Structured Review

Antibodies Inc mouse monoclonal anti kv7 2 primary antibodies
Functional characterization of heteromeric <t>Kv7.2</t> + Kv7.3 channels incorporating Kv7.2 R325G subunits. ( a ) Macroscopic currents recorded in response to the indicated voltage protocol (cDNA transfection ratios are in parenthesis). Current scale: 500 pA; time scale: 200 ms. ( b,c ) Quantification of retigabine (RTG)-induced effects on activation V ½ ( b ) and current density at 0 mV ( c ). Asterisks indicate values significantly different (p
Mouse Monoclonal Anti Kv7 2 Primary Antibodies, supplied by Antibodies Inc, used in various techniques. Bioz Stars score: 80/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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80/100 stars

Images

1) Product Images from "Early-onset epileptic encephalopathy caused by a reduced sensitivity of Kv7.2 potassium channels to phosphatidylinositol 4,5-bisphosphate"

Article Title: Early-onset epileptic encephalopathy caused by a reduced sensitivity of Kv7.2 potassium channels to phosphatidylinositol 4,5-bisphosphate

Journal: Scientific Reports

doi: 10.1038/srep38167

Functional characterization of heteromeric Kv7.2 + Kv7.3 channels incorporating Kv7.2 R325G subunits. ( a ) Macroscopic currents recorded in response to the indicated voltage protocol (cDNA transfection ratios are in parenthesis). Current scale: 500 pA; time scale: 200 ms. ( b,c ) Quantification of retigabine (RTG)-induced effects on activation V ½ ( b ) and current density at 0 mV ( c ). Asterisks indicate values significantly different (p
Figure Legend Snippet: Functional characterization of heteromeric Kv7.2 + Kv7.3 channels incorporating Kv7.2 R325G subunits. ( a ) Macroscopic currents recorded in response to the indicated voltage protocol (cDNA transfection ratios are in parenthesis). Current scale: 500 pA; time scale: 200 ms. ( b,c ) Quantification of retigabine (RTG)-induced effects on activation V ½ ( b ) and current density at 0 mV ( c ). Asterisks indicate values significantly different (p

Techniques Used: Functional Assay, Transfection, Mass Spectrometry, Activation Assay

TEA-sensitivity of heteromeric channels carrying Kv7.2 R325G subunits. ( a , b , c ) Representative current responses to voltage ramps from −80 mV to 0 mV from the indicated channels recorded in control solution C, upon perfusion for 2 min with 30 mM ( a , b ) or 3 mM ( c ) TEA, and upon drug washout (W). Current scale: 50 pA; time scale: 200 ms. ( d ) Average data from experiments such as those shown in panels a (white bars), b (light grey bars), or c (dark grey bars). Asterisks indicate values significantly different from respective controls (leftmost bar in each group; p
Figure Legend Snippet: TEA-sensitivity of heteromeric channels carrying Kv7.2 R325G subunits. ( a , b , c ) Representative current responses to voltage ramps from −80 mV to 0 mV from the indicated channels recorded in control solution C, upon perfusion for 2 min with 30 mM ( a , b ) or 3 mM ( c ) TEA, and upon drug washout (W). Current scale: 50 pA; time scale: 200 ms. ( d ) Average data from experiments such as those shown in panels a (white bars), b (light grey bars), or c (dark grey bars). Asterisks indicate values significantly different from respective controls (leftmost bar in each group; p

Techniques Used: Mass Spectrometry

Effect of PIP5K, CaM, and CaM 1234 on homomeric Kv7.2 and Kv7.2 R325G channels. ( a ) Macroscopic currents recorded in response to the voltage protocol shown, in the absence (left) or presence (right) of PIP5K. Current scale: 500 pA (upper panels) or 50 pA (lower panels); time scale: 200 ms. ( b ) Current densities (0 mV) from cells expressing Kv7.2 or Kv7.2 R325G channels alone or in combination with PIP5K, CaM or CaM 1234 , as indicated. In the last row, the number of experiments (n), each from a separate cell, is indicated. ( c ) Effect of retigabine (RTG, 10 μM) on activation gating (V ½ ) for the indicated channels. In all panels, asterisks indicate values significantly different (p
Figure Legend Snippet: Effect of PIP5K, CaM, and CaM 1234 on homomeric Kv7.2 and Kv7.2 R325G channels. ( a ) Macroscopic currents recorded in response to the voltage protocol shown, in the absence (left) or presence (right) of PIP5K. Current scale: 500 pA (upper panels) or 50 pA (lower panels); time scale: 200 ms. ( b ) Current densities (0 mV) from cells expressing Kv7.2 or Kv7.2 R325G channels alone or in combination with PIP5K, CaM or CaM 1234 , as indicated. In the last row, the number of experiments (n), each from a separate cell, is indicated. ( c ) Effect of retigabine (RTG, 10 μM) on activation gating (V ½ ) for the indicated channels. In all panels, asterisks indicate values significantly different (p

Techniques Used: Chick Chorioallantoic Membrane Assay, Mass Spectrometry, Expressing, Activation Assay

