Journal: PLoS ONE

Article Title: Loss of the V-ATPase B1 Subunit Isoform Expressed in Non-Neuronal Cells of the Mouse Olfactory Epithelium Impairs Olfactory Function

doi: 10.1371/journal.pone.0045395

Figure Lengend Snippet: (A) A schematic diagram showing the subunit composition of the V-ATPase. The cytosolic V 1 domain is composed of subunits A through H (shown in white or light gray, marked with blue letters). The transmembrane V 0 domain is composed of subunits a, c, c” (or b), d, e, and Ac45 (shown in blue, marked with white letters). Some of the subunit interactions are putative. (B) Section from a 3-D image reconstruction showing that B1 V-ATPase (red) localizes to the microvilli of olfactory sustentacular cells in a 2-week old female mouse pup. Apical cilia of olfactory sensory neurons are immunostained for CNGA2 (green). DAPI (blue) stains cell nuclei. Bar = 30 µm.

Article Snippet: We used the following commercially available antibodies as olfactory markers: a goat polyclonal antibody against olfactory marker protein (OMP) (WAKO Chemicals USA, Richmond, VA), a rabbit polyclonal antibody against cytokeratin-18 (CK-18) (Abcam, Cambridge, MA), a rabbit polyclonal antibody against cyclic nucleotide gated cation channel 2 (CNGA2) (Alomone Labs, Jerusalem, Israel), a mouse monoclonal anti-tubulin antibody and a rat monoclonal anti-L-CAM/E-cadherin antibody (Sigma-Aldrich, St. Louis, MO).

Techniques:

Journal: PLoS ONE

Article Title: Glutamate Excitotoxicity Inflicts Paranodal Myelin Splitting and Retraction

doi: 10.1371/journal.pone.0006705

Figure Lengend Snippet: (A) Time-lapse images showing the leakage of dextran-FITC into the split myelin after glutamate application. The myelin (red) and dextran-FITC (green) were monitored by CARS and TPEF, simultaneously. (B) and (C) CARS images of myelin sheath (red) and confocal fluorescence images of degraded MBP (green) in spinal tissues after incubation in 1 mM glutamate solution (Glut) or normal Krebs' solution (Normal). The curves on the left of the images are intensity profiles of the lines indicated in the images. (D) EM images in a normal tissue show the paranodal myelin held in tight contact with the axolemma. The right panel is the magnified image of the dash frame in the left panel. (E) EM images show that glutamate induces paranodal myelin splitting (arrow), disruption and retraction. The right panel is the magnified image of the dash frame in the left panel. An elongated node, detachment of paranodal myelin from axolemma (arrow head), and disrupted myelin debris (star) were observed. (F)–(H) CARS images of myelin sheath (red) and TPEF images of Kv1.2 channels (green) at the juxtaparanodes after normal Krebs' solution (F, Normal) and application of glutamate (G–H, Glut). Both the exposure of Kv1.2 channels (G) and displacement of Kv1.2 channels into paranodes and node (H) were observed. For (D) and (E), bar = 1 µm. For (A)–(C) and (F)–(H), bar = 10 µm.

Article Snippet: Antiserum against Kv1.2 antibody (Alomone Lab, Jerusalem, Israel) was used to locate the K + channels at the juxtaparanodes.

Techniques: Fluorescence, Incubation

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Genetic ablation of smooth muscle K IR 2.1 is inconsequential to the function of mouse cerebral arteries

doi: 10.1177/0271678X221093432

Figure Lengend Snippet: Genetic ablation of K IR 2.1 does not eliminate inward K + currents in cerebral arterial smooth muscle cells (SMCs). Whole-cell patch clamp electrophysiology was used to measure K IR current with voltage ramps from −100 to +20 mV in the absence and presence of Ba 2+ in 60 mM K + . (a,b) Representative recordings of whole-cell and Ba 2+ -subtracted K IR currents in myocytes isolated from SMC K IR 2.1 −/− mice and non-induced Cre SMC controls. (c) Summary data compare peak inward current at −100 mV between groups ( n = 9 SMCs from 6 mice in control group and n = 9 SMCs from 8 mice in knockout group; nested t -test).

Article Snippet: Rabbit polyclonal primary antibodies against K IR 2.1 (APC-026, 1:200; Alomone) and K IR 2.2 (APC-042, 1:200; Alomone) were diluted in quench solution and applied to cells for overnight incubation (4 °C).

