Journal: Cell Calcium

Article Title: Vascular Kv7 channels control intracellular Ca 2+ dynamics in smooth muscle

doi: 10.1016/j.ceca.2020.102283

Figure Lengend Snippet: Kv7 channel inhibition induces Ca 2+ oscillations linked to L- and T-type VGCCs activity. (A) Representative example trace showing Ca 2+ oscillations (indicated as F340/F380 ratio units; r.u.) evoked by Kv7 channel inhibitor XE991 (10 μM). (B) Comparison of amplitude of Ca 2+ signals induced by XE991 and 50 mM K + , estimated as area under the curve during first 50 s of stimulus application (corresponding to the duration of high-K + stimulation; exemplified in the inset on the right). (C-E) Example traces of XE991-induced Ca 2+ transients recorded in the presence of Kv7 channel opener, retigabine (10 μM; C), L-type (nifedipine; 2 μM; D) or T-type (NNC 55-0396; 3 μM; E) Ca 2+ channel blockers (as indicated). (F) Upper panel: superimposed are Fura2 ratiometric Ca 2+ recording (black) and FuoVolt membrane potential recording (measured as ΔF/F 0 ; red) during application of XE991 (10 μM) and retigabine (10 μM) during periods indicated by horizontal bars. Lower panel: Cross correlation of the normalised ratiometric Ca 2+ signal with the normalised FluoVolt signal indicated the time lag between the signals at which the peak correlation occurred. (G-I) Bar graphs summarising the effects of retigabine (G), nifedipine (H) or NNC 55-0396 (I) on the XE991-induced Ca 2+ spike frequency (spikes/s). Control is the spike frequency in the presence of XE991 measured from the onset of the first spike. For quantification of drug effect spike frequency was calculated from the onset of the drug application and until the end of the application of XE991. In panels G-I data are presented as mean ± S.E.M.; **P < 0.01, ***P < 0.001 (n = 5).

Article Snippet: Blocking buffer was removed prior to addition of primary antibody and coverslips were then treated with the primary antibodies against Kv7.1 (anti-Mouse 1:500; Santa Cruz), Kv7.2 (anti-Mouse 1:500; Santa Cruz), Kv7.3 (anti-Rabbit 1:500; Alomone), Kv7.4 (anti-Rabbit 1:200; Neuromab) or Kv7.5 (anti-Rabbit 1:500; Abcam) for 1 h 15 min (Supplemental Table II).

Techniques: Inhibition, Activity Assay

Journal: Cell Calcium

Article Title: Vascular Kv7 channels control intracellular Ca 2+ dynamics in smooth muscle

doi: 10.1016/j.ceca.2020.102283

Figure Lengend Snippet: Expression of Kcnq genes and Kv7 proteins in A7r5 cells. (A) Agarose gels stained with SYBR safe to visualise the RT-PCR products of Kcnq1 , Kcnq2 , Kcnq3 , Kcnq4 and Kcnq5 . (B) Quantification of RT-PCR results exemplified in panel A, expression is normalised to that of a housekeeping gene, Hprt1 (hypoxanthine phosphoribosyltransferase 1). (C) Immunofluorescence labelling of Kv7.1 – Kv7.5 channel subunits in A7r5 cells, scale bars are 20 μm. In panel B data are presented as mean ± S.E.M.; ***P < 0.001 (n = 6).

Article Snippet: Blocking buffer was removed prior to addition of primary antibody and coverslips were then treated with the primary antibodies against Kv7.1 (anti-Mouse 1:500; Santa Cruz), Kv7.2 (anti-Mouse 1:500; Santa Cruz), Kv7.3 (anti-Rabbit 1:500; Alomone), Kv7.4 (anti-Rabbit 1:200; Neuromab) or Kv7.5 (anti-Rabbit 1:500; Abcam) for 1 h 15 min (Supplemental Table II).

