Journal: bioRxiv

Article Title: Divergent regulation of KCNQ1/E1 by targeted recruitment of protein kinase A to distinct sites on the channel complex

doi: 10.1101/2022.09.12.507626

Figure Lengend Snippet: (a) Cartoon showing Q1/E1-YFP complex co-expressed with or without free PKA Cα subunit. (b) Representative immunoblots of lysates from HEK293 cells co-expressing Q1/E1-YFP with either empty pcDNA3.1 vector or free Cα. Anti-pKCNQ1 (top) detects phosphorylated KCNQ1-S27, anti-KCNQ1 (middle) detects total KCNQ1, and anti-Actin (bottom) detects total actin. (c) I Ks activation curves in CHO cells co-expressing Q1, E1-YFP with either empty pcDNA3.1 vector (black symbols, n = 13) or free PKA Cα (red symbols, n = 13). (d) Tail-decay times for currents recorded from cells co-expressing Q1/E-YFP + yotiao and either nano or free PKA Cα, or cells co-expressing Q1[S27A]/E1-YFP + yotiao and free PKA Cα. (e-h) Cartoon, immunoblots, I Ks activation curves, and population current densities of Q1/E1-YFP complex expressed with either nano (n = 10) or nanoCα ( n = 10). (i) Cartoon showing targeting of nanoCα to Q1/E1 complex via YFP tag on Q1 C-terminus. (j) Exemplar current traces I Ks traces from CHO cells co-expressing Q1-YFP/E1 with either nanoCα ( left ) or catalytically inactive nanoCα [T198A] mutant ( right ). (k) Population current densities (nano, n = 26; nanoCα, n = 19; nanoCα[T198A], n = 10).

Article Snippet: Membranes were pre-treated with 0.5% glutaraldehyde and re-blotted with rabbit anti-KCNQ1 antibody (1:1,000, Alomone labs, Israel) and rabbit anti-actin antibody (1:2000, Abcam, USA).

Techniques: Western Blot, Expressing, Plasmid Preparation, Activation Assay, Mutagenesis

Journal: bioRxiv

Article Title: Divergent regulation of KCNQ1/E1 by targeted recruitment of protein kinase A to distinct sites on the channel complex

doi: 10.1101/2022.09.12.507626

Figure Lengend Snippet: (a) Cartoon of FKBP/FRB heterodimerization strategy utilized for rapamycin-induced recruitment of engineered Cα to BBS-Q1-YFP/E1. (B) Exemplar flow cytometry contour plots showing surface expression (BTX-647 fluorescence) and CFP fluorescence in cells expressing BBS-Q1-YFP/E1 with FRB-Cα and FKBP-nano at times t = 0 ( left ), t = 6h ( middle ) and t = 24h ( right ) after rapamycin addition. (c) Normalized mean Q1 surface density (BTX-647 fluorescence) plotted as a function of time after rapamycin induction. (d) Normalized mean Q1 total expression (YFP fluorescence) plotted as a function of time after rapamycin induction. (e) Exemplar I Ks traces recorded in CHO cells co-expressing KCNQ1-YFP/KCNE1/Nano-FKBP-FRB-Cα incubated 20 hours either without ( left ) or with ( right ) rapamycin. (f) Mean current densities in CHO cells co-expressing KCNQ1-YFP/KCNE1/Nano-FKBP-FRB-Cα without rapamycin (black, n = 10) or after 20 h rapamycin incubation (red, n = 14). *** P < 0.001, paired t test. (g) Mean current densities in control cells co-expressing KCNQ1-YFP/KCNE1 without rapamycin (black, n = 8) or after 20 h rapamycin incubation (red, n = 9).

Article Snippet: Membranes were pre-treated with 0.5% glutaraldehyde and re-blotted with rabbit anti-KCNQ1 antibody (1:1,000, Alomone labs, Israel) and rabbit anti-actin antibody (1:2000, Abcam, USA).

