Alomone Labs kcnq1 rabbit ab

Figure 1. Fluorescent protein- and epitope-tagged <t>KCNQ1</t> and KCNE1 retain their distribution patterns in cardiac myocytes and normal IKs channel function A, properties of Q1-GFP and HA-E1-dsR expressed in rat ventricular myocytes. Left: confocal images of Q1-GFP and HA-E1-dsR in a rat ventricular myocyte. Shown are the XY plane view at the central z-plane and a selected XZ plane view (locations noted by the white dashed lines in the XY plane view). Overlap between Q1-GFP and HA-E1-dsR in the XZ plane view is marked by asterisks in the ‘merged’ image. Right: currents recorded from rat ventricular myocytes transduced with Q1-GFP and HA-E1-dsR (upper) and time-control (myocyte isolated from the same heart and cultured for the same duration without adenoviruses, middle). The voltage clamp protocol is indicated in the inset. Lower: Boltzmann fit to the voltage dependence of channel activation in transduced myocytes, estimated by the relationship between the test pulse voltages (Vt) and the peak tail current amplitudes (Ipeak): degree of activation = (Imax/Ipeak)/(1 + exp[(V0.5 – Vt)/k]), where Imax, V0.5 and k are the estimated maximal peak tail current amplitude of the cell, half-maximum activation voltage and slope factor, respectively. The parameter values are listed in the inset. Data were pooled from four myocytes of one heart. B, properties of native KCNQ1 and KCNE1 in guinea pig ventricular myocytes. Left: confocal images of immunofluorescence of native KCNQ1 and KCNE1 in a guinea ventricular myocyte. KCNQ1 and KCNE1 were labelled with goat and mouse primary antibodies, followed by Alexa-488 donkey anti-goat and Alexa-568 donkey anti-mouse secondary Abs. The format here is the same as that for (A, left). Right: native IKs recorded from guinea pig ventricular myocytes. Representative current traces are shown at the top, with a diagram of the voltage clamp protocol, and Boltzmann fit to the voltage dependence of channel activation is shown at the bottom, with the estimated parameter values in the inset. Data were pooled from nine myocytes of three hearts.

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Figure 1. Fluorescent protein- and epitope-tagged KCNQ1 and KCNE1 retain their distribution patterns in cardiac myocytes and normal IKs channel function A, properties of Q1-GFP and HA-E1-dsR expressed in rat ventricular myocytes. Left: confocal images of Q1-GFP and HA-E1-dsR in a rat ventricular myocyte. Shown are the XY plane view at the central z-plane and a selected XZ plane view (locations noted by the white dashed lines in the XY plane view). Overlap between Q1-GFP and HA-E1-dsR in the XZ plane view is marked by asterisks in the ‘merged’ image. Right: currents recorded from rat ventricular myocytes transduced with Q1-GFP and HA-E1-dsR (upper) and time-control (myocyte isolated from the same heart and cultured for the same duration without adenoviruses, middle). The voltage clamp protocol is indicated in the inset. Lower: Boltzmann fit to the voltage dependence of channel activation in transduced myocytes, estimated by the relationship between the test pulse voltages (Vt) and the peak tail current amplitudes (Ipeak): degree of activation = (Imax/Ipeak)/(1 + exp[(V0.5 – Vt)/k]), where Imax, V0.5 and k are the estimated maximal peak tail current amplitude of the cell, half-maximum activation voltage and slope factor, respectively. The parameter values are listed in the inset. Data were pooled from four myocytes of one heart. B, properties of native KCNQ1 and KCNE1 in guinea pig ventricular myocytes. Left: confocal images of immunofluorescence of native KCNQ1 and KCNE1 in a guinea ventricular myocyte. KCNQ1 and KCNE1 were labelled with goat and mouse primary antibodies, followed by Alexa-488 donkey anti-goat and Alexa-568 donkey anti-mouse secondary Abs. The format here is the same as that for (A, left). Right: native IKs recorded from guinea pig ventricular myocytes. Representative current traces are shown at the top, with a diagram of the voltage clamp protocol, and Boltzmann fit to the voltage dependence of channel activation is shown at the bottom, with the estimated parameter values in the inset. Data were pooled from nine myocytes of three hearts.

Journal: The Journal of Physiology

Article Title: Delayed KCNQ1/KCNE1 assembly on the cell surface helps I_Ks fulfil its function as a repolarization reserve in the heart

doi: 10.1113/jp281773

Figure Lengend Snippet: Figure 1. Fluorescent protein- and epitope-tagged KCNQ1 and KCNE1 retain their distribution patterns in cardiac myocytes and normal IKs channel function A, properties of Q1-GFP and HA-E1-dsR expressed in rat ventricular myocytes. Left: confocal images of Q1-GFP and HA-E1-dsR in a rat ventricular myocyte. Shown are the XY plane view at the central z-plane and a selected XZ plane view (locations noted by the white dashed lines in the XY plane view). Overlap between Q1-GFP and HA-E1-dsR in the XZ plane view is marked by asterisks in the ‘merged’ image. Right: currents recorded from rat ventricular myocytes transduced with Q1-GFP and HA-E1-dsR (upper) and time-control (myocyte isolated from the same heart and cultured for the same duration without adenoviruses, middle). The voltage clamp protocol is indicated in the inset. Lower: Boltzmann fit to the voltage dependence of channel activation in transduced myocytes, estimated by the relationship between the test pulse voltages (Vt) and the peak tail current amplitudes (Ipeak): degree of activation = (Imax/Ipeak)/(1 + exp[(V0.5 – Vt)/k]), where Imax, V0.5 and k are the estimated maximal peak tail current amplitude of the cell, half-maximum activation voltage and slope factor, respectively. The parameter values are listed in the inset. Data were pooled from four myocytes of one heart. B, properties of native KCNQ1 and KCNE1 in guinea pig ventricular myocytes. Left: confocal images of immunofluorescence of native KCNQ1 and KCNE1 in a guinea ventricular myocyte. KCNQ1 and KCNE1 were labelled with goat and mouse primary antibodies, followed by Alexa-488 donkey anti-goat and Alexa-568 donkey anti-mouse secondary Abs. