Journal: International Journal of Molecular Sciences

Article Title: Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

doi: 10.3390/ijms22094772

Figure Lengend Snippet: TRIP8b-deficient mice have altered atrial electrophysiology. ( A ) In vitro electrophysiological measurements from Langendorff-perfused hearts show an increase of atrial refractory period (ARP) and atrioventricular-nodal refractory period (AVNRP) in TRIP8b-deficient mice, without changes in sino-nodal activity (sino-nodal recovery time, SNRT) and heart rate (HR). ( B ) Representative tracings are shown for wild-type and TRIP8b-deficient mice. A, atrial activity; atrium electrophysiological tracings from the atrium; V, ventricular activity; ventricle electrophysiological tracings from the ventricle; black arrowheads mark atrial or ventricular stimulation. ( C ) Ganglionic blockade with 0.5 mM hexamethonium leads to a reduction of AVNRP in TRIP8b-deficient mice. Data are presented as box plots (minimum to maximum, n = 5–11 per genotype) and were compared using an unpaired t -test or Mann–Whitney, as appropriate.

Article Snippet: This was verified using a different TRIP8b antibody (Alomone Labs, Jerusalem, Israel) with the same result.

Techniques: In Vitro, Activity Assay, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

doi: 10.3390/ijms22094772

Figure Lengend Snippet: Trip8b mRNA is present in the cardiac nervous system. ( A ) Exon 6–7 can be amplified in cDNA of ganglia-containing atrial tissue of wild-type but not of TRIP8b-deficient mice (left panel). Quantitative PCR analyses show that exon 8–9, 9–10, and 13–14 of Trip8b are still detectable in knockout mice. Data (normalized to Cdkn1b) are presented as individual data points with SEM ( n = 3, right panel) and were compared via one-way ANOVA followed by Sidaks’ multiple comparison test; ns, not significant. ( B ) Trip8b mRNA can be visualized with RNAscope in situ hybridization in neuronal cell bodies of cardiac ganglia. Black arrows in magnifications point to single neurons with Trip8b mRNAs. ( C ) Trip8b mRNA (black arrows) can be visualized with RNAscope in situ hybridization in cardiac nerves of wild-type mice.

Article Snippet: This was verified using a different TRIP8b antibody (Alomone Labs, Jerusalem, Israel) with the same result.

Techniques: Amplification, Real-time Polymerase Chain Reaction, Knock-Out, In Situ Hybridization

Journal: International Journal of Molecular Sciences

Article Title: Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

doi: 10.3390/ijms22094772

Figure Lengend Snippet: Trip8b mRNAs are detectable in the cardiac conduction system to a lower amount than in the intracardiac nervous system. ( A ) Sinus node and ( B ) atrioventricular node (AV node) were identified via hematoxylin and eosin (H&E) staining and Hcn4 RNAscope in situ hybridization. Subsequent sections treated with a probe specific for Trip8b show solitary mRNA spots (black arrows) surrounding the sinus node artery and in the AV node. Nuclei are counterstained with hematoxylin in blue. ( C ) The histogram shows the distribution of Trip8b mRNA spots per cell in the intracardiac nervous system (ICNS, nerves, and ganglia), sinus node, and AV node of wild-type and TRIP8b-deficient mice. Overall, 279–404 cells were analyzed for each region of interest per genotype, n = 2–3 images/genotype. ( D ) Trip8b in situ hybridization (red) detects mRNA in wild-type mice but also, to a lower amount, in knockout mice.

Article Snippet: This was verified using a different TRIP8b antibody (Alomone Labs, Jerusalem, Israel) with the same result.

Techniques: Staining, In Situ Hybridization, Knock-Out

Journal: International Journal of Molecular Sciences

Article Title: Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

doi: 10.3390/ijms22094772

Figure Lengend Snippet: TRIP8b protein is not detectable in the atrial lysates and the cardiac autonomic nervous system. ( A ) Western blot analysis of brain tissue as positive control detects specific bands for TRIP8b (NBP2-38840, Novusbio) already at 2.5 µg total protein. For the heart, 50 µg atrial or ventricular lysate did not show any specific bands, while HCN4 ( B ) is detectable in both genotypes. ( C ) Immunohistochemistry for TRIP8b (APR-070, Alomone Labs) on paraffin sections was established in the central nervous system, more specifically, the cerebral cortex. Neurons positive for TRIP8b are detectable in the wild-type animals but not cortex of TRIP8b-deficient animals. ( D ) To increase the sensitivity of detection, atrial whole-mount preparations (upper panel shows exemplary staining with αTH ab152, Merck Millipore) were stained and ganglia cut out for confocal microscopy (bottom panel with αTH ab76442, Abcam). No specific signal was obtained for TRIP8b (APR-070, Alomone Labs), and no differences were detectable between the genotypes.