Techniques: Expressing, Staining, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence

Journal: Cell Calcium

Article Title: Vascular Kv7 channels control intracellular Ca 2+ dynamics in smooth muscle

doi: 10.1016/j.ceca.2020.102283

Figure Lengend Snippet: XE991 elicits a partial blockade of current through Kv7.5/Kv7.3 heteromeric channels that can be fully recovered by retigabine. (A) Current voltage relationship of Kv7.4 (n = 8) and Kv7.5/3 (n = 10) channels prior to and post XE991 (10 μM) treatment. (B,C) Representative voltage clamp recordings at −20 mV showing effects of XE991 (10 μM) and retigabine (10 μM; applied in the presence of XE991) on Kv7.4 (B) or Kv7.5/3) (C) channel currents. (D) Retigabine induced recovery (Ir) of Kv7 (M) current at −20 mV after XE991 application (Ix) expressed as a percentage of the control (vehicle) current (Iv). Recovery calculated as (Ir-Ix)/(Iv-Ix). Data are presented as mean ± S.E.M.; ***P < 0.001 (independent measures two-tailed t -test).

Article Snippet: Blocking buffer was removed prior to addition of primary antibody and coverslips were then treated with the primary antibodies against Kv7.1 (anti-Mouse 1:500; Santa Cruz), Kv7.2 (anti-Mouse 1:500; Santa Cruz), Kv7.3 (anti-Rabbit 1:500; Alomone), Kv7.4 (anti-Rabbit 1:200; Neuromab) or Kv7.5 (anti-Rabbit 1:500; Abcam) for 1 h 15 min (Supplemental Table II).

Techniques: Two Tailed Test

Journal: Cell Calcium

Article Title: Vascular Kv7 channels control intracellular Ca 2+ dynamics in smooth muscle

doi: 10.1016/j.ceca.2020.102283

Figure Lengend Snippet: AVP-induced Ca 2+ oscillations can be abolished by Kv7 activator, L- and T-type Ca 2+ channel blockers. (A) Representative example trace showing Ca 2+ oscillations (indicated as F340/F380 ratio units; r.u.) evoked by the physiological concentration of vasoactive hormone, vasopressin (AVP; 100 pM) in A7r5 cells. (B-D) Example traces of AVP-induced Ca 2+ transients recorded in the presence of Kv7 channel opener, retigabine (10 μM; B), L-type (nifedipine; 2 μM; C) or T-type (NNC 55-0396; 3 μM; D) Ca 2+ channel blockers (as indicated). (E) Upper panel: superimposed are Fura2 ratiometric Ca 2+ recording (black) and FuoVolt membrane potential recording (measured as ΔF/F 0 ; red) during application of AVP (100 pM) and retigabine (10 μM) during periods indicated by horizontal bars. Lower panel: Cross correlation of the normalised ratiometric Ca 2+ signal with the normalised FluoVolt signal indicated the time lag between the signals at which the peak correlation occurred. (F-H) Bar graphs summarising the effects of retigabine (F), nifedipine (G) or NNC 55-0396 (H) on the AVP-induced Ca 2+ spike frequency (spikes/s). Control is the spike frequency in the presence of AVP measured from the onset of the first spike. For quantification of drug effect spike frequency was calculated from the onset of the drug application and until the end of the application of AVP. In panels F-H data are presented as mean ± S.E.M.; n.s., not significant; **P < 0.01 (n = 5).

Article Snippet: Blocking buffer was removed prior to addition of primary antibody and coverslips were then treated with the primary antibodies against Kv7.1 (anti-Mouse 1:500; Santa Cruz), Kv7.2 (anti-Mouse 1:500; Santa Cruz), Kv7.3 (anti-Rabbit 1:500; Alomone), Kv7.4 (anti-Rabbit 1:200; Neuromab) or Kv7.5 (anti-Rabbit 1:500; Abcam) for 1 h 15 min (Supplemental Table II).

Techniques: Concentration Assay

Journal: Cell Calcium

Article Title: Vascular Kv7 channels control intracellular Ca 2+ dynamics in smooth muscle

doi: 10.1016/j.ceca.2020.102283

Figure Lengend Snippet: Ca 2+ oscillations induced by Kv7 channel inhibition are insensitive to PLC inhibition. (A) Representative example trace showing Ca 2+ oscillations (indicated as F340/F380 ratio units; r.u.) evoked by XE991 (10 μM) in A7r5 cells pretreated with PLC inhibitor, edelfosine (10 μM). (B-D) Bar graphs summarising the effects of edelfosine on the peak Ca 2+ level (B), Ca 2+ response amplitude (ΔR; C) and Ca 2+ spike frequency (spikes/s) (D) induced by XE991. In panels B-D data are presented as mean ± S.E.M.; n.s., not significant; (n = 5).