Techniques: Patch Clamp, Isolation, Knock-Out

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Genetic ablation of smooth muscle K IR 2.1 is inconsequential to the function of mouse cerebral arteries

doi: 10.1177/0271678X221093432

Figure Lengend Snippet: K I R 2.1 is negligibly expressed in cerebral vascular smooth muscle cells (SMCs) while K IR 2.2 is highly expressed at the cell membrane. Tamoxifen-induced K IR 2.1 knockout significantly reduced subunit expression but levels remained detectable by immunofluorescence. (a) Fluorescent anti-K IR 2.1 (green) exhibited a faint labeling pattern in cerebral arterial myocytes from SMC K IR 2.1 −/− and control mice with nuclei stained with DAPI (blue). (b) Summary of data compares fluorescence intensity (background subtracted) of K IR 2.1 signal between groups ( n = 10 cells from 5 animals in control group and n = 9 cells from 5 animals in knockout group; unpaired t -test). (c) Immunofluorescence labeling of SMCs for K IR 2.2. (d) Summary of data compares background-subtracted fluorescence intensity of K IR 2.2 signal between groups ( n = 8 cells pooled from 4 animals/group; unpaired t -test). Two cells were analyzed per animal with background signal subtracted using 2° antibody control.

Article Snippet: Rabbit polyclonal primary antibodies against K IR 2.1 (APC-026, 1:200; Alomone) and K IR 2.2 (APC-042, 1:200; Alomone) were diluted in quench solution and applied to cells for overnight incubation (4 °C).

Techniques: Knock-Out, Expressing, Immunofluorescence, Labeling, Staining, Fluorescence

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Genetic ablation of smooth muscle K IR 2.1 is inconsequential to the function of mouse cerebral arteries

doi: 10.1177/0271678X221093432

Figure Lengend Snippet: K IR 2.2 protein expression is unaltered in smooth muscle cell (SMC) K IR 2.1 −/− cerebral arteries. (a) Western blot of intact cerebral arteries confirmed that protein levels of K IR 2.2 are not impacted by deletion of SMC K IR 2.1. (b) Summary of data compares K IR 2.2 protein levels between control and knockout mice (normalized to actin; n = 6 mice; unpaired t -test).

Article Snippet: Rabbit polyclonal primary antibodies against K IR 2.1 (APC-026, 1:200; Alomone) and K IR 2.2 (APC-042, 1:200; Alomone) were diluted in quench solution and applied to cells for overnight incubation (4 °C).

Techniques: Expressing, Western Blot, Knock-Out

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Genetic ablation of smooth muscle K IR 2.1 is inconsequential to the function of mouse cerebral arteries

doi: 10.1177/0271678X221093432

Figure Lengend Snippet: K IR 2.1 and K IR 2.2 subunits are inversely expressed in cerebral endothelial and vascular smooth muscle cells. (a) K IR 2.1 and (b) K IR 2.2 subunit expression is shown as average cellular transcript counts per cell, as determined by single-cell RNA sequencing of the mouse brain vasculature. Data highlight differences in the dominant subunit between cell types. Abbreviations: PC – pericytes; SMC – smooth muscle cells; EC – endothelial cells; v – venous; c – capillary; a – arterial; aa – arteriolar. Figures provided by http://betsholtzlab.org/VascularSingleCells/database.html . 33,34

Article Snippet: Rabbit polyclonal primary antibodies against K IR 2.1 (APC-026, 1:200; Alomone) and K IR 2.2 (APC-042, 1:200; Alomone) were diluted in quench solution and applied to cells for overnight incubation (4 °C).

Techniques: Expressing, RNA Sequencing Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Genetic ablation of smooth muscle K IR 2.1 is inconsequential to the function of mouse cerebral arteries

doi: 10.1177/0271678X221093432

Figure Lengend Snippet: Myogenic responses and K + -induced dilation are intact in cerebral arteries of smooth muscle cell (SMC) K IR 2.1 −/− mice. Cerebral arteries from control and SMC K IR 2.1 −/− mice were cannulated and intravascular pressure was elevated stepwise while vasomotor responses were measured. (a) Representative diameter traces from endothelium-denuded vessels of control and SMC K IR 2.1 −/− mice show the effect of increasing pressure on myogenic tone. (b) Summary of data highlights limited impact of smooth muscle K IR 2.1 knockout on myogenic tone development. Paired t -test was performed for 0 vs. 100 μM Ba 2+ treatment ( n = 10 vessels in the control group and n = 11 vessels in the SMC K IR 2.1 −/− group with 1 vessel/mouse; *P < 0.05). (c) K + -induced dilation, elicited by increasing extracellular K + from 5 mM to 10 mM before and after treatment with 100-μM Ba 2+ , was intact in the knockout group ( n = 6 vessels in the control group and SMC K IR 2.1 −/− group; one-way ANOVA with Sidak’s multiple comparisons test). (d) K + -induced dilation (5 mM K + to 10 mM K + ) was abrogated with exposure to low concentrations of Ba 2+ , implicating K IR 2.2 as the mediator of this response based on its Ba 2+ sensitivity profile ( n = 7 vessels from control mice; repeated measures one-way ANOVA with Sidak’s multiple comparisons test). Myogenic tone (%) was calculated as: [(passive diameter – active diameter)/(passive diameter – minimal diameter)] × 100 at each pressure step. K + -induced dilation (%) was calculated as difference between diameter at 10 mM [K + ] and 5 mM [K + ] divided by the dilatory range (passive diameter – minimal diameter).