Article Snippet: This was verified using a different TRIP8b antibody (Alomone Labs, Jerusalem, Israel) with the same result.

Techniques: Western Blot, Positive Control, Immunohistochemistry, Staining, Confocal Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

doi: 10.3390/ijms22094772

Figure Lengend Snippet: TRIP8b protein is not detectable in the cardiac conduction system in wild-type mice. Sinus node (upper panel) and atrioventricular node (AV node, bottom panel) were identified by anatomical landmarks and HCN4 staining (green). No staining for TRIP8b (red, APR-070, Alomone Labs) was detectable beyond the background.

Article Snippet: This was verified using a different TRIP8b antibody (Alomone Labs, Jerusalem, Israel) with the same result.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

doi: 10.3390/ijms22094772

Figure Lengend Snippet: HCN channel expression in intracardiac ganglia. ( A ) In situ hybridization of two exemplary wild-type ganglia for Hcn2 (green) and Hcn4 (red). Both mRNAs are present within the ganglia. Boxed area is magnified in the inlay. ( B ) Gene expression analysis of Hcn2 and Hcn4 in TRIP8b-deficient mice and wild-type littermates. Data are presented as normalized gene expression to Cdkn1b using the formula 2 −ΔCt (box plots, minimum to maximum, n = 6 per genotype) and were compared using Mann–Whitney test.

Article Snippet: This was verified using a different TRIP8b antibody (Alomone Labs, Jerusalem, Israel) with the same result.

Techniques: Expressing, In Situ Hybridization, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of the HCN channel auxiliary subunit TRIP8b is altered in an animal model of temporal lobe epilepsy and modulates channel function

doi: 10.1074/jbc.RA119.010027

Figure Lengend Snippet: Identification of in vivo TRIP8b phosphorylation at Ser237. A and B, TRIP8b was immunopurified from hippocampal lysates prepared from saline (control) or KA-seized (Racine scale level 5) rats. TRIP8b immunoprecipitation products were analyzed by MS/MS. Spectral coverage of TRIP8b immunopurified from hippocampal lysates from saline-treated (A) and KA-treated (B) rats is highlighted in yellow. pSer237 is highlighted in green within the recovered tryptic peptide fragment, as indicated by the double underline. C, deconvolution of a representative pSer237-containing peptide MS/MS spectrum of the phosphopeptide NHpSLEEEFER derived from TRIP8b immunopurified from a hippocampal lysate prepared from a saline-treated rat. The fragmentation pattern of the peptide is shown, highlighting that the y8 peptide includes the phosphorylated Ser237 residue yielding a 98 m/z shift on the x axis. An m/z shift of +2 for Ser237 was detected with a confidence score of 10−7.6, indicating a low probability of mistaken identification of Ser237 phosphorylation. D, summary of the overall spectral coverage of TRIP8b from saline-treated and KA-treated rats, including the overall sequence coverage as shown in A and B.

Article Snippet: Phospho-specific antibody pSer 237 Phosphorylated TRIP8b antibody was generated by YenZym Antibodies, LLC (South San Francisco, CA).

Techniques: In Vivo, Phospho-proteomics, Saline, Control, Immunoprecipitation, Tandem Mass Spectroscopy, Derivative Assay, Residue, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of the HCN channel auxiliary subunit TRIP8b is altered in an animal model of temporal lobe epilepsy and modulates channel function

doi: 10.1074/jbc.RA119.010027

Figure Lengend Snippet: Location of Ser237 on TRIP8b and phosphospecific antibody validation. A, schematic of one subunit of an HCN channel and TRIP8b. The intracellular CNBD of the HCN subunit and the C-terminal residues SNL are labeled. The unbound cAMP binding site is depicted on the CNBD. On TRIP8b, the N-terminal variable domain, the nano domain (yellow shape), and the C-terminal TPR domains are indicated. Serine 237 is indicated (purple star) within the nano domain of TRIP8b that interacts with the HCN CNBD. B, Ser237 (red text) is conserved in select vertebrates: Homo sapiens (human), Pan troglodytes (chimpanzee), Macaca mulatta (rhesus monkey), Mus musculus (mouse), Rattus norvegicus (rat), Sus scrofa (pig), and Ovis aries (sheep). TRIP8b residue numbers are based on isoform 1a-4 (UniProt Q925N3) as a reference. C, WT and TKO mouse hippocampal lysates incubated with or without CIP for 2 h at 37 °C, n = 4. Samples were immunoblotted with antibodies against TRIP8b, pSer237, and α-tubulin. D, purified TRIP8b(219–602) protein incubated with CaMKIIα or PKA with or without ATP at 30 °C for 30 min; n = 3. Molecular mass markers are shown in kilodaltons.