Location of the R325 residue within a PIP 2 -binding pocket in a Kv7.2 subunit. ( a ) Partial primary sequence alignment (from the end of S 6 to the beginning of the A helix) among Kv7 subunits. ( b ) A single PIP 2 molecule docked onto a Kv7.2 subunit. Kv7.2 α-carbon traces are in grey; the PIP 2 molecule is shown as sticks, and colored according to atom type (carbon, green; phosphorous, orange; oxygen, red). The CaM molecule is in purple; Ca 2+ ions are in yellow. Dashed yellow lines indicate Kv7.2/PIP 2 interactions occurring at distances
Figure Legend Snippet: Location of the R325 residue within a PIP 2 -binding pocket in a Kv7.2 subunit. ( a ) Partial primary sequence alignment (from the end of S 6 to the beginning of the A helix) among Kv7 subunits. ( b ) A single PIP 2 molecule docked onto a Kv7.2 subunit. Kv7.2 α-carbon traces are in grey; the PIP 2 molecule is shown as sticks, and colored according to atom type (carbon, green; phosphorous, orange; oxygen, red). The CaM molecule is in purple; Ca 2+ ions are in yellow. Dashed yellow lines indicate Kv7.2/PIP 2 interactions occurring at distances

Techniques Used: Binding Assay, Sequencing, Chick Chorioallantoic Membrane Assay

Effect of VSP on Kv7.2 + Kv7.3 and Kv7.2 + Kv7.2 R325G + Kv7.3 currents. ( a ) Currents recorded in response to the indicated voltage protocol in cells expressing Kv7.2 + Kv7.3 (control), Kv7.2 + Kv7.3 + VSP, or Kv7.2 + Kv7.3 + VSP + PIP5K, as indicated. ( b ) Currents recorded in response to the indicated voltage protocol in cells expressing VSP and Kv7.2 + Kv7.3 (black trace) or Kv7.2 + Kv7.2 R325G + Kv7.3 (gray trace) channels. ( c,d ) Time-dependence of current decrease ( c ) and recovery ( d ) in cells co-expressing the indicated channels and VSP, in the absence or in the presence of PIP5K. VSP-dependent current inhibition ( c ) was expressed as the ratio between the current values recorded at 0 mV immediately after and before the +100 mV step. Recovery ( d ) was expressed as the ratio between currents measured every second at the end and before the +100 mV depolarizing pulse. For the data shown in panels c and d, the number of experiments (n) is 11 for Kv7.2 + Kv7.3 + VSP, 11 for Kv7.2 + Kv7.3 + VSP + PIP5K, 12 for Kv7.2 + Kv7.2 R325G + Kv7.3 + VSP, and 12 for Kv7.2 + Kv7.2 R325G + Kv7.3 + VSP + PIP5K.
Figure Legend Snippet: Effect of VSP on Kv7.2 + Kv7.3 and Kv7.2 + Kv7.2 R325G + Kv7.3 currents. ( a ) Currents recorded in response to the indicated voltage protocol in cells expressing Kv7.2 + Kv7.3 (control), Kv7.2 + Kv7.3 + VSP, or Kv7.2 + Kv7.3 + VSP + PIP5K, as indicated. ( b ) Currents recorded in response to the indicated voltage protocol in cells expressing VSP and Kv7.2 + Kv7.3 (black trace) or Kv7.2 + Kv7.2 R325G + Kv7.3 (gray trace) channels. ( c,d ) Time-dependence of current decrease ( c ) and recovery ( d ) in cells co-expressing the indicated channels and VSP, in the absence or in the presence of PIP5K. VSP-dependent current inhibition ( c ) was expressed as the ratio between the current values recorded at 0 mV immediately after and before the +100 mV step. Recovery ( d ) was expressed as the ratio between currents measured every second at the end and before the +100 mV depolarizing pulse. For the data shown in panels c and d, the number of experiments (n) is 11 for Kv7.2 + Kv7.3 + VSP, 11 for Kv7.2 + Kv7.3 + VSP + PIP5K, 12 for Kv7.2 + Kv7.2 R325G + Kv7.3 + VSP, and 12 for Kv7.2 + Kv7.2 R325G + Kv7.3 + VSP + PIP5K.

Techniques Used: Expressing, Inhibition

Functional and biochemical characterization of Kv7.2 R325G subunits. ( a ) Macroscopic currents from CHO cells in response to the voltage protocol shown; NT: non-transfected cells. Current scale: 200 pA; time scale: 200 ms. ( b ) Western blot analysis of proteins from total lysates (left) or biotinylated plasma membrane fractions (right) from CHO cells transfected with the indicated constructs. Higher and lower blots were probed with anti-Kv7.2 or anti-α-tubulin antibodies, as indicated. Numbers on the left correspond to the molecular masses of the protein markers. For clarity, the images shown are cropped from full-length gels; these are shown as Supplementary Information ( Supplementary Fig. 2 ). ( c ) Current densities (0 mV) in cells co-expressing Kv7.2 R325G subunits and Kv7.2 (n = 11 and 20 in the absence and in the presence of Kv7.2 R325G subunits, respectively), Kv7.3 (n = 15 and 13 in the absence and in the presence of Kv7.2 R325G subunits, respectively), or Kv7.3* (n = 18 and 17 in the absence and in the presence of Kv7.2 R325G subunits, respectively) subunits. Asterisks indicate values significantly different (p
Figure Legend Snippet: Functional and biochemical characterization of Kv7.2 R325G subunits. ( a ) Macroscopic currents from CHO cells in response to the voltage protocol shown; NT: non-transfected cells. Current scale: 200 pA; time scale: 200 ms. ( b ) Western blot analysis of proteins from total lysates (left) or biotinylated plasma membrane fractions (right) from CHO cells transfected with the indicated constructs. Higher and lower blots were probed with anti-Kv7.2 or anti-α-tubulin antibodies, as indicated. Numbers on the left correspond to the molecular masses of the protein markers. For clarity, the images shown are cropped from full-length gels; these are shown as Supplementary Information ( Supplementary Fig. 2 ). ( c ) Current densities (0 mV) in cells co-expressing Kv7.2 R325G subunits and Kv7.2 (n = 11 and 20 in the absence and in the presence of Kv7.2 R325G subunits, respectively), Kv7.3 (n = 15 and 13 in the absence and in the presence of Kv7.2 R325G subunits, respectively), or Kv7.3* (n = 18 and 17 in the absence and in the presence of Kv7.2 R325G subunits, respectively) subunits. Asterisks indicate values significantly different (p