Article Snippet: Blocking buffer was removed prior to addition of primary antibody and coverslips were then treated with the primary antibodies against Kv7.1 (anti-Mouse 1:500; Santa Cruz), Kv7.2 (anti-Mouse 1:500; Santa Cruz), Kv7.3 (anti-Rabbit 1:500; Alomone), Kv7.4 (anti-Rabbit 1:200; Neuromab) or Kv7.5 (anti-Rabbit 1:500; Abcam) for 1 h 15 min (Supplemental Table II).

Techniques: Inhibition

Journal: Cell Calcium

Article Title: Vascular Kv7 channels control intracellular Ca 2+ dynamics in smooth muscle

doi: 10.1016/j.ceca.2020.102283

Figure Lengend Snippet: AVP-induced Ca 2+ transients in human IMA SMCs is reduced by Kv7 activator, L- and T-type Ca 2+ channel blockers. (A) Representative example traces showing rises in [Ca 2+ ] i (indicated as F340/F380 ratio units; r.u.) evoked by AVP (100 pM; the application period is indicated by the solid bar) in control conditions (black) or in the presence of Kv7 channel opener, retigabine (10 μM; green), L-type (nifedipine; 2 μM; blue) or T-type (NNC 55-0396; 3 μM; red) Ca 2+ channel blockers (as indicated). (B,C) Bar graphs showing the peak Ca 2+ level (B) and mean area under the curve of the response (C) to AVP. (D) Agarose gels stained with SYBR safe to visualise the RT-PCR products corresponding to L-type ( CACNA1C , CACNA1D ) and T-type ( CACNA1 G , CACNA1H , CACNA1I ) VGCCs genes. (E) Quantification of RT-PCR results exemplified in panel D, expression is normalised to that of a housekeeping gene, HPRT1 (hypoxanthine phosphoribosyltransferase 1). In panels B, C and E data are presented as mean ± S.E.M.; **P < 0.01, ***P < 0.001 (panels B,C, n≥4; panel E, n≥3).

Article Snippet: Blocking buffer was removed prior to addition of primary antibody and coverslips were then treated with the primary antibodies against Kv7.1 (anti-Mouse 1:500; Santa Cruz), Kv7.2 (anti-Mouse 1:500; Santa Cruz), Kv7.3 (anti-Rabbit 1:500; Alomone), Kv7.4 (anti-Rabbit 1:200; Neuromab) or Kv7.5 (anti-Rabbit 1:500; Abcam) for 1 h 15 min (Supplemental Table II).

Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Cell Calcium

Article Title: Vascular Kv7 channels control intracellular Ca 2+ dynamics in smooth muscle

doi: 10.1016/j.ceca.2020.102283

Figure Lengend Snippet: Functional expression of Kv7 channels in primary human IMA cells. (A) Representative example trace showing rises in [Ca 2+ ] i (indicated as F340/F380 ratio units; r.u.) evoked by XE991 (10 μM; the application period is indicated by the solid bar). (B) Comparison of Ca 2+ signals (area under the curve) induced by AVP and XE991 in IMA cells. (C) Agarose gels stained with SYBR safe to visualise the RT-PCR products of KCNQ1 , KCNQ2 , KCNQ3 , KCNQ4 and KCNQ5 . (D) Quantification of RT-PCR results exemplified in panel C, expression is normalised to that of a housekeeping gene, HPRT1 (hypoxanthine phosphoribosyltransferase 1). In panels B and D data are presented as mean ± S.E.M.; ***P < 0.001, ****P < 0.0001 (n≥4).

Article Snippet: Blocking buffer was removed prior to addition of primary antibody and coverslips were then treated with the primary antibodies against Kv7.1 (anti-Mouse 1:500; Santa Cruz), Kv7.2 (anti-Mouse 1:500; Santa Cruz), Kv7.3 (anti-Rabbit 1:500; Alomone), Kv7.4 (anti-Rabbit 1:200; Neuromab) or Kv7.5 (anti-Rabbit 1:500; Abcam) for 1 h 15 min (Supplemental Table II).

