Journal: Cell Reports Medicine

Article Title: MicroRNA-dependent suppression of biological pacemaker activity induced by TBX18

doi: 10.1016/j.xcrm.2022.100871

Figure Lengend Snippet:

Article Snippet: Rabbit Polyclonal anti-HCN2 , Alomone Labs , Cat# APC-030 RRID: AB_2313726.

Techniques: Recombinant, Staining, Luciferase, Reporter Assay, Western Blot, Isolation, Plasmid Preparation, Software

Journal: PLoS ONE

Article Title: HCN Channels Are Not Required for Mechanotransduction in Sensory Hair Cells of the Mouse Inner Ear

doi: 10.1371/journal.pone.0008627

Figure Lengend Snippet: (A) Conventional RT-PCR was used to examine the expression of HCN1-4 in vestibular epithelia of wild-type mice. Arrows indicate the expected size of the PCR product: HCN1: 491 bp; HCN2: 337 bp; HCN3: 339 bp and HCN4: 419 bp. Lane 1 in each gel contained markers. Each subsequent lane contained the PCR product obtained using template cDNA harvested from the following sources. Lane 2: wild-type mouse brain; Lane 3: HCN1-deficient mouse brain; Lane 4: wild-type utricle; Lane 5: HCN1-deficient mouse utricle. (B) Quantitative RT-PCR was used to estimate total number of copies of messenger RNA for each of the four HCN subunits using wild-type mouse cochlea as template. Total copies in thousands of HCN mRNA transcripts per cochlea. Results were from a pool of 4 cochlea obtained from mice at P4.

Article Snippet: For HCN2, the primary antibody was a rabbit polyclonal directed against the N-terminus (Alomone, Israel, APC-030, 1∶100 dilution).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: HCN Channels Are Not Required for Mechanotransduction in Sensory Hair Cells of the Mouse Inner Ear

doi: 10.1371/journal.pone.0008627

Figure Lengend Snippet: Representative mechanotransduction currents evoked by hair bundle deflections. The left column of data were recorded from mouse utricle type II hair cells at P3 - P6. The data in the right column were recorded from mouse cochlear outer hair cells at P6 - P7. The scale bars at the top apply to all data in the column. The deflection protocol is shown at the bottom of trace panels I and J. Data were recorded under the following conditions: (A & B) wild-type control; (C & D) in the presence of 500 µM ZD7288; (E & F) HCN1 −/− ; (G & H) HCN2 −/− ; (I& J) HCN1 −/− and HCN2 −/− . Panels K-P show fluorescent images of hair cells following application of the transduction channel permeable dye, FM1-43, in utricle (P5 –P7) and organ of Corti (P6 – P9) sensory epithelia under the following conditions: (K & L) HCN1 −/− ; (M & N) HCN2 −/− ; (O& P) HCN1 −/− and HCN2 −/− . Uptake of FM1-43 appeared normal in all tissues examined. The scale bar in panel (O) equals 10 µm and also applies to panels K and M. The scale bar in panel (P) equals 5 µm and also applies to panels L and N. (Q) Summary of transduction currents recorded from vestibular and auditory hair cells. Maximum current amplitudes under each condition were averaged and were normalized relative to wild-type controls. Error bars show standard deviation; the number of samples is indicated above each bar.

Article Snippet: For HCN2, the primary antibody was a rabbit polyclonal directed against the N-terminus (Alomone, Israel, APC-030, 1∶100 dilution).