Techniques Used: Functional Assay, Transfection, Mass Spectrometry, Western Blot, Construct, Expressing

AIS localization of Kv7.2 R325G subunits. ( a ) Representative images of primary rat hippocampal neurons transfected with Kv7.3 and the EGFP-Kv7.2-HA-, EGFP-Kv7.2 R325G-HA-, or EGFP-Kv7.2 A294V-HA-expressing plasmids revealed by anti-HA (red; before permeabilization) or anti-ank-G (blue; after permeabilization) antibodies. In green is the EGFP fluorescence. Lower panels are magnifications of the boxed regions. ( b ) Quantification of the intensity (expressed as arbitrary units, A.U.) of the HA (red) and Ank-G (blue) fluorescence signals for Kv7.2 (n = 20), Kv7.2 R325G (n = 18), and Kv7.2 A294V (n = 15) subunits, measured on a 40 μm-long axonal region starting from the soma, as described in Methods . ( c ) Quantification of AIS/Soma and AIS/Dendrite fluorescence ratios for Kv7.2, Kv7.2 R325G, and Kv7.2 A294V subunits, calculated as described in Methods . Asterisks indicate values significantly different (p
Figure Legend Snippet: AIS localization of Kv7.2 R325G subunits. ( a ) Representative images of primary rat hippocampal neurons transfected with Kv7.3 and the EGFP-Kv7.2-HA-, EGFP-Kv7.2 R325G-HA-, or EGFP-Kv7.2 A294V-HA-expressing plasmids revealed by anti-HA (red; before permeabilization) or anti-ank-G (blue; after permeabilization) antibodies. In green is the EGFP fluorescence. Lower panels are magnifications of the boxed regions. ( b ) Quantification of the intensity (expressed as arbitrary units, A.U.) of the HA (red) and Ank-G (blue) fluorescence signals for Kv7.2 (n = 20), Kv7.2 R325G (n = 18), and Kv7.2 A294V (n = 15) subunits, measured on a 40 μm-long axonal region starting from the soma, as described in Methods . ( c ) Quantification of AIS/Soma and AIS/Dendrite fluorescence ratios for Kv7.2, Kv7.2 R325G, and Kv7.2 A294V subunits, calculated as described in Methods . Asterisks indicate values significantly different (p

Techniques Used: Transfection, Expressing, Fluorescence

2) Product Images from "Functional Characterization of Two Variants at the Intron 6—Exon 7 Boundary of the KCNQ2 Potassium Channel Gene Causing Distinct Epileptic Phenotypes"

Article Title: Functional Characterization of Two Variants at the Intron 6—Exon 7 Boundary of the KCNQ2 Potassium Channel Gene Causing Distinct Epileptic Phenotypes

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2022.872645

Functional and biochemical characterization of Kv7.2 G310S and Kv7.2 G310Δ10 subunits. (A) Maximal current densities measured in non transfected cells (NT) or in cells expressing wild-type or mutant Kv7.2 subunits, as indicated. * = p
Figure Legend Snippet: Functional and biochemical characterization of Kv7.2 G310S and Kv7.2 G310Δ10 subunits. (A) Maximal current densities measured in non transfected cells (NT) or in cells expressing wild-type or mutant Kv7.2 subunits, as indicated. * = p

Techniques Used: Functional Assay, Transfection, Expressing, Mutagenesis

Localization of KCNQ2 variants herein investigated and effects on KCNQ2 mRNAs expression. (A) Localization of the two variants herein investigated in the KCNQ2 gene, falling at the last nucleotide of intron 6 (KCNQ2 (C) 928-1G > C; blue circle; already reported in Soldovieri et al., 2014 ) or at the first nucleotide of exon 7 (KCNQ2 (C) 928G > (A) ; already reported in Soldovieri et al., 2020 ). (B) Representative agarose gel run and (C) electrophoresis profiles of major bands obtained from RT-PCR experiments performed on Kv7.2 mRNAs expressed in father- or probands-deriving lymphoblasts, as indicated; the mRNA fragment of 295 bp (B) or 218 bp (C) are expected when the ag acceptor site at the end of intron 6 is used, while those of 265 bp (B) or 188 bp (C) bands are obtained using the alternative splice site in the exon 7. Panels in C have been obtained cropping the regions of interest from the complete electrophoresis profiles, reported in Supplementary Figure S2 Topology of a Kv7.2 subunit showing the 6 transmembrane segments (called from S1 to S6) and four intracellular domains at the C-terminus (called from A to D), binding many regulatory molecules. Middle and left panels show the structural alterations prompted by the Kv7.2 G310Δ10 in-frame deletion or by the missense Kv7.2 G310S variant, respectively.
Figure Legend Snippet: Localization of KCNQ2 variants herein investigated and effects on KCNQ2 mRNAs expression. (A) Localization of the two variants herein investigated in the KCNQ2 gene, falling at the last nucleotide of intron 6 (KCNQ2 (C) 928-1G > C; blue circle; already reported in Soldovieri et al., 2014 ) or at the first nucleotide of exon 7 (KCNQ2 (C) 928G > (A) ; already reported in Soldovieri et al., 2020 ). (B) Representative agarose gel run and (C) electrophoresis profiles of major bands obtained from RT-PCR experiments performed on Kv7.2 mRNAs expressed in father- or probands-deriving lymphoblasts, as indicated; the mRNA fragment of 295 bp (B) or 218 bp (C) are expected when the ag acceptor site at the end of intron 6 is used, while those of 265 bp (B) or 188 bp (C) bands are obtained using the alternative splice site in the exon 7. Panels in C have been obtained cropping the regions of interest from the complete electrophoresis profiles, reported in Supplementary Figure S2 Topology of a Kv7.2 subunit showing the 6 transmembrane segments (called from S1 to S6) and four intracellular domains at the C-terminus (called from A to D), binding many regulatory molecules. Middle and left panels show the structural alterations prompted by the Kv7.2 G310Δ10 in-frame deletion or by the missense Kv7.2 G310S variant, respectively.

Techniques Used: Expressing, Agarose Gel Electrophoresis, Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Variant Assay

Functional characterization of heteromeric Kv7.2 + Kv7.3 channels incorporating Kv7.2 G310S or Kv7.2 G310Δ10 subunits expressed in neurons. (A) Macroscopic currents from neurons expressing wild-type or mutant Kv7.2 + Kv7.3 subunits, as indicated. (B,C) Current density/voltage curves measured in non transfected neurons (NT) or expressing the indicated channels incorporating Kv7.2 G310Δ10 (B) or Kv7.2 G310S (C) subunits, as indicated.
Figure Legend Snippet: Functional characterization of heteromeric Kv7.2 + Kv7.3 channels incorporating Kv7.2 G310S or Kv7.2 G310Δ10 subunits expressed in neurons. (A) Macroscopic currents from neurons expressing wild-type or mutant Kv7.2 + Kv7.3 subunits, as indicated. (B,C) Current density/voltage curves measured in non transfected neurons (NT) or expressing the indicated channels incorporating Kv7.2 G310Δ10 (B) or Kv7.2 G310S (C) subunits, as indicated.

Techniques Used: Functional Assay, Expressing, Mutagenesis, Transfection

Functional study of the modulation of Kv7.2 G310S or Kv7.2 G310Δ10 subunits by PIP 2 and calmodulin. (A,B) Representative traces (A) and quantification (B) of currents measured in CHO cells co-expressing homomeric wild-type or mutant Kv7.2 subunits with PIP5K, CaM, and/or CaM 1234 , as indicated. * = p
Figure Legend Snippet: Functional study of the modulation of Kv7.2 G310S or Kv7.2 G310Δ10 subunits by PIP 2 and calmodulin. (A,B) Representative traces (A) and quantification (B) of currents measured in CHO cells co-expressing homomeric wild-type or mutant Kv7.2 subunits with PIP5K, CaM, and/or CaM 1234 , as indicated. * = p

Techniques Used: Functional Assay, Expressing, Mutagenesis, Chick Chorioallantoic Membrane Assay

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    Antibodies Inc mouse monoclonal anti kv7 2 primary antibodies
    Functional characterization of heteromeric <t>Kv7.2</t> + Kv7.3 channels incorporating Kv7.2 R325G subunits. ( a ) Macroscopic currents recorded in response to the indicated voltage protocol (cDNA transfection ratios are in parenthesis). Current scale: 500 pA; time scale: 200 ms. ( b,c ) Quantification of retigabine (RTG)-induced effects on activation V ½ ( b ) and current density at 0 mV ( c ). Asterisks indicate values significantly different (p
    Mouse Monoclonal Anti Kv7 2 Primary Antibodies, supplied by Antibodies Inc, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse monoclonal anti kv7 2 primary antibodies/product/Antibodies Inc
    Average 80 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse monoclonal anti kv7 2 primary antibodies - by Bioz Stars, 2022-09
    80/100 stars
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    Functional characterization of heteromeric Kv7.2 + Kv7.3 channels incorporating Kv7.2 R325G subunits. ( a ) Macroscopic currents recorded in response to the indicated voltage protocol (cDNA transfection ratios are in parenthesis). Current scale: 500 pA; time scale: 200 ms. ( b,c ) Quantification of retigabine (RTG)-induced effects on activation V ½ ( b ) and current density at 0 mV ( c ). Asterisks indicate values significantly different (p

    Journal: Scientific Reports

    Article Title: Early-onset epileptic encephalopathy caused by a reduced sensitivity of Kv7.2 potassium channels to phosphatidylinositol 4,5-bisphosphate

    doi: 10.1038/srep38167

    Figure Lengend Snippet: Functional characterization of heteromeric Kv7.2 + Kv7.3 channels incorporating Kv7.2 R325G subunits. ( a ) Macroscopic currents recorded in response to the indicated voltage protocol (cDNA transfection ratios are in parenthesis). Current scale: 500 pA; time scale: 200 ms. ( b,c ) Quantification of retigabine (RTG)-induced effects on activation V ½ ( b ) and current density at 0 mV ( c ). Asterisks indicate values significantly different (p

    Article Snippet: Channel subunits were identified using mouse monoclonal anti-Kv7.2 primary antibodies (clone N26A/23, dilution 1:1000; Antibodies Inc., Davis, CA), followed by horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibodies (clone NA931V; dilution 1:5,000; GE Healthcare, Little Chalfont, UK).