Article Snippet: Rabbit polyclonal primary antibodies against K IR 2.1 (APC-026, 1:200; Alomone) and K IR 2.2 (APC-042, 1:200; Alomone) were diluted in quench solution and applied to cells for overnight incubation (4 °C).

Techniques: Knock-Out

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Genetic ablation of smooth muscle K IR 2.1 is inconsequential to the function of mouse cerebral arteries

doi: 10.1177/0271678X221093432

Figure Lengend Snippet: Region-specific brain perfusion is not altered in smooth muscle cell (SMC) K IR 2.1 −/− mice at rest and with increased systemic blood pressure. (a) Representative arterial spin-labeled MR brain perfusion maps. Scans were done in a posterior-to-anterior direction and the volume of brain scanned was divided into 5 coronal slices. Resting cerebral blood flow was measured in control and SMC K IR 2.1 −/− mice. Scans were repeated after blood pressure challenge with an intraperitoneal phenylephrine injection. Figure shows slices from 2 regions of the brain (red boxes) spanning cerebral nuclei, hippocampus, thalamus, and hypothalamus. (b) Baseline perfusion in several major brain structures was not significantly different between control and tamoxifen-induced mice. The blood pressure challenge caused a modest but significant rise in cerebral blood flow to a similar extent in control and SMC K IR 2.1 −/− animals. Unpaired t -test was performed for control ( n = 7 mice) vs. SMC K IR 2.1 −/− ( n = 11 mice) comparison; paired t -test was performed for baseline vs. phenylephrine-treatment. *P < 0.05 compared to baseline control.

Article Snippet: Rabbit polyclonal primary antibodies against K IR 2.1 (APC-026, 1:200; Alomone) and K IR 2.2 (APC-042, 1:200; Alomone) were diluted in quench solution and applied to cells for overnight incubation (4 °C).

Techniques: Labeling, Injection

Journal: Translational Psychiatry

Article Title: Identification of ultra-rare disruptive variants in voltage-gated calcium channel-encoding genes in Japanese samples of schizophrenia and autism spectrum disorder

doi: 10.1038/s41398-022-01851-y

Figure Lengend Snippet: a Three-dimensional (3D) model structure of the Ca v 1.2 channel with the folded N-terminal structure. b 3D model structure of the Ca v 1.2 channel with the CaM-binding structure, shown in complex with the N-lobe of CaM. c Enlarged view around the alanine 36 (A36) residue of the folded N-terminal structure. The A36 site is highlighted by green dotted circles. The N-terminal spatial Ca 2+ -transforming element (NSCaTE) region (47–68), Ca 2+ , and CaM are indicated in yellow, orange, and magenta, respectively. Molecular graphics were created using UCSF Chimera . d A schematic illustration of the hypothesis that the A36V mutation attenuates Ca 2+ -dependent inactivation (CDI) by conformational equilibrium shift favoring the folded structure.

Article Snippet: The antibodies used for immunoblotting were purchased commercially as follows: a rabbit polyclonal antibody against Ca v 1.2 (ACC-003, Alomone Labs, Jerusalem, Israel), a mouse monoclonal antibody against β-actin (A5441, Merck), and secondary antibodies conjugated to HRP (ab97051 and ab97023, abcam, Cambridge, UK).

Techniques: Binding Assay, Mutagenesis

Journal: Translational Psychiatry

Article Title: Identification of ultra-rare disruptive variants in voltage-gated calcium channel-encoding genes in Japanese samples of schizophrenia and autism spectrum disorder

doi: 10.1038/s41398-022-01851-y

Figure Lengend Snippet: a Sanger sequencing results for the de novo variant p.A36V (left) and schematic illustration of the primary structure of the Ca v 1.2 channel (right). The red asterisk indicates the A36V mutation near the N-terminal spatial Ca 2+ -transforming element (NSCaTE). b The N-terminal amino acid sequences for Ca v 1.2 channels (short isoforms). The A36V mutation and the A39V Brugada mutation are indicated in red letters. c Expression of wild-type (WT) and A36V Ca v 1.2 channels in HEK293T cells as detected by anti-Ca v 1.2 antibody. d Membrane localization of WT and A36V Ca v 1.2 channels overexpressed in BHK cells. The plasma membrane was visualized by membrane-tethering red fluorescent protein (RFP-KRasCT). e The fluorescence intensity profiles of the line shown in Fig. 1d. f Plasma membrane to cytoplasm intensity ratio of Ca v 1.2. Statistical comparison was performed by two-tailed Welch’s t test (n.s., not significant). Data are presented as mean ± s.e.m.

Article Snippet: The antibodies used for immunoblotting were purchased commercially as follows: a rabbit polyclonal antibody against Ca v 1.2 (ACC-003, Alomone Labs, Jerusalem, Israel), a mouse monoclonal antibody against β-actin (A5441, Merck), and secondary antibodies conjugated to HRP (ab97051 and ab97023, abcam, Cambridge, UK).

Techniques: Sequencing, Variant Assay, Mutagenesis, Expressing, Fluorescence, Two Tailed Test

Journal: Translational Psychiatry

Article Title: Identification of ultra-rare disruptive variants in voltage-gated calcium channel-encoding genes in Japanese samples of schizophrenia and autism spectrum disorder

doi: 10.1038/s41398-022-01851-y

Figure Lengend Snippet: a Families of Ba 2+ currents evoked by 30-ms depolarizing pulses from −30 to 60 mV with increments of 10 mV for wild-type (WT) and A36V neuronal Ca v 1.2 channels. b Current density–voltage ( I – V ) relationships. Data are expressed as mean ± s.e.m., WT: n = 18, A36V: n = 12. The values of G, Erev, V 0.5 , and k were −0.40, 63.0 mV, 7.6 mV, and 5.6 mV for WT channels, and −0.50, 61.3 mV, 6.7 mV, and 4.9 mV for A36V Ca v 1.2 channels. c Inactivation curves for WT (○, n = 9) and A36V (●, n = 4) neuronal Ca v 1.2 channels. Data are expressed as mean ± s.e.m. The values of V 0.5 , and k were (respectively) −37.6 mV and 11.5 mV for WT channels, and −41.6 mV and 12.1 mV for A36V Ca v 1.2 channels. d , g Ca 2+ -dependent inactivation (CDI) of neuronal ( d ) and cardiac ( g ) Ca v 1.2 channels. Ba 2+ (blue) and Ca 2+ (black) currents evoked by 350-ms step depolarization to 30 mV were normalized at their peak current amplitudes for WT and A36V Ca v 1.2 channels. e , f, h, i , Ratios of current amplitude to the peak amplitude were plotted against depolarizing time in the Ba 2+ ( e, h ) and the Ca 2+ ( f, i ) external solutions. The numbers of recorded cells were 10 and 15 for WT and A36V neuronal Ca v 1.2 channels ( e , f ), and 8 and 6 for WT and A36V cardiac Ca v 1.2 channels ( h – i ), respectively. Statistical comparison was performed by two-tailed non-paired Student’s t test (* p < 0.05). Data are presented as mean ± s.e.m.

Article Snippet: The antibodies used for immunoblotting were purchased commercially as follows: a rabbit polyclonal antibody against Ca v 1.2 (ACC-003, Alomone Labs, Jerusalem, Israel), a mouse monoclonal antibody against β-actin (A5441, Merck), and secondary antibodies conjugated to HRP (ab97051 and ab97023, abcam, Cambridge, UK).