Techniques: Transduction, Standard Deviation

Journal: PLoS ONE

Article Title: HCN Channels Are Not Required for Mechanotransduction in Sensory Hair Cells of the Mouse Inner Ear

doi: 10.1371/journal.pone.0008627

Figure Lengend Snippet: (A & B) Representative mechanotransduction currents recorded from mouse utricle type II hair cells at P3 - P6. Bundle deflections were evoked using the protocol shown at the bottom of . Panel A shows data from a non-transfected control cell and panel B shows data from a GFP+ cell transfected with the HCN2-AYA construct. The scale bars in B apply to panels A and B. (C & D) Representative currents recorded in response to families of voltage steps that ranged between −124 mV and −64 mV in 10 mV increments. Capacitive transients and leak currents were subtracted for clarity. The scale bars in panel D apply to both panels C and D. Panel C shows I h recorded from a non-transfected utricle type II hair cell from the same epithelium as that shown in panel A. Panel D shows a family of currents recorded from the same GFP+ shown in panel B. A family of voltage steps was used that was identical to those used to evoke the data shown in panel C. In this case, expression of the HCN2-AYA construct inhibited I h . (E) A fluorescence image that revealed GFP expression was superimposed on a DIC image of the same field of cells. The recording pipette is visible to the right of the cell. Scale bar equals 5 µm.

Article Snippet: For HCN2, the primary antibody was a rabbit polyclonal directed against the N-terminus (Alomone, Israel, APC-030, 1∶100 dilution).

Techniques: Transfection, Construct, Expressing, Fluorescence, Transferring

Journal: PLoS ONE

Article Title: The cGMP-Dependent Protein Kinase II Is an Inhibitory Modulator of the Hyperpolarization-Activated HCN2 Channel

doi: 10.1371/journal.pone.0017078

Figure Lengend Snippet: ( A ) Coimmunoprecipitation of HCN2 and cGKII in HEK293 cells. Lysates of HEK293 cells transfected with HCN2 and cGKII or cGKII alone were immunoprecipitated (IP) using a cGKII antibody and stained for HCN2 and cGKII as loading control. 500 µg protein was applied per lane. ( B ) Protein extracts of hypothalamic brain tissue from WT and HCN2-KO mice were immunoprecipitated using a cGKII antibody and analyzed in immunoblots (IB) for HCN2. Anti-cGKII served as loading control. ( C ) Schematic representation of full length HCN2 (862 amino acids) and myc-tagged HCN2-domains used for interaction studies. The calculated molecular size of the proteins is indicated. NT, N-terminus; TMR, transmembrane region; CT, complete HCN2 C-terminus; L, C-linker; CNBD, cyclic nucleotide-binding domain; dC, distal C-terminus. ( D ) GFP-Trap. Lysates of HEK293 cells coexpressing cGKII-GFP and myc-tagged portions of the HCN2 C-terminus were bound to GFP-tagged beads. Co-immunoprecipitated proteins were detected by immunoblotting with an anti-myc antibody. Anti-cGKII was used as loading control.

Article Snippet: Primary neurons were incubated over night at 4°C with anti-HCN2 (Alomone) and anti-myc (clone 9B11, Cell Signaling) diluted in 5% Chemiblocker (Chemicon).

Techniques: Transfection, Immunoprecipitation, Staining, Western Blot, Binding Assay

Journal: PLoS ONE

Article Title: The cGMP-Dependent Protein Kinase II Is an Inhibitory Modulator of the Hyperpolarization-Activated HCN2 Channel

doi: 10.1371/journal.pone.0017078

Figure Lengend Snippet: ( A–D ) Colocalization in primary neurons. Hippocampal neurons of neonatal mice (E16.5) were cotransduced with lentivirus expressing HCN2 and cGKII-myc, respectively. Neurons were stained with antibodies against myc ( A ) and HCN2 ( B ). Counterstaining was performed with Hoechst dye. ( C ) Merge of ( A ) and ( B ). ( D ) Negative control (nc). Merge of stainings in the absence of primary antibodies. Scale bar corresponds to 100 µm. ( E–H ) Immunohistochemical staining of coronal brain slices. Consecutive slices from wild-type mice were stained with anti-cGKII ( E ) or anti-HCN2 ( F ). The signal was amplified by tyramide signal amplification. Counter stain was performed with Hoechst 33342 nuclear dye. As negative control, coronal slices of cGKII-KO ( G ) and HCN2-KO mice ( H ) were used. Scale bar corresponds to 500 µm. ( I, J ) Higher magnification of cGKII ( I ) and HCN2 ( J ) staining in the hypothalamic region corresponding to the dotted white box as indicated in ( E ). Scale bar corresponds to 50 µm.