    Techniques: Functional Assay, Transfection, Mass Spectrometry, Activation Assay

    TEA-sensitivity of heteromeric channels carrying Kv7.2 R325G subunits. ( a , b , c ) Representative current responses to voltage ramps from −80 mV to 0 mV from the indicated channels recorded in control solution C, upon perfusion for 2 min with 30 mM ( a , b ) or 3 mM ( c ) TEA, and upon drug washout (W). Current scale: 50 pA; time scale: 200 ms. ( d ) Average data from experiments such as those shown in panels a (white bars), b (light grey bars), or c (dark grey bars). Asterisks indicate values significantly different from respective controls (leftmost bar in each group; p

    Journal: Scientific Reports

    Article Title: Early-onset epileptic encephalopathy caused by a reduced sensitivity of Kv7.2 potassium channels to phosphatidylinositol 4,5-bisphosphate

    doi: 10.1038/srep38167

    Figure Lengend Snippet: TEA-sensitivity of heteromeric channels carrying Kv7.2 R325G subunits. ( a , b , c ) Representative current responses to voltage ramps from −80 mV to 0 mV from the indicated channels recorded in control solution C, upon perfusion for 2 min with 30 mM ( a , b ) or 3 mM ( c ) TEA, and upon drug washout (W). Current scale: 50 pA; time scale: 200 ms. ( d ) Average data from experiments such as those shown in panels a (white bars), b (light grey bars), or c (dark grey bars). Asterisks indicate values significantly different from respective controls (leftmost bar in each group; p

    Article Snippet: Channel subunits were identified using mouse monoclonal anti-Kv7.2 primary antibodies (clone N26A/23, dilution 1:1000; Antibodies Inc., Davis, CA), followed by horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibodies (clone NA931V; dilution 1:5,000; GE Healthcare, Little Chalfont, UK).

    Techniques: Mass Spectrometry

    Effect of PIP5K, CaM, and CaM 1234 on homomeric Kv7.2 and Kv7.2 R325G channels. ( a ) Macroscopic currents recorded in response to the voltage protocol shown, in the absence (left) or presence (right) of PIP5K. Current scale: 500 pA (upper panels) or 50 pA (lower panels); time scale: 200 ms. ( b ) Current densities (0 mV) from cells expressing Kv7.2 or Kv7.2 R325G channels alone or in combination with PIP5K, CaM or CaM 1234 , as indicated. In the last row, the number of experiments (n), each from a separate cell, is indicated. ( c ) Effect of retigabine (RTG, 10 μM) on activation gating (V ½ ) for the indicated channels. In all panels, asterisks indicate values significantly different (p

    Journal: Scientific Reports

    Article Title: Early-onset epileptic encephalopathy caused by a reduced sensitivity of Kv7.2 potassium channels to phosphatidylinositol 4,5-bisphosphate

    doi: 10.1038/srep38167

    Figure Lengend Snippet: Effect of PIP5K, CaM, and CaM 1234 on homomeric Kv7.2 and Kv7.2 R325G channels. ( a ) Macroscopic currents recorded in response to the voltage protocol shown, in the absence (left) or presence (right) of PIP5K. Current scale: 500 pA (upper panels) or 50 pA (lower panels); time scale: 200 ms. ( b ) Current densities (0 mV) from cells expressing Kv7.2 or Kv7.2 R325G channels alone or in combination with PIP5K, CaM or CaM 1234 , as indicated. In the last row, the number of experiments (n), each from a separate cell, is indicated. ( c ) Effect of retigabine (RTG, 10 μM) on activation gating (V ½ ) for the indicated channels. In all panels, asterisks indicate values significantly different (p

    Article Snippet: Channel subunits were identified using mouse monoclonal anti-Kv7.2 primary antibodies (clone N26A/23, dilution 1:1000; Antibodies Inc., Davis, CA), followed by horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibodies (clone NA931V; dilution 1:5,000; GE Healthcare, Little Chalfont, UK).

    Techniques: Chick Chorioallantoic Membrane Assay, Mass Spectrometry, Expressing, Activation Assay

    Location of the R325 residue within a PIP 2 -binding pocket in a Kv7.2 subunit. ( a ) Partial primary sequence alignment (from the end of S 6 to the beginning of the A helix) among Kv7 subunits. ( b ) A single PIP 2 molecule docked onto a Kv7.2 subunit. Kv7.2 α-carbon traces are in grey; the PIP 2 molecule is shown as sticks, and colored according to atom type (carbon, green; phosphorous, orange; oxygen, red). The CaM molecule is in purple; Ca 2+ ions are in yellow. Dashed yellow lines indicate Kv7.2/PIP 2 interactions occurring at distances

    Journal: Scientific Reports

    Article Title: Early-onset epileptic encephalopathy caused by a reduced sensitivity of Kv7.2 potassium channels to phosphatidylinositol 4,5-bisphosphate

    doi: 10.1038/srep38167

    Figure Lengend Snippet: Location of the R325 residue within a PIP 2 -binding pocket in a Kv7.2 subunit. ( a ) Partial primary sequence alignment (from the end of S 6 to the beginning of the A helix) among Kv7 subunits. ( b ) A single PIP 2 molecule docked onto a Kv7.2 subunit. Kv7.2 α-carbon traces are in grey; the PIP 2 molecule is shown as sticks, and colored according to atom type (carbon, green; phosphorous, orange; oxygen, red). The CaM molecule is in purple; Ca 2+ ions are in yellow. Dashed yellow lines indicate Kv7.2/PIP 2 interactions occurring at distances

    Article Snippet: Channel subunits were identified using mouse monoclonal anti-Kv7.2 primary antibodies (clone N26A/23, dilution 1:1000; Antibodies Inc., Davis, CA), followed by horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibodies (clone NA931V; dilution 1:5,000; GE Healthcare, Little Chalfont, UK).

