomone, APC-030), anti-HCN4 (Alomone, APC-052), or anti-Trip8b (N212/7, Neuromab) using Novex Protein G magnetic beads (Thermo Fisher Scientific) according to the manufacturer’s protocol. qRT-PCR.

Techniques: Plasmid Preparation, Injection, Transfection, Staining, Generated

Journal:

Article Title: Functional stabilization of weakened thalamic pacemaker channel regulation in rat absence epilepsy

doi: 10.1113/jphysiol.2006.110486

Figure Lengend Snippet: A, representative coronal brain sections at the level of the thalamus that have been subjected to quantitative in situ hybridization for the HCN2 channel isoform. The preferential expression of this isoform in thalamus is apparent. B and C, quantitative analysis of mRNA expression levels of HCN2 channels comparing GAERS to NE rats. The strains are not distinguishable in the expression of this isoform. D, representative sections of GAERS and NE rat brain for HCN4 mRNA expression. Note the relatively lower expression of this isoform in the Rtn, and the typical signal of HCN4 in the habenula, supporting the specificity of the probes. E and F, quantitative analyses of HCN4 mRNA expression indicate the absence of significant differences in GAERS versus NE. Hb = habenula.

Article Snippet: Rabbit anti-HCN1 and anti-HCN2 antibodies were obtained from Chemicon, Temecula, CA, USA.

Techniques: In Situ Hybridization, Expressing

Journal:

Article Title: Functional stabilization of weakened thalamic pacemaker channel regulation in rat absence epilepsy

doi: 10.1113/jphysiol.2006.110486

Figure Lengend Snippet: A, a schematic diagram showing the level where coronal sections from brains of GAERS and NE control rats were obtained for in situ hybridization. B, representative sections from GAERS and NE rats. Although HCN1 channel expression in the thalamus is significantly lower than that of the HCN2 isoform, the darker in situ hybridization signal over the Vpm and Rtn is apparent in the GAERS brain. Note the robust expression of HCN1 in the principal cell layers of the hippocampus of both strains. C, quantitative analysis reveals a 59% increase in HCN1 mRNA levels in GAERS Vpm. D, quantitative analysis shows a more modest increase of HCN1 mRNA in the Rtn (n = 5 per group). *P < 0.05 (see Methods).

Article Snippet: Rabbit anti-HCN1 and anti-HCN2 antibodies were obtained from Chemicon, Temecula, CA, USA.

Techniques: In Situ Hybridization, Expressing