Techniques: Flow Cytometry, Expressing, Fluorescence, Incubation

Journal: bioRxiv

Article Title: Divergent regulation of KCNQ1/E1 by targeted recruitment of protein kinase A to distinct sites on the channel complex

doi: 10.1101/2022.09.12.507626

Figure Lengend Snippet: (a) Top , schematic of Q1 showing positions of Ser and Thr residues where phosphorylation was increased when nanoCα was targeted to Q1 C-terminus. Bottom , relative abundance of phosphorylated KCNQ1-YFP peptides identified using mass spectrometry in cells co-expressing nano (black), nanoCα (red), or free Cα (cyan). (b) Exemplar CDF plots showing channel surface density in cells expressing WT BBS-Q1-YFP ( left ), BBS-3S/A-YFP ( middle ), or BBS-3S/D-YFP ( right ) in the absence (black traces) or presence (red traces) of nanoCα. (c) Channel surface density (BTX-647 fluorescence) in cells expressing WT BBS-Q1-YFP, BBS-3S/A-YFP, or BBS-3S/D-YFP in the presence of either nano or nanoCα. WT BBS-Q1-YFP (nano, n = 15,411, N = 2; nanoCα, n = 9,053, N = 2). BBS-3S/A-YFP (nano, n = 6,158, N = 2; nanoCα, n = 4,798, N = 2). BBS-3S/D-YFP (nano, n = 11,823, N = 2; nanoCα, n = 5,685, N = 2).

Article Snippet: Membranes were pre-treated with 0.5% glutaraldehyde and re-blotted with rabbit anti-KCNQ1 antibody (1:1,000, Alomone labs, Israel) and rabbit anti-actin antibody (1:2000, Abcam, USA).

Techniques: Mass Spectrometry, Expressing, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: Pharmacological suppression of Nedd4-2 rescues the reduction of Kv11.1 channels in pathological cardiac hypertrophy

doi: 10.3389/fphar.2022.942769

Figure Lengend Snippet: I Kr currents and Kv11.1 protein were reduced in the myocardium in pCH. (A) Representative tail traces of I Kr before (left) and after application of E-4031 (2 μM) (right) in upper panel. Ventricular myocyte was pulsed as shown protocol. The lower panel showing representative tail traces recorded in myocytes from control (CON) and pCH (Ang II), respectively. (B) Summary data for I Kr tail current density-voltage relationship in control and pCH ( n = 10–14 cardiomyocytes from 3 to 5 hearts, * p < 0.05 versus CON). (C) Normalized current-voltage relationship for I Kr tail current. Curves were fit by Boltzmann function. (D) Representative I Ks traces recorded using the pulse protocol shown in the inset before (left) and after application of HMR1556 (2 μM) (right) in upper panel. The lower panel showing representative traces recorded in myocytes from control (CON) and pCH (Ang II), respectively. (E) Summary data for I Ks tail current density-voltage relationship in control and pCH ( n = 11–15 cardiomyocytes from 3 to 5 hearts). (F) Normalized current-voltage relationship for I Ks tail current. Curves were fit by Boltzmann function. (G) Representative immunoblots bands for mature and immature Kv11.1 proteins and corresponding summary data (CON n = 5, Ang II n = 7). (H) Representative immunoblots bands for Kv7.1 and KCNE1 proteins and corresponding summary data (CON n = 5, Ang II n = 7). GADPH was used as an internal control to normalize these bands. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus CON. ns: not statistically significant.

Article Snippet: Antibodies against Kv7.1 (APC-022), KCNE1 (APC-163), or Kv11.1 (APC-016) were obtained from Alomone Labs, Jerusalem, Israel.