    Techniques: Binding Assay, Sequencing, Chick Chorioallantoic Membrane Assay

    Effect of VSP on Kv7.2 + Kv7.3 and Kv7.2 + Kv7.2 R325G + Kv7.3 currents. ( a ) Currents recorded in response to the indicated voltage protocol in cells expressing Kv7.2 + Kv7.3 (control), Kv7.2 + Kv7.3 + VSP, or Kv7.2 + Kv7.3 + VSP + PIP5K, as indicated. ( b ) Currents recorded in response to the indicated voltage protocol in cells expressing VSP and Kv7.2 + Kv7.3 (black trace) or Kv7.2 + Kv7.2 R325G + Kv7.3 (gray trace) channels. ( c,d ) Time-dependence of current decrease ( c ) and recovery ( d ) in cells co-expressing the indicated channels and VSP, in the absence or in the presence of PIP5K. VSP-dependent current inhibition ( c ) was expressed as the ratio between the current values recorded at 0 mV immediately after and before the +100 mV step. Recovery ( d ) was expressed as the ratio between currents measured every second at the end and before the +100 mV depolarizing pulse. For the data shown in panels c and d, the number of experiments (n) is 11 for Kv7.2 + Kv7.3 + VSP, 11 for Kv7.2 + Kv7.3 + VSP + PIP5K, 12 for Kv7.2 + Kv7.2 R325G + Kv7.3 + VSP, and 12 for Kv7.2 + Kv7.2 R325G + Kv7.3 + VSP + PIP5K.

    Journal: Scientific Reports

    Article Title: Early-onset epileptic encephalopathy caused by a reduced sensitivity of Kv7.2 potassium channels to phosphatidylinositol 4,5-bisphosphate

    doi: 10.1038/srep38167

    Figure Lengend Snippet: Effect of VSP on Kv7.2 + Kv7.3 and Kv7.2 + Kv7.2 R325G + Kv7.3 currents. ( a ) Currents recorded in response to the indicated voltage protocol in cells expressing Kv7.2 + Kv7.3 (control), Kv7.2 + Kv7.3 + VSP, or Kv7.2 + Kv7.3 + VSP + PIP5K, as indicated. ( b ) Currents recorded in response to the indicated voltage protocol in cells expressing VSP and Kv7.2 + Kv7.3 (black trace) or Kv7.2 + Kv7.2 R325G + Kv7.3 (gray trace) channels. ( c,d ) Time-dependence of current decrease ( c ) and recovery ( d ) in cells co-expressing the indicated channels and VSP, in the absence or in the presence of PIP5K. VSP-dependent current inhibition ( c ) was expressed as the ratio between the current values recorded at 0 mV immediately after and before the +100 mV step. Recovery ( d ) was expressed as the ratio between currents measured every second at the end and before the +100 mV depolarizing pulse. For the data shown in panels c and d, the number of experiments (n) is 11 for Kv7.2 + Kv7.3 + VSP, 11 for Kv7.2 + Kv7.3 + VSP + PIP5K, 12 for Kv7.2 + Kv7.2 R325G + Kv7.3 + VSP, and 12 for Kv7.2 + Kv7.2 R325G + Kv7.3 + VSP + PIP5K.

    Article Snippet: Channel subunits were identified using mouse monoclonal anti-Kv7.2 primary antibodies (clone N26A/23, dilution 1:1000; Antibodies Inc., Davis, CA), followed by horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibodies (clone NA931V; dilution 1:5,000; GE Healthcare, Little Chalfont, UK).

    Techniques: Expressing, Inhibition

    Functional and biochemical characterization of Kv7.2 R325G subunits. ( a ) Macroscopic currents from CHO cells in response to the voltage protocol shown; NT: non-transfected cells. Current scale: 200 pA; time scale: 200 ms. ( b ) Western blot analysis of proteins from total lysates (left) or biotinylated plasma membrane fractions (right) from CHO cells transfected with the indicated constructs. Higher and lower blots were probed with anti-Kv7.2 or anti-α-tubulin antibodies, as indicated. Numbers on the left correspond to the molecular masses of the protein markers. For clarity, the images shown are cropped from full-length gels; these are shown as Supplementary Information ( Supplementary Fig. 2 ). ( c ) Current densities (0 mV) in cells co-expressing Kv7.2 R325G subunits and Kv7.2 (n = 11 and 20 in the absence and in the presence of Kv7.2 R325G subunits, respectively), Kv7.3 (n = 15 and 13 in the absence and in the presence of Kv7.2 R325G subunits, respectively), or Kv7.3* (n = 18 and 17 in the absence and in the presence of Kv7.2 R325G subunits, respectively) subunits. Asterisks indicate values significantly different (p

    Journal: Scientific Reports

    Article Title: Early-onset epileptic encephalopathy caused by a reduced sensitivity of Kv7.2 potassium channels to phosphatidylinositol 4,5-bisphosphate

    doi: 10.1038/srep38167

    Figure Lengend Snippet: Functional and biochemical characterization of Kv7.2 R325G subunits. ( a ) Macroscopic currents from CHO cells in response to the voltage protocol shown; NT: non-transfected cells. Current scale: 200 pA; time scale: 200 ms. ( b ) Western blot analysis of proteins from total lysates (left) or biotinylated plasma membrane fractions (right) from CHO cells transfected with the indicated constructs. Higher and lower blots were probed with anti-Kv7.2 or anti-α-tubulin antibodies, as indicated. Numbers on the left correspond to the molecular masses of the protein markers. For clarity, the images shown are cropped from full-length gels; these are shown as Supplementary Information ( Supplementary Fig. 2 ). ( c ) Current densities (0 mV) in cells co-expressing Kv7.2 R325G subunits and Kv7.2 (n = 11 and 20 in the absence and in the presence of Kv7.2 R325G subunits, respectively), Kv7.3 (n = 15 and 13 in the absence and in the presence of Kv7.2 R325G subunits, respectively), or Kv7.3* (n = 18 and 17 in the absence and in the presence of Kv7.2 R325G subunits, respectively) subunits. Asterisks indicate values significantly different (p

    Article Snippet: Channel subunits were identified using mouse monoclonal anti-Kv7.2 primary antibodies (clone N26A/23, dilution 1:1000; Antibodies Inc., Davis, CA), followed by horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibodies (clone NA931V; dilution 1:5,000; GE Healthcare, Little Chalfont, UK).