Article Snippet: Phospho-specific antibody pSer 237 Phosphorylated TRIP8b antibody was generated by YenZym Antibodies, LLC (South San Francisco, CA).

Techniques: Biomarker Discovery, Labeling, Binding Assay, Residue, Incubation, Purification

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of the HCN channel auxiliary subunit TRIP8b is altered in an animal model of temporal lobe epilepsy and modulates channel function

doi: 10.1074/jbc.RA119.010027

Figure Lengend Snippet: Immunohistochemical localization of pSer237 in the CA1 and neocortical distal dendrites. A, the CA1 region of WT and TKO mice probed with antibodies against HCN1, TRIP8b, pSer237, and TRIP8b exon 4. B, the distal dendritic region was quantified in each stain: HCN1 (WT: 1, TKO: 0.52 ± 0.03, Student's t test, p < 0.001, n = 5), TRIP8b (WT: 1, TKO: 0.27 ± 0.02, p < 0.001, n = 5), pSer237 (WT: 1, TKO: 0.56 ± 0.1, p = 0.01, n = 5), and exon 4 (WT: 1, TKO: 0.5 ± 0.1, p < 0.001, n = 6). C, mouse hippocampal extracts before (input) and after (eluate) immunoprecipitation (IP) with an α-TRIP8b exon 4 antibody. Lysates were immunoblotted with antibodies against TRIP8b and pSer237 (n = 3). D, neocortex region of WT and TKO mice probed with antibodies against HCN1, TRIP8b, and pSer237. E, the distal dendritic region was quantified in each stain: HCN1 (WT: 1, TKO: 0.5 ± 0.1, Student's t test, p = 0.01, n = 4), TRIP8b (WT: 1, TKO: 0.35 ± 0.06, p = 0.002, n = 4), and pSer237 (WT: 1, TKO: 0.7 ± 0.1, p = 0.04, n = 5). Scale bars = 100 μm; asterisks, distal dendritic region; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; SLM, stratum lacunosum moleculare. Molecular mass markers are shown in kilodaltons. All error bars represent mean ± S.E.

Article Snippet: Phospho-specific antibody pSer 237 Phosphorylated TRIP8b antibody was generated by YenZym Antibodies, LLC (South San Francisco, CA).

Techniques: Immunohistochemical staining, Staining, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of the HCN channel auxiliary subunit TRIP8b is altered in an animal model of temporal lobe epilepsy and modulates channel function

doi: 10.1074/jbc.RA119.010027

Figure Lengend Snippet: Phosphorylated TRIP8b is absent from the CA3 and cerebellum but highly expressed in the CA1 region of the hippocampus. A and B, CA3 (A) and the cerebellum region (B) stained with antibodies against TRIP8b and pSer237 (n = 3). SP, stratum pyramidale; WM, white matter; GL, granular layer; PC, Purkinje cells; ML, molecular layer. C, protein lysate was loaded onto the gel from various mouse brain regions: 6 μg total lysate from the cornu ammonis 1 (CA1), dentate gyrus (DG), thalamus (Thal), and neocortex (Ctx) and 30 μg of total lysate from the cerebellum (CB). The brain regions were immunoblotted with the indicated antibodies. Note that different quantities of protein had to be loaded to obtain any pSer237 signal, and this led to predictable differences in the total TRIP8b loading control. D, quantification of C, with the pSer237 level normalized to total TRIP8b; one-way analysis of variance with Tukey's post hoc test (CA1, 1.0; dentate gyrus, 0.2 ± 0.1; thalamus, 0.3 ± 0.1; neocortex, 0.5 ± 0.1; cerebellum, 0.05 ± 0.04) with significant differences between the CA1 and dentate gyrus (p < 0.001), CA1 and thalamus (p = 0.001), CA1 and neocortex (p = 0.02), CA1 and cerebellum (p < 0.001), and neocortex and cerebellum (p = 0.03). n = 3; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Scale bars = 100 μm. Molecular mass markers are shown in kilodaltons. All error bars represent mean ± S.E.

Article Snippet: Phospho-specific antibody pSer 237 Phosphorylated TRIP8b antibody was generated by YenZym Antibodies, LLC (South San Francisco, CA).