Techniques: Western Blot

Journal: Frontiers in Pharmacology

Article Title: Pharmacological suppression of Nedd4-2 rescues the reduction of Kv11.1 channels in pathological cardiac hypertrophy

doi: 10.3389/fphar.2022.942769

Figure Lengend Snippet: Nedd4-2-mediated ubiquitination of Kv11.1 was increased in pCH. (A) Representative immunoblots showing Nedd4-2, phosphorylated Nedd4-2 (p-Nedd4-2) and Rab11 proteins and corresponding quantifications of band densities in myocardium from control (CON) ( n = 5) and pCH (Ang II, n = 7). GADPH was used as an internal control to normalize the bands. (B) Representative blots immunoprecipitated (IP) by anti-Kv11.1, and western immunoblotting (IB) was performed by using anti-Nedd4-2 antibody. Quantification of band densities was shown as the ratio of Nedd4-2 to Kv11.1. (C) Representative blots immunoprecipitated by anti-Kv11.1, and western immunoblotting (IB) was performed by using anti-ubiquitin antibody. Quantification of band densities was shown as the ratio of ubiquitin to Kv11.1. (D) Proteins from control and pCH myocardium were immunoprecipitated with an anti-Kv7.1 or anti-IgG antibody, and IB was performed by using anti-Nedd4-2 antibody. IgG was used as negative controls. n = 3–4. * p < 0.05 and *** p < 0.001 versus CON. ns: not statistically significant.

Article Snippet: Antibodies against Kv7.1 (APC-022), KCNE1 (APC-163), or Kv11.1 (APC-016) were obtained from Alomone Labs, Jerusalem, Israel.

Techniques: Western Blot, Immunoprecipitation

Journal: Stem Cells Translational Medicine

Article Title: Heterogeneity of Potassium Channels in Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium

doi: 10.1093/stcltm/szac029

Figure Lengend Snippet: M-currents in the hESC-RPE. ( A ) Representative example of the sustained currents as responses to 1000 ms voltage steps from −70 mV to 40 mV in 10 mV increments ( B ) and the normalized and averaged IV-curve (mean ± SEM, n = 9). ( C ) Current responses evoked by 1000 ms voltage pulses from −10 mV holding potential to −100 mV and up to 40 mV test potentials in 10 mV steps ( D ) and the normalized and averaged GV-curve (mean ± SEM, n = 5) that has been obtained using tail current analysis (see Methods). ( E ) The effect of 300 nM linopirdine on the currents evoked by 1000 ms voltage pulses from −70 mV to 40 mV ( F ) and the averaged and normalized IV-curve (mean ± SEM, n = 3). Immunostainings of paraffin-embedded vertical sections shown as xy -maximum intensity projections (apical side upwards) for KCNQ channels (gray): ( G ) KCNQ1, ( H ) KCNQ2, ( I ) KCNQ3, ( J ) KCNQ4, and ( K ) KCNQ5. Scale bars 10 µm. (A-F) present patch-clamp data from the cell line 11/013 and (G-K) show representative images of the cell lines 08/023 and 08/017. Abbreviations: hESC, human embryonic stem cell; RPE, retinal pigment epithelium.

Article Snippet: In this study, we used primary antibodies Kir4.1 (1:100; ab80959; Abcam, UK), Kir7.1 (1:100; ab170631; Abcam), K V 1.4 (1:50; ab99332; Abcam), K V 4.2 (1:50; ab46797; Abcam), KCNQ1 (1:100; APC-022; Alomone Labs, Jerusalem, Israel), KCNQ2 (1:100; APC-050; Alomone Labs), KCNQ3 (1:100; APC-051; Alomone Labs), KCNQ4 (1:100; APC-164; Alomone Labs), KCNQ5 (1:100; APC-155; Alomone Labs), cellular retinaldehyde-binding protein (CRALBP; ab15051; 1:500; Abcam), zonula occludens (ZO-1; 339100; 1:50; Life Technologies), claudin-3 (1:80; 34-1700; Invitrogen, USA), Na + /K + -ATPase (1:200; ab7671; Abcam) and Bestrophin-1 (1:500; 016-Best1-01; Lagen Laboratories, USA) (see ).