Techniques: Functional Assay, Expressing, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

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

doi: 10.1101/2021.01.21.427626

Figure Lengend Snippet: ( A, B ) Immunostaining of 1-month-old somatosensory cortex for AnkR (red) and Kv3.1b (green). Low magnification images are shown in ( A ) and high magnification images in ( B ). The genotypes analyzed are shown. Scalebars, 50 µm in ( A ) and 10 µm in ( B ). (C) Immunostaining of human cortical biopsies from two separate patients using antibodies against AnkR (red) and Kv3.1b (green), and DAPI (blue) to label nuclei (Nu). Scalebars, 10 µm. (D) Quantification of Kv3.1b immunofluorescence intensity in control and Ank1 F/F ;Dlx5/6-Cre mice. (E) Immunoblots of brain homogenates from 3 one-month-old control and 3 one-month-old AnkR-deficient brains using antibodies against Kv3.1b, AnkR, and NFM. (F) Quantification of Kv3.1b protein normalized to NFM. ( G, H ). Immunoblots of AnkR-GFP immunoprecipitations in cells co-expressing AnkR-GFP with Myc-tagged β 1 spectrin, full length Flag-tagged Kv3.1b, or truncated versions of Flag-tagged Kv3.1b. The amino acids included in the Flag-tagged Kv3.1b truncation mutants are indicated. (I) The consensus AnkR-binding motif present in Kv3.1b and Kv3.3, but not Kv3.2. (J) Immunoblots of Kv3.1b, AnkR, and Kv2.1 immunoprecipitation reactions using antibodies against AnkR and Kv3.1b. (K) Immunostaining of ventral root nodes of Ranvier in Ank3 F/+ and Ank3 F/F ;ChAT-Cre mice using antibodies against AnkG (green), Kv7.2 (red), and neurofascin (NFasc, blue) on the left, and AnkR (green), Kv3.1b (red), and NFasc (blue) on the right. Scalebars, 1μm. (L) Quantification of the percentage of nodes of Ranvier labeled for Kv7.2, Kv3.1b, AnkG, and AnkR in Ank3 F/+ and Ank3 F/F ;ChAT-Cre mice. 60-116 nodes/group. Error bars indicate mean ± SEM.

Article Snippet: The primary antibodies used here include: mouse monoclonal antibodies against AnkR (UC Davis/NIH NeuroMab Facility Cat# 75-380, RRID:AB_2491109), β 1 spectrin (UC Davis/NIH NeuroMab Facility Cat# 73-374, RRID:AB_2315814), AnkG (UC Davis/NIH NeuroMab Facility Cat# 73-146, RRID:AB_10697718), parvalbumin (UC Davis/NIH NeuroMab Facility Cat# 73-455, RRID:AB_2629420), actin (Millipore Cat# MAB1501, RRID:AB_2223041), tenascinR (R and D Systems Cat# MAB1624, RRID:AB_2207001), aggrecan (Millipore Cat# AB1031, RRID:AB_90460), brevican (UC Davis/NIH NeuroMab Facility Cat# 75-294, RRID:AB_2315824), NrCAM (R and D Systems Cat# MAB2034, RRID:AB_2267411), Kv3.1b (UC Davis/NIH NeuroMab Facility Cat# N16B/8, RRID:AB_2750730 and Thermo Fisher Cat# MA5-27684, RRID:AB_2735238), Kv3.3 (Antibodies-Online Cat# ABIN572016, RRID:AB_10782137), Kv7.2 (James Trimmer, University of California at Davis Cat# N26A/23, RRID:AB_2750761), Flag-tag or DDDDK-tag (MBL International Cat# M185-3L, RRID:AB_11123930); 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), versican (Millipore Cat# AB1032, RRID:AB_11213831), PlexinA4 (Abcam Cat# ab39350, RRID:AB_944890), and neuropilin-1 (GeneTex Cat# GTX16786, RRID:AB_422398), 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); and chicken polyclonal antibody against Neurofascin (R and D Systems Cat# AF3235, RRID:AB_10890736).

Techniques: Immunostaining, Immunofluorescence, Western Blot, Expressing, Binding Assay, Immunoprecipitation, Labeling

Journal: Molecular Pain

Article Title: Accumulation of Kv7.2 channels in putative ectopic transduction zones of mice nerve-end neuromas

doi: 10.1186/1744-8069-7-58

Figure Lengend Snippet: Antibodies, dilutions and sources

Article Snippet: Kv7.2 , aa 578-593 at the C-terminal domain , Sigma, rabbit, polyclonal , 1:200.