Techniques: Staining, Control

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of the HCN channel auxiliary subunit TRIP8b is altered in an animal model of temporal lobe epilepsy and modulates channel function

doi: 10.1074/jbc.RA119.010027

Figure Lengend Snippet: Phosphorylated TRIP8b at Ser237 enhances HCN1 CNBD binding. A, pictured is one subunit of an HCN channel with TRIP8b, indicating the TRIP8b mutant constructs used: TRIP8b(N382A), TRIP8b(S237A,N382A), TRIP8b(R234A,N382A), and TRIP8b(Δ58,N382A). The N382A mutation was described previously as TPR3-N13A (21). B, HEK293T cells were transiently transfected with TRIP8b(WT), TRIP8b(N382A), TRIP8b(S237A, N382A), or TRIP8b(R234A, N382A) constructs, and cell lysates were immunoblotted with antibodies against pSer237 and TRIP8b. The pSer237 antibody recognizes TRIP8b(WT) and TRIP8b(N382A), but both phosphoablative constructs demonstrate a reduced signal (n = 3). C, HEK293T cells were transiently transfected with the indicated constructs. HCN1 was immunoprecipitated (IP), and HCN1 and TRIP8b were immunoblotted. D, relative immunoprecipitation of HCN1 + TRIP8b(N382A): 100, +TRIP8b(S237A,N382A): 78.2 ± 7.0, +TRIP8b(R234A,N382A): 76.1 ± 5.0, +TRIP8b(Δ58,N382A): 2.1 ± 1.0. One-way analysis of variance with Tukey's post hoc test, n = 4. Significant differences identified between +TRIP8b(N382A) and +TRIP8b(S237A,N382A): p = 0.02, +TRIP8b(N382A) and +TRIP8b(R234A,N382A): p = 0.0098, +TRIP8b(N382A) and +TRIP8b(Δ58,N382A): p < 0.001, +TRIP8b(S237A, N382A) and +TRIP8b(Δ58,N382A), p < 0.001, +TRIP8b(R234A, N382A) and +TRIP8b(Δ58,N382A): p < 0.001. E, a fluorescence polarization assay was performed with fixed concentrations of CNBD387–591 (0.625 μm) and 8-f-cAMP (10 nm). Purified TRIP8b protein was incubated with CaMKIIα, with ATP (phosphorylated TRIP8b), or without ATP (nonphosphorylated TRIP8b) and titrated. F, quantification of IC50 with phosphorylated (6.3 ± 0.6 μm) and nonphosphorylated (8.2 ± 0.8 μm) TRIP8b, n = 6, p = 0.001; matched samples were analyzed with a paired Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001. All error bars represent mean ± S.E. Molecular mass markers are shown in kilodaltons.

Article Snippet: Phospho-specific antibody pSer 237 Phosphorylated TRIP8b antibody was generated by YenZym Antibodies, LLC (South San Francisco, CA).

Techniques: Binding Assay, Mutagenesis, Construct, Transfection, Immunoprecipitation, Fluorescence, Purification, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of the HCN channel auxiliary subunit TRIP8b is altered in an animal model of temporal lobe epilepsy and modulates channel function

doi: 10.1074/jbc.RA119.010027

Figure Lengend Snippet: TRIP8b with a phosphoablative mutation prevents hyperpolarization of HCN2 gating. A, representative current traces from HEK293 cells stably expressing HCN2 and transiently transfected with GFP and TRIP8b(WT), TRIP8b(S237A), TRIP8b(R234A), or control vector. Whole-cell recordings were performed with cells held at −40 mV in voltage clamp and stepped from −40 to −120 mV. B, quantification of V50; HCN2 + vector: −90.1 ± 1.6 mV (n = 15), HCN2 +TRIP8b(WT): −95.3 ± 1.6 mV (n = 13), HCN2 + TRIP8b(S237A): −88.3 ± 2.2 mV (n = 14), HCN2 + TRIP8b(R234A): −83.9 ± 2.3 mV (n = 15). Independent Student's t tests were performed to compare TRIP8b(WT) to each condition; +TRIP8b(WT) and + vector: p = 0.03, +TRIP8b(WT) and +TRIP8b(S237A): p = 0.02, +TRIP8b(WT) and +TRIP8b(R234A): p < 0.001. C, maximum tail current was divided by the cell capacitance to obtain Ih current amplitude for each condition. HCN2 + vector: 7.2 ± 1.2 pA/pF (n = 15), HCN2 + TRIP8b(WT): 27.3 ± 3.9 pA/pF (n = 13), HCN2 + TRIP8b(S237A): 15.4 ± 4.3 pA/pF (n = 14), HCN2 + TRIP8b(R234A): 18.3 ± 4.0 pA/pF (n = 15). Student's t tests were similarly performed to compare TRIP8b(WT) with each condition; +TRIP8b(WT) and +vector: p < 0.001, +TRIP8b(WT) and +TRIP8b(S237A): p = 0.054, +TRIP8b(WT) and +TRIP8b(R234A): p = 0.1. *, p < 0.05; **, p < 0.01; ***, p < 0.001. All error bars represent mean ± S.E.