Techniques: Patch Clamp

Journal: Stem Cells Translational Medicine

Article Title: Heterogeneity of Potassium Channels in Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium

doi: 10.1093/stcltm/szac029

Figure Lengend Snippet: Localization of the K + channels in the mouse RPE. Confocal images of the mouse RPE-eyecup whole mount preparations illustrate the xy -maximum intensity projections and yz -confocal sections (apical side upwards, localization of the section highlighted with a white bar), where actin cytoskeleton (phalloidin, magenta) is stained together with ( A ) Kir4.1 (green), ( B ) Kir7.1 (green), and ( C ) K V 1.4 (green). Confocal images of paraffin-embedded vertical sections of mouse eyecups shown as xy -maximum intensity projections (apical side upwards), where BF images are presented together with the K + channels (green). ( D ) Kir7.1, ( E ) K V 4.2, ( F ) KCNQ1, ( G ) KCNQ2, ( H ) KCNQ3, ( I ) KCNQ4, and ( J ) KCNQ5. Scale bars 10 µm. Abbreviations: BF, bright field; Kir, inwardly rectifying K + channel; K V , voltage-gated K + channel.

Article Snippet: In this study, we used primary antibodies Kir4.1 (1:100; ab80959; Abcam, UK), Kir7.1 (1:100; ab170631; Abcam), K V 1.4 (1:50; ab99332; Abcam), K V 4.2 (1:50; ab46797; Abcam), KCNQ1 (1:100; APC-022; Alomone Labs, Jerusalem, Israel), KCNQ2 (1:100; APC-050; Alomone Labs), KCNQ3 (1:100; APC-051; Alomone Labs), KCNQ4 (1:100; APC-164; Alomone Labs), KCNQ5 (1:100; APC-155; Alomone Labs), cellular retinaldehyde-binding protein (CRALBP; ab15051; 1:500; Abcam), zonula occludens (ZO-1; 339100; 1:50; Life Technologies), claudin-3 (1:80; 34-1700; Invitrogen, USA), Na + /K + -ATPase (1:200; ab7671; Abcam) and Bestrophin-1 (1:500; 016-Best1-01; Lagen Laboratories, USA) (see ).

Techniques: Staining

Journal: Nature cardiovascular research

Article Title: Sigma non-opioid receptor 1 is a potential therapeutic target for long QT syndrome

doi: 10.1038/s44161-021-00016-2

Figure Lengend Snippet: ( a ) Representative confocal fluorescent images of Timothy syndrome cardiomyocytes without treatment (left, TS) and with dextromethorphan (+Dxm). Scale bar, 5μm. ( b ) Quantification of fraction of green (hERG) over red (WGA) fluorescence signals in TS cardiomyocytes without treatment ( n =38) and with Dxm ( n =31). ( c ) Quantification of KCNH2 transcript in TS cardiomyocytes with and without Dxm ( n =4/group). ( d ) Representative immunoblots of human hERG and GAPDH protein using lysates from non-treated and Dxm-treated TS cardiomyocytes. ( e ) Quantification of hERG protein expressions (normalized to GAPDH) in non-treated and Dxm-treated TS cardiomyocytes ( n =3/group). The trend towards a decreased hERG protein expression in the treated group might be driven by one sample ( # ) with a reduced loading amount compared with others. ( f ) I Ks current steady-state amplitudes were significantly reduced in TS cardiomyocytes ( n =10) compared to isogenic control ( n =10) at 20 and 50 mV steps from −40mV hold. ( g ) Representative traces of I Ks currents (Chromanol 293B-sensitive) in isogenic Ctrl cardiomyocytes (black) and TS cardiomyocytes treated with Dxm (purple), Dxm and NE-100 (green) or without treatment (red, TS). The I Ks traces are shown in . ( h ) Representative confocal fluorescent images of Timothy syndrome cardiomyocyte without treatment (left, TS) and treated with dextromethorphan (+Dxm). Scale bar, 5μm. ( i ) Quantification of fraction of green (K V 7.1) over red (WGA) fluorescence signals in TS cardiomyocytes without treatment ( n =21) and treated with Dxm ( n =31). ( j ) Quantification of KCNQ1 transcripts (isoform 1 and 2) in TS cardiomyocytes without treatment and treated with Dxm ( n =4/group). ( k ) Representative immunoblots of human K V 7.1 and GAPDH protein using lysates from non-treated and Dxm-treated TS cardiomyocytes. ( l-m ) Quantification of K V 7.1 isoform 1 ( l ) and isoform 2 protein expressions ( m , normalized to GAPDH) in non-treated and Dxm-treated TS cardiomyocytes ( n =3/group). All data are mean ± s.d. The treatment of Dxm or NE-100 for all experiments was 5μM, 2hrs. Unpaired two-tailed Student’s t -test was used for b, c, e, i, j, l, m between the groups and used for f at each voltage step. * P <0.05. n.s., not significant. The cell samples were from at least two independent differentiations.