    Techniques: Functional Assay, Transfection, Mass Spectrometry, Western Blot, Construct, Expressing

    AIS localization of Kv7.2 R325G subunits. ( a ) Representative images of primary rat hippocampal neurons transfected with Kv7.3 and the EGFP-Kv7.2-HA-, EGFP-Kv7.2 R325G-HA-, or EGFP-Kv7.2 A294V-HA-expressing plasmids revealed by anti-HA (red; before permeabilization) or anti-ank-G (blue; after permeabilization) antibodies. In green is the EGFP fluorescence. Lower panels are magnifications of the boxed regions. ( b ) Quantification of the intensity (expressed as arbitrary units, A.U.) of the HA (red) and Ank-G (blue) fluorescence signals for Kv7.2 (n = 20), Kv7.2 R325G (n = 18), and Kv7.2 A294V (n = 15) subunits, measured on a 40 μm-long axonal region starting from the soma, as described in Methods . ( c ) Quantification of AIS/Soma and AIS/Dendrite fluorescence ratios for Kv7.2, Kv7.2 R325G, and Kv7.2 A294V subunits, calculated as described in Methods . Asterisks indicate values significantly different (p

    Journal: Scientific Reports

    Article Title: Early-onset epileptic encephalopathy caused by a reduced sensitivity of Kv7.2 potassium channels to phosphatidylinositol 4,5-bisphosphate

    doi: 10.1038/srep38167

    Figure Lengend Snippet: AIS localization of Kv7.2 R325G subunits. ( a ) Representative images of primary rat hippocampal neurons transfected with Kv7.3 and the EGFP-Kv7.2-HA-, EGFP-Kv7.2 R325G-HA-, or EGFP-Kv7.2 A294V-HA-expressing plasmids revealed by anti-HA (red; before permeabilization) or anti-ank-G (blue; after permeabilization) antibodies. In green is the EGFP fluorescence. Lower panels are magnifications of the boxed regions. ( b ) Quantification of the intensity (expressed as arbitrary units, A.U.) of the HA (red) and Ank-G (blue) fluorescence signals for Kv7.2 (n = 20), Kv7.2 R325G (n = 18), and Kv7.2 A294V (n = 15) subunits, measured on a 40 μm-long axonal region starting from the soma, as described in Methods . ( c ) Quantification of AIS/Soma and AIS/Dendrite fluorescence ratios for Kv7.2, Kv7.2 R325G, and Kv7.2 A294V subunits, calculated as described in Methods . Asterisks indicate values significantly different (p

    Article Snippet: Channel subunits were identified using mouse monoclonal anti-Kv7.2 primary antibodies (clone N26A/23, dilution 1:1000; Antibodies Inc., Davis, CA), followed by horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibodies (clone NA931V; dilution 1:5,000; GE Healthcare, Little Chalfont, UK).

    Techniques: Transfection, Expressing, Fluorescence

    Functional and biochemical characterization of Kv7.2 G310S and Kv7.2 G310Δ10 subunits. (A) Maximal current densities measured in non transfected cells (NT) or in cells expressing wild-type or mutant Kv7.2 subunits, as indicated. * = p

    Journal: Frontiers in Pharmacology

    Article Title: Functional Characterization of Two Variants at the Intron 6—Exon 7 Boundary of the KCNQ2 Potassium Channel Gene Causing Distinct Epileptic Phenotypes

    doi: 10.3389/fphar.2022.872645

    Figure Lengend Snippet: Functional and biochemical characterization of Kv7.2 G310S and Kv7.2 G310Δ10 subunits. (A) Maximal current densities measured in non transfected cells (NT) or in cells expressing wild-type or mutant Kv7.2 subunits, as indicated. * = p

    Article Snippet: Channel subunits were identified using mouse monoclonal anti-Kv7.2 primary antibodies (clone N26A/23, dilution 1:1,000; Antibodies Inc., Davis, CA), followed by horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibodies (clone NA931V; dilution 1:5,000; GE Healthcare, Little Chalfont, United Kingdom).

    Techniques: Functional Assay, Transfection, Expressing, Mutagenesis

    Localization of KCNQ2 variants herein investigated and effects on KCNQ2 mRNAs expression. (A) Localization of the two variants herein investigated in the KCNQ2 gene, falling at the last nucleotide of intron 6 (KCNQ2 (C) 928-1G > C; blue circle; already reported in Soldovieri et al., 2014 ) or at the first nucleotide of exon 7 (KCNQ2 (C) 928G > (A) ; already reported in Soldovieri et al., 2020 ). (B) Representative agarose gel run and (C) electrophoresis profiles of major bands obtained from RT-PCR experiments performed on Kv7.2 mRNAs expressed in father- or probands-deriving lymphoblasts, as indicated; the mRNA fragment of 295 bp (B) or 218 bp (C) are expected when the ag acceptor site at the end of intron 6 is used, while those of 265 bp (B) or 188 bp (C) bands are obtained using the alternative splice site in the exon 7. Panels in C have been obtained cropping the regions of interest from the complete electrophoresis profiles, reported in Supplementary Figure S2 Topology of a Kv7.2 subunit showing the 6 transmembrane segments (called from S1 to S6) and four intracellular domains at the C-terminus (called from A to D), binding many regulatory molecules. Middle and left panels show the structural alterations prompted by the Kv7.2 G310Δ10 in-frame deletion or by the missense Kv7.2 G310S variant, respectively.