Techniques: Two Tailed Test

Journal: Lipids in Health and Disease

Article Title: Atherogenic L5 LDL induces cardiomyocyte apoptosis and inhibits K ATP channels through CaMKII activation

doi: 10.1186/s12944-020-01368-7

Figure Lengend Snippet: Decreased I KATP density in NRCMs treated with L5. a , Representative traces of I KATP in NRCMs treated with Pin-baseline (20 μM pinacidil, IKATP opener), Pin+L5 (7.5 μg/mL), Pin-washout, and Pin+Gli (100 μM glibenclamide, I KATP blocker). b , Time-course analysis showing the effect of L5 on I KATP . c , The perfusion of NRCMs with L5 reduced I KATP , which partially recovered after 5 min of washout (n = 5 per group, ** P < 0.01,* P < 0.05 vs. Pin-baseline, # P < 0.05 vs. Pin+L5). d , The overnight incubation of NRCMs with L5 decreased the expression level of Kir6.2, but no change was seen after overnight incubation with L1, n = 3 per group. * P < 0.05 vs. PBS

Article Snippet: Monoclonal antibodies against Kir6.2 (rabbit polyclonal, 1:200; Alomone Labs Ltd, Jerusalem, Israel), Nox2/gp91phox (rabbit polyclonal, 1:1000, Abcam, Cambridge, United Kingdom), or CaMKIIδ (rabbit polyclonal, 1:200; Santa Cruz Biotechnology, Santa Cruz, California, USA) were used for immunoblotting overnight at 4 °C.

Techniques: Incubation, Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: Hesperetin improves diabetic coronary arterial vasomotor responsiveness by upregulating myocyte voltage-gated K + channels

doi: 10.3892/etm.2020.8670

Figure Lengend Snippet: Chronic administration of HSP upregulated mRNA and protein expression of Kv1.2 in diabetic RCASMCs. The mRNA levels of myocyte (A) Kv 1.2 and (B) Kv 1.5 channels in the three experimental groups, which were expressed as the percentage relative to NDB. Representative western blot images showing (C) Kv1.2 and (D) Kv1.5 channel protein expression. The β-actin-normalized densitometric values of (E) Kv1.2 and (F) Kv1.5 channel protein expression in rat coronary arteries. Each value was presented as a mean ± SD from 6 measurements of pooled samples from 6-12 animals. * P<0.05 vs. NDB and # P<0.05 vs. DB. RCASMCs, rat coronary artery smooth muscle cells; Kv, voltage-dependent K + channels; HSP, hesperetin; NDB, non-diabetic control; DB, vehicle-treated diabetic control; DB + HSP, vehicle-treated diabetic control treated with hesperetin.

Article Snippet: The membranes were blocked in 5% non-fat milk containing TBS-0.02% Tween-20 (TBS-T) at room temperature for 2 h. The membranes were then incubated with polyclonal antibodies against Kv1.2 (1:400; cat. no. APC-004; Alomone Labs), Kv1.5 (1:400; cat. no. APC-010; Alomone Labs) or β-actin (1:4,000; cat. no. D110001-0100; Sangon Biotech Co., Ltd.) overnight at 4˚C.

Techniques: Expressing, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: Hesperetin improves diabetic coronary arterial vasomotor responsiveness by upregulating myocyte voltage-gated K + channels

doi: 10.3892/etm.2020.8670

Figure Lengend Snippet: Co-incubation of RCASMCs with HSP reversed high glucose-induced reductions in Kv1.2 channel expression. (A) Representative western blotting images of Kv1.2 channel protein expression. (B) β-actin-normalized densitometric values of Kv1.2 channel protein expression in RCASMCs. Each value was presented as a mean ± SD from 6 measurements of pooled samples from 6-12 animals. * P<0.05 vs. NG and # P<0.05 vs. HG. RCASMCs, rat coronary artery smooth muscle cells; HSP, hesperetin; HLG, osmotic control; NG, normal glucose; HG, high glucose.

Article Snippet: The membranes were blocked in 5% non-fat milk containing TBS-0.02% Tween-20 (TBS-T) at room temperature for 2 h. The membranes were then incubated with polyclonal antibodies against Kv1.2 (1:400; cat. no. APC-004; Alomone Labs), Kv1.5 (1:400; cat. no. APC-010; Alomone Labs) or β-actin (1:4,000; cat. no. D110001-0100; Sangon Biotech Co., Ltd.) overnight at 4˚C.

Techniques: Incubation, Expressing, Western Blot

Journal: eNeuro

Article Title: Homeostatic Recovery of Embryonic Spinal Activity Initiated by Compensatory Changes in Resting Membrane Potential

doi: 10.1523/ENEURO.0526-19.2020

Figure Lengend Snippet: Changes in voltage-gated channel expression following in ovo gabazine treatment. Western blottings showing changes in the expression of voltage-dependent Na + channels (Nav1.2) and inactivating K + channels (Kv4.2) in the chick embryo spinal cord following 12 h ( A ) or 6 h ( B ) of GABAR blockade in ovo .

Article Snippet: The primary antibodies against Nav1.2 and Kv4.2 were from Alomone Labs.

Techniques: Expressing, In Ovo, Western Blot