Article Snippet: The following day the membranes were incubated with the primary antibody solution overnight at 4 °C (KCNQ1/K V 7.1 antibody, Alomone lab, # APC-022, 1:2,000 dilution; GAPDH antibody, Abcam, Catalog # ab181602, 1:10,000 dilution; beta-tubulin antibody, Sigma Aldrich, T5201, 1:8,000 dilution).

Techniques: Fluorescence, Western Blot, Expressing, Two Tailed Test

Journal: Nature cardiovascular research

Article Title: Sigma non-opioid receptor 1 is a potential therapeutic target for long QT syndrome

doi: 10.1038/s44161-021-00016-2

Figure Lengend Snippet: ( a ) Representative immunoblots of K V 7.1, β-tubulin and Gapdh protein using mouse heart lysates from TS (non-treated and Dxm-treated) mice and the control littermates (Ctrl) at day 11 (each, n =3, using the same samples with the global proteomics shown in ). #, bands indicate putative degradation of K V 7.1 in TS mouse hearts. ( b-c ) Quantification of β-tubulin ( b ) and Kcnq1/K V 7.1 protein expression ( c , normalized to Gapdh) in TS (non-treated and Dxm-treated) and Ctrl mouse hearts ( n =3/group). Altered β-tubulin expression has been reported previously in animal model of myocardial infarction and Gapdh was recommended as a more reliable loading control for Western blot analyses . ( d ) Kcnq1/ K V 7.1 mRNA expression (normalized to Gapdh) in TS (non-treated and Dxm-treated) and Ctrl mouse hearts ( n =3/group). All data are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons was used. ** P <0.01, n.s., not significant.

Article Snippet: The following day the membranes were incubated with the primary antibody solution overnight at 4 °C (KCNQ1/K V 7.1 antibody, Alomone lab, # APC-022, 1:2,000 dilution; GAPDH antibody, Abcam, Catalog # ab181602, 1:10,000 dilution; beta-tubulin antibody, Sigma Aldrich, T5201, 1:8,000 dilution).

Techniques: Western Blot, Expressing, Animal Model

Journal: Nature cardiovascular research

Article Title: Sigma non-opioid receptor 1 is a potential therapeutic target for long QT syndrome