Article Snippet: Primary neurons were incubated over night at 4°C with anti-HCN2 (Alomone) and anti-myc (clone 9B11, Cell Signaling) diluted in 5% Chemiblocker (Chemicon).

Techniques: Expressing, Staining, Negative Control, Immunohistochemical staining, Amplification

Journal: PLoS ONE

Article Title: The cGMP-Dependent Protein Kinase II Is an Inhibitory Modulator of the Hyperpolarization-Activated HCN2 Channel

doi: 10.1371/journal.pone.0017078

Figure Lengend Snippet: ( A ) In vitro phosphorylation of HCN2 by cGKII. Lysates of COS-7 cells expressing HCN2 and cGKII were incubated with [γ-32P]-ATP for the times indicated. After incubation, proteins were separated on SDS page and analyzed by autoradiography. The first lane represents a control reaction with a cell lysate lacking cGKII. ( B ) HCN channel constructs used for phosphorylation studies. The positions of the three putative cGKII phosphorylation sites (S641, S786 and S840) are indicated. The calculated molecular mass is given for each construct. ( C ) Phosphorylation assay of a HCN2 mutant lacking S786 and S840 (first lane) and the HCN2-S641A mutant. ( D ) Pulldown of phosphoproteins by TiO 2 beads. Lysates of cells expressing HCN2-CT or HCN2-CT-S641A in the presence or absence of cGKII, respectively, were incubated with TiO 2 beads. Proteins specifically bound to the beads were analyzed with an anti-myc antibody.

Article Snippet: Primary neurons were incubated over night at 4°C with anti-HCN2 (Alomone) and anti-myc (clone 9B11, Cell Signaling) diluted in 5% Chemiblocker (Chemicon).

Techniques: In Vitro, Expressing, Incubation, SDS Page, Autoradiography, Construct, Phosphorylation Assay, Mutagenesis

Journal: PLoS ONE

Article Title: The cGMP-Dependent Protein Kinase II Is an Inhibitory Modulator of the Hyperpolarization-Activated HCN2 Channel

doi: 10.1371/journal.pone.0017078

Figure Lengend Snippet: ( A ) Voltage step protocol and family of current traces of a HEK293 cell transiently transfected with HCN2. ( B–D ) Normalized current-voltage (IV) dependence of HCN2 activation in the presence and absence of cGKII. The voltage-dependence was determined in the presence of 10 µM intracellular cGMP ( B ), 100 µM intracellular cGMP ( C ) and 1 µM intracellular cGMP ( D ). ( E ) IV curves of HCN2 in the presence or absence of cGKII at 2 µM intracellular cAMP. ( F ) IV curves determined at 10 µM intracellular cGMP from cells coexpressing cGKII and HCN2 or HCN2-S641A. ( G ) IV curves of HCN2 compared to the IV curve of an HCN2 mutant with functionally impaired cyclic nucleotide binding domain (HCN2-RT>EA) that was coexpressed with cGKII. Currents were measured in the presence of 10 µM cGMP. ( H ) Comparison of midpoint potentials (V 0.5 ) of wild type (WT) and HCN2 mutants (HCN2-S641A, HCN2-RT>EA). Channels were expressed alone or together with either wild type or catalytically inactive GKII (cGKII-D576A). V 0.5 was determined from the normalized IV curves in the presence (+) or absence (−) of 10 µM cGMP as indicated. In one set of experiments the cGKII was inhibited by the pharmacological blocker KT5823. *** = p<0.001.

Article Snippet: Primary neurons were incubated over night at 4°C with anti-HCN2 (Alomone) and anti-myc (clone 9B11, Cell Signaling) diluted in 5% Chemiblocker (Chemicon).

Techniques: Transfection, Activation Assay, Mutagenesis, Binding Assay

Journal: PLoS ONE

Article Title: The cGMP-Dependent Protein Kinase II Is an Inhibitory Modulator of the Hyperpolarization-Activated HCN2 Channel

doi: 10.1371/journal.pone.0017078

Figure Lengend Snippet: cGMP shifts the voltage-dependence of HCN2 activation to more positive voltage (+ΔV) via direct interaction with the CNBD of HCN2 and induces a hyperpolarizing shift (−ΔV) by activating cGKII that is bound to the channel.

Article Snippet: Primary neurons were incubated over night at 4°C with anti-HCN2 (Alomone) and anti-myc (clone 9B11, Cell Signaling) diluted in 5% Chemiblocker (Chemicon).

Techniques: Activation Assay

Journal: PLoS ONE

Article Title: K + Channel Regulator KCR1 Suppresses Heart Rhythm by Modulating the Pacemaker Current I f

doi: 10.1371/journal.pone.0001511

Figure Lengend Snippet: (A) KCR1 and HCN2 can associate in protein complexes. Coimmunoprecipitation of HCN2 and KCR1 from mammalian cell extracts. Protein extracts from cells transfected with pCFLAG 3 -KCR1 (lane 0), HCN2 plus pCFLAG 3 -KCR1 (lanes 1), HCN2 plus p3xFLAG-CMV (lanes 2) or non-transfected cells (lanes 3) assayed by Western blot using anti-HCN2 antibody. Input: input lysates were blotted to show that HCN2 protein was successfully produced and detected. Anti-FLAG-Sepharose: 700 µg of total lysate immunoprecipitated with anti-FLAG-Sepharose shows that HCN2 and KCR1 (pCFLAG 3 -KCR1) can be coimmunoprecipitated (lane 1). A-Sepharose: mock immunoprecipitation using protein A-Sepharose beads unlinked to FLAG-antibody to exclude any unspecific antibody binding. (B) KCR1 message (218 bp) can be detected in all cell types tested, except for non-transfected CHO cells (upper panel). Representative electrophoresis gel illustrating results obtained in single-cell RT-PCR experiments (as described in ) with three different cells types. CHO: Non-transfected Chinese hamster ovary cells; CHO+KCR1: CHO cells transfected with KCR1 cDNA; Rat NN : Neonatal rat cardiomyocytes; Rat A : Adult rat cardiomyocytes. GAPDH mRNA (318 bp) was used as a control and is found in all cell types tested, including non-transfected, KCR1-negative CHO cells (lower panel). Negative controls did not include RNA or reverse transcriptase and gave no amplicons (not shown). (C) Regional differences in expression of KCR1 in adult rat and pig heart. KCR1 mRNA levels determined by quantitative real-time PCR (qPCR; n = 3–4). RA = right atrium, RV = right ventricular free wall, LA = left atrium, LV = left ventricular free wall, Se = septum, SAN = sinoatrial node, AV = atrioventricular node. Values are mean±SEM. *p<0.05 vs. AV node, # p<0.05 vs. SAN. Upper panel: Representative electrophoresis gel obtained from qPCR products of different cardiac regions (the AV-node region was divided in: AV S = superior part and AV I = inferior part), samples were loaded and normalized to GAPDH.

Article Snippet: The gel was transferred to Hybond ECL membrane (Amersham) for Western blotting with an anti-HCN2 antibody (Alomone Labs, Israel).

Techniques: Transfection, Western Blot, Produced, Immunoprecipitation, Binding Assay, Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: K + Channel Regulator KCR1 Suppresses Heart Rhythm by Modulating the Pacemaker Current I f

doi: 10.1371/journal.pone.0001511

Figure Lengend Snippet: (A) Representative HCN2 whole-cell currents in absence and presence of KCR1. (B) Mean current densities of heterologously expressed HCN2 alone or together with KCR1 demonstrate that KCR1 significantly ( * , p<0.001) reduced current density of recombinant I HCN2 . For data see text.

Article Snippet: The gel was transferred to Hybond ECL membrane (Amersham) for Western blotting with an anti-HCN2 antibody (Alomone Labs, Israel).