    Journal: Frontiers in Pharmacology

    Article Title: Functional Characterization of Two Variants at the Intron 6—Exon 7 Boundary of the KCNQ2 Potassium Channel Gene Causing Distinct Epileptic Phenotypes

    doi: 10.3389/fphar.2022.872645

    Figure Lengend Snippet: Localization of KCNQ2 variants herein investigated and effects on KCNQ2 mRNAs expression. (A) Localization of the two variants herein investigated in the KCNQ2 gene, falling at the last nucleotide of intron 6 (KCNQ2 (C) 928-1G > C; blue circle; already reported in Soldovieri et al., 2014 ) or at the first nucleotide of exon 7 (KCNQ2 (C) 928G > (A) ; already reported in Soldovieri et al., 2020 ). (B) Representative agarose gel run and (C) electrophoresis profiles of major bands obtained from RT-PCR experiments performed on Kv7.2 mRNAs expressed in father- or probands-deriving lymphoblasts, as indicated; the mRNA fragment of 295 bp (B) or 218 bp (C) are expected when the ag acceptor site at the end of intron 6 is used, while those of 265 bp (B) or 188 bp (C) bands are obtained using the alternative splice site in the exon 7. Panels in C have been obtained cropping the regions of interest from the complete electrophoresis profiles, reported in Supplementary Figure S2 Topology of a Kv7.2 subunit showing the 6 transmembrane segments (called from S1 to S6) and four intracellular domains at the C-terminus (called from A to D), binding many regulatory molecules. Middle and left panels show the structural alterations prompted by the Kv7.2 G310Δ10 in-frame deletion or by the missense Kv7.2 G310S variant, respectively.

    Article Snippet: Channel subunits were identified using mouse monoclonal anti-Kv7.2 primary antibodies (clone N26A/23, dilution 1:1,000; Antibodies Inc., Davis, CA), followed by horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibodies (clone NA931V; dilution 1:5,000; GE Healthcare, Little Chalfont, United Kingdom).

    Techniques: Expressing, Agarose Gel Electrophoresis, Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Variant Assay

    Functional characterization of heteromeric Kv7.2 + Kv7.3 channels incorporating Kv7.2 G310S or Kv7.2 G310Δ10 subunits expressed in neurons. (A) Macroscopic currents from neurons expressing wild-type or mutant Kv7.2 + Kv7.3 subunits, as indicated. (B,C) Current density/voltage curves measured in non transfected neurons (NT) or expressing the indicated channels incorporating Kv7.2 G310Δ10 (B) or Kv7.2 G310S (C) subunits, as indicated.

    Journal: Frontiers in Pharmacology

    Article Title: Functional Characterization of Two Variants at the Intron 6—Exon 7 Boundary of the KCNQ2 Potassium Channel Gene Causing Distinct Epileptic Phenotypes

    doi: 10.3389/fphar.2022.872645

    Figure Lengend Snippet: Functional characterization of heteromeric Kv7.2 + Kv7.3 channels incorporating Kv7.2 G310S or Kv7.2 G310Δ10 subunits expressed in neurons. (A) Macroscopic currents from neurons expressing wild-type or mutant Kv7.2 + Kv7.3 subunits, as indicated. (B,C) Current density/voltage curves measured in non transfected neurons (NT) or expressing the indicated channels incorporating Kv7.2 G310Δ10 (B) or Kv7.2 G310S (C) subunits, as indicated.

    Article Snippet: Channel subunits were identified using mouse monoclonal anti-Kv7.2 primary antibodies (clone N26A/23, dilution 1:1,000; Antibodies Inc., Davis, CA), followed by horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibodies (clone NA931V; dilution 1:5,000; GE Healthcare, Little Chalfont, United Kingdom).

    Techniques: Functional Assay, Expressing, Mutagenesis, Transfection

    Functional study of the modulation of Kv7.2 G310S or Kv7.2 G310Δ10 subunits by PIP 2 and calmodulin. (A,B) Representative traces (A) and quantification (B) of currents measured in CHO cells co-expressing homomeric wild-type or mutant Kv7.2 subunits with PIP5K, CaM, and/or CaM 1234 , as indicated. * = p

    Journal: Frontiers in Pharmacology

    Article Title: Functional Characterization of Two Variants at the Intron 6—Exon 7 Boundary of the KCNQ2 Potassium Channel Gene Causing Distinct Epileptic Phenotypes

    doi: 10.3389/fphar.2022.872645

    Figure Lengend Snippet: Functional study of the modulation of Kv7.2 G310S or Kv7.2 G310Δ10 subunits by PIP 2 and calmodulin. (A,B) Representative traces (A) and quantification (B) of currents measured in CHO cells co-expressing homomeric wild-type or mutant Kv7.2 subunits with PIP5K, CaM, and/or CaM 1234 , as indicated. * = p

    Article Snippet: Channel subunits were identified using mouse monoclonal anti-Kv7.2 primary antibodies (clone N26A/23, dilution 1:1,000; Antibodies Inc., Davis, CA), followed by horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibodies (clone NA931V; dilution 1:5,000; GE Healthcare, Little Chalfont, United Kingdom).

    Techniques: Functional Assay, Expressing, Mutagenesis, Chick Chorioallantoic Membrane Assay