Article Snippet: Phospho-specific antibody pSer 237 Phosphorylated TRIP8b antibody was generated by YenZym Antibodies, LLC (South San Francisco, CA).

Techniques: Mutagenesis, Stable Transfection, Expressing, Transfection, Control, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of the HCN channel auxiliary subunit TRIP8b is altered in an animal model of temporal lobe epilepsy and modulates channel function

doi: 10.1074/jbc.RA119.010027

Figure Lengend Snippet: Hippocampal HCN1 protein expression and neocortical phosphorylated TRIP8b expression are unchanged during epileptogenesis. A, HCN1 protein was immunoblotted in hippocampal lysates harvested 1 h (i), 1 day (ii), and 28 days (iii) post-SE. HCN1 levels from Sal- and KA-treated rats were normalized to β3-tubulin. Immunoblot band density was quantified using an unpaired Student's t test; 1 Sal and 1 KA animal are shown. HCN1/β3-tubulin; 1 h post-SE: Sal (1.0 ± 0.1, n = 5), KA (1.1 ± 0.1, n = 6), p > 0.05; 1 day post-SE: Sal (1.0 ± 0.1, n = 6), KA (0.9 ± 0.2, n = 5), p > 0.05; 28 days post-SE: Sal (1.00 ± 0.04, n = 5), KA (1.1 ± 0.1, n = 5), p > 0.05) (Table S1). Full blots are shown in Fig. S2. B, the neocortex was harvested from Sal- or KA-treated rats 28 days post-SE. Immunoblots were probed with antibodies against pSer237, TRIP8b, and β3-tubulin. Immunoblot band density was quantified using an unpaired Student's t test; 1 Sal and 1 KA animal are shown. pSer237/TRIP8b; 28 days post-SE: Sal (1.0 ± 0.1, n = 5), KA (0.9 ± 0.1, n = 5), p > 0.05 (Table S2). Full blots are shown in Fig. S2. All error bars represent mean ± S.E. Molecular mass markers are shown in kilodaltons.

Article Snippet: Phospho-specific antibody pSer 237 Phosphorylated TRIP8b antibody was generated by YenZym Antibodies, LLC (South San Francisco, CA).

Techniques: Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of the HCN channel auxiliary subunit TRIP8b is altered in an animal model of temporal lobe epilepsy and modulates channel function

doi: 10.1074/jbc.RA119.010027

Figure Lengend Snippet: Reduction in TRIP8b phosphorylation and CaMKIIα activity in the KA model of TLE. A and B, hippocampi were harvested from Sal- or KA-treated rats 1 h (i), 1 day (ii), or 28 days (iii) post-SE. Immunoblots were probed with antibodies against pSer237, TRIP8b, and β3-tubulin (A) or pCaMKIIα and CaMKIIα (B). Immunoblot band density was quantified using unpaired Student's t test; 3 Sal and 3 KA animals are shown. A, pSer237/TRIP8b: 1 h post-SE: Sal (1.00 ± 0.04, n = 5), KA (0.81 ± 0.04, n = 6), p < 0.01; 1 day post-SE: Sal (1.0 ± 0.1, n = 6), KA (0.7 ± 0.1, n = 5), p < 0.01; 28 days post-SE: Sal (1.0 ± 0.1, n = 10), KA (0.7 ± 0.1, n = 11), p < 0.01. B, pCaMKIIα/CaMKIIα, 1 h post-SE: Sal (1.0 ± 0.2, n = 5), KA (0.9 ± 0.1, n = 6), p > 0.05; 1 day post-SE: Sal (1.0 ± 0.1, n = 6), KA (0.5 ± 0.2, n = 5), p < 0.05); 28 days post-SE: Sal (1.0 ± 0.1, n = 5), KA (0.63 ± 0.04, n = 5), p < 0.01) (Table S3). *, p < 0.05; **, p < 0.01. Full blots are shown in Fig. S3. All error bars represent mean ± S.E. Molecular mass markers are shown in kilodaltons.

Article Snippet: Phospho-specific antibody pSer 237 Phosphorylated TRIP8b antibody was generated by YenZym Antibodies, LLC (South San Francisco, CA).

Techniques: Phospho-proteomics, Activity Assay, Western Blot