Techniques:

Journal: Molecular Pain

Article Title: Accumulation of Kv7.2 channels in putative ectopic transduction zones of mice nerve-end neuromas

doi: 10.1186/1744-8069-7-58

Figure Lengend Snippet: Expression of Nav and Kv7.2 channels in nodes of Ranvier of intact fibres. A and B show teased fibers form normal saphenous nerves immunostained for Myelin P2 and Pan Nav ( A ) or Kv7.2 ( B ). In this and following figures, color codes for antibodies are specified on images. Note that the fibers are well structured and nodes are easily identifiable. The following figures show high magnification images of nodal Nav channels flanked by depositions of Myelin P2 ( C 1 ), Caspr ( D ), Pan NF ( E ). C 2 shows the profile of immunofluorescence intensity along the nodal/paranodal zone shown in C 1 (in this and following figures intensity curves are given in arbitrary units). F and G show nodal zones costained for Kv7.2 and Myelin P2 ( F 1 ) or Caspr ( G ). F 2 shows the profile of immunofluorescence intensity along the nodal/paranodal zone shown in F 1 . Scale bars: for A and B , 20 μm (shown in B ); for C 1 , D, F 1 and G , 5 μm (shown in G ); for E , 5 μm.

Article Snippet: Kv7.2 , aa 578-593 at the C-terminal domain , Sigma, rabbit, polyclonal , 1:200.

Techniques: Expressing, Immunofluorescence

Journal: Molecular Pain

Article Title: Accumulation of Kv7.2 channels in putative ectopic transduction zones of mice nerve-end neuromas

doi: 10.1186/1744-8069-7-58

Figure Lengend Snippet: Measurements on nodes of Ranvier

Article Snippet: Kv7.2 , aa 578-593 at the C-terminal domain , Sigma, rabbit, polyclonal , 1:200.

Techniques:

Journal: Molecular Pain

Article Title: Accumulation of Kv7.2 channels in putative ectopic transduction zones of mice nerve-end neuromas

doi: 10.1186/1744-8069-7-58

Figure Lengend Snippet: Increase in Nav and Kv7.2 co-expression after axotomy. A shows low magnification fields containing nodal accumulations of Nav ( A 1 ) and Kv7.2 ( A 2 ) channels in control nerve. A 3 shows a merge of A1 and A2 showing co-staining of some nodes (marked by arrows). B and C follow the same scheme as A but fields were obtained from the proximal end ( B ) or the distal end of the neuroma ( C ). Note the large proportion of co-staining in neuromatose fibers. D 1 shows a larger scale picture of a long nodal formation which corresponds to the area in the red square of B 3 . D 2 shows the immunostaining intensity profile corresponding to the nodal zone marked with an asterisk in D 1 . Scale bars: for A and B 10 μm (shown in B 3 ); for C , 10 μm (shown in C 3 ); for D 2 , 5 μm.

Article Snippet: Kv7.2 , aa 578-593 at the C-terminal domain , Sigma, rabbit, polyclonal , 1:200.

Techniques: Expressing, Staining, Immunostaining

Journal: Molecular Pain

Article Title: Accumulation of Kv7.2 channels in putative ectopic transduction zones of mice nerve-end neuromas

doi: 10.1186/1744-8069-7-58

Figure Lengend Snippet: Abnormal expression of Nav and Kv7.2 channels in distal neuromas. A and B are medium scale magnification fields obtained from distal areas of the neuroma. In both instances there is a clear lack of structure and myelin loss. In addition, large accumulations of Nav and Kv7.2 channels are shown in A and B respectively. C and D contain field images of transition areas in which normal nodes (marked by arrows) are detected close to larger accumulations of Nav ( C ) or Kv7.2 ( D ). E shows the co-staining of Nav and Kv7.2 in a dotted line-like structure, probably an indication of a demyelination/remyelination process within the same fiber. F 1 shows several individual nodes at a high magnification (contained in red square in E 3 ). The corresponding immnofluoresce intensity profiles are shown in F 2 . Scale bars: for A and B , 20 μm (shown in B) ; C and D , 5 μm (shown in D ); for E , 5 μm (shown in E 3 ) and for F 1 , 5 μm.

Article Snippet: Kv7.2 , aa 578-593 at the C-terminal domain , Sigma, rabbit, polyclonal , 1:200.

Techniques: Expressing, Staining