Techniques: Recombinant

Journal: PLoS ONE

Article Title: K + Channel Regulator KCR1 Suppresses Heart Rhythm by Modulating the Pacemaker Current I f

doi: 10.1371/journal.pone.0001511

Figure Lengend Snippet: (A) Representative HCN2 single-channel currents (sampling frequency: 5 kHz, corner frequency: 1 kHz) of inside-out recordings from one- and multi-channel (n∼50 channels) patches at different test-potentials (−90 to −130 mV). (B) Pharmacological characteristics of HCN2 single-channels (test potential: −90 mV, holding potential: −35 mV). Ivabradine (50 µM) blocks HCN2 single-channel current during repetitive activation/deactivation steps (−90 mV, 150 ms/+10 mV, 600 ms). The observations that ivabradine significantly reduced the open probability (25.8±7.07% vs.6.08±3.45%, n = 4, p<0.05), the mean open time (1.03±0.12 ms vs. 0.61±0.16 ms, n = 4, p<0.05) and the availability (75.9±10.1% vs. 25.3±6.98%, n = 4, p<0.05) suggests an open-channel blockade by a fast and a slow gating mechanism. cAMP induced an increase of the channel activity (for data, see text). The data were sampled at 10 kHz and filtered at 2 kHz. (C) Effect of ivabradine (Iva) and cAMP on single-channel activity. Open probability (P open ) decreased after ivabradine (50 µM) and increased after cAMP (1 mM) application, respectively. Data recorded as in .

Article Snippet: The gel was transferred to Hybond ECL membrane (Amersham) for Western blotting with an anti-HCN2 antibody (Alomone Labs, Israel).

Techniques: Sampling, Activation Assay, Activity Assay

Journal: PLoS ONE

Article Title: K + Channel Regulator KCR1 Suppresses Heart Rhythm by Modulating the Pacemaker Current I f

doi: 10.1371/journal.pone.0001511

Figure Lengend Snippet: (A) Comparison of single recombinant HCN2 channels transfected in CHO cells alone (left) and with KCR1 (right). Middle, 20 consecutive single traces of each channel without and with KCR1. Single channels were hyperpolarized at continuous pulse mode for a total duration of 3 s (20×150 ms sweeps), with a holding potential of −35 mV and a test potential of −90 mV. Bottom, ensemble average current of one consecutive sweep of 3 s pulse duration. Scale bars, 50 ms, 6 pA (unitary current traces) for HCN2 alone (left) and 2 pA when co-transfected with KCR1 (right), or 1 s, 0.5 pA (ensemble average current) for HCN2 and HCN2+KCR1. (B) KCR1 significantly shifted I HCN2 activation to more negative potentials. Channel activation was measured by the parameter availability, plotted against the test potential and then determined by using the Boltzmann function. For data see text. (C) Open-time histograms: KCR1 reduced the number of HCN2 open states. Number of open events (square root) were plotted against the logarithmically binned open time durations for HCN2 alone and HCN2+KCR1 (pooled one- and multi [n≤3]-channel experiments). (D) Closed-time histograms: KCR1 did not affect the number of HCN2 closed states. Number of closed events (square root) were plotted against the logarithmically binned closed time durations for HCN2 alone and HCN2+KCR1 (pooled one-channel experiments only).

Article Snippet: The gel was transferred to Hybond ECL membrane (Amersham) for Western blotting with an anti-HCN2 antibody (Alomone Labs, Israel).

Techniques: Recombinant, Transfection, Activation Assay

Journal: PLoS ONE

Article Title: K + Channel Regulator KCR1 Suppresses Heart Rhythm by Modulating the Pacemaker Current I f

doi: 10.1371/journal.pone.0001511

Figure Lengend Snippet: Gating of single recombinant HCN2 and KCR1-cotransfected I HCN2

Article Snippet: The gel was transferred to Hybond ECL membrane (Amersham) for Western blotting with an anti-HCN2 antibody (Alomone Labs, Israel).

Techniques: Recombinant