doi: 10.1038/s44161-021-00016-2

Figure Lengend Snippet: ( a ) Representative confocal fluorescent images of Timothy syndrome cardiomyocytes without treatment (left, TS) and with dextromethorphan (+Dxm). Scale bar, 5μm. ( b ) Quantification of fraction of green (hERG) over red (WGA) fluorescence signals in TS cardiomyocytes without treatment ( n =38) and with Dxm ( n =31). ( c ) Quantification of KCNH2 transcript in TS cardiomyocytes with and without Dxm ( n =4/group). ( d ) Representative immunoblots of human hERG and GAPDH protein using lysates from non-treated and Dxm-treated TS cardiomyocytes. ( e ) Quantification of hERG protein expressions (normalized to GAPDH) in non-treated and Dxm-treated TS cardiomyocytes ( n =3/group). The trend towards a decreased hERG protein expression in the treated group might be driven by one sample ( # ) with a reduced loading amount compared with others. ( f ) I Ks current steady-state amplitudes were significantly reduced in TS cardiomyocytes ( n =10) compared to isogenic control ( n =10) at 20 and 50 mV steps from −40mV hold. ( g ) Representative traces of I Ks currents (Chromanol 293B-sensitive) in isogenic Ctrl cardiomyocytes (black) and TS cardiomyocytes treated with Dxm (purple), Dxm and NE-100 (green) or without treatment (red, TS). The I Ks traces are shown in . ( h ) Representative confocal fluorescent images of Timothy syndrome cardiomyocyte without treatment (left, TS) and treated with dextromethorphan (+Dxm). Scale bar, 5μm. ( i ) Quantification of fraction of green (K V 7.1) over red (WGA) fluorescence signals in TS cardiomyocytes without treatment ( n =21) and treated with Dxm ( n =31). ( j ) Quantification of KCNQ1 transcripts (isoform 1 and 2) in TS cardiomyocytes without treatment and treated with Dxm ( n =4/group). ( k ) Representative immunoblots of human K V 7.1 and GAPDH protein using lysates from non-treated and Dxm-treated TS cardiomyocytes. ( l-m ) Quantification of K V 7.1 isoform 1 ( l ) and isoform 2 protein expressions ( m , normalized to GAPDH) in non-treated and Dxm-treated TS cardiomyocytes ( n =3/group). All data are mean ± s.d. The treatment of Dxm or NE-100 for all experiments was 5μM, 2hrs. Unpaired two-tailed Student’s t -test was used for b, c, e, i, j, l, m between the groups and used for f at each voltage step. * P <0.05. n.s., not significant. The cell samples were from at least two independent differentiations.

Article Snippet: The samples were next incubated in primary antibody overnight at 4°C (KCNQ1/K V 7.1, Alomone lab, # APC-022, 1:500; SIGMAR1, Santa Cruz, # sc-137075 (mouse), 1:500; SIGMAR1, Abcam, # ab53852 (rabbit), 1:500; KCNH2/hERG, Santa Cruz, # sc-377388, 1:500; CDK5, Abcam, # ab40773, 1:500; Ca V 1.2, Alomone labs, # ACC-003, 1:100).

Techniques: Fluorescence, Western Blot, Expressing, Two Tailed Test

Journal: Nature cardiovascular research

Article Title: Sigma non-opioid receptor 1 is a potential therapeutic target for long QT syndrome

doi: 10.1038/s44161-021-00016-2

Figure Lengend Snippet: ( a ) Representative immunoblots of K V 7.1, β-tubulin and Gapdh protein using mouse heart lysates from TS (non-treated and Dxm-treated) mice and the control littermates (Ctrl) at day 11 (each, n =3, using the same samples with the global proteomics shown in ). #, bands indicate putative degradation of K V 7.1 in TS mouse hearts. ( b-c ) Quantification of β-tubulin ( b ) and Kcnq1/K V 7.1 protein expression ( c , normalized to Gapdh) in TS (non-treated and Dxm-treated) and Ctrl mouse hearts ( n =3/group). Altered β-tubulin expression has been reported previously in animal model of myocardial infarction and Gapdh was recommended as a more reliable loading control for Western blot analyses . ( d ) Kcnq1/ K V 7.1 mRNA expression (normalized to Gapdh) in TS (non-treated and Dxm-treated) and Ctrl mouse hearts ( n =3/group). All data are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons was used. ** P <0.01, n.s., not significant.

Article Snippet: The samples were next incubated in primary antibody overnight at 4°C (KCNQ1/K V 7.1, Alomone lab, # APC-022, 1:500; SIGMAR1, Santa Cruz, # sc-137075 (mouse), 1:500; SIGMAR1, Abcam, # ab53852 (rabbit), 1:500; KCNH2/hERG, Santa Cruz, # sc-377388, 1:500; CDK5, Abcam, # ab40773, 1:500; Ca V 1.2, Alomone labs, # ACC-003, 1:100).

Techniques: Western Blot, Expressing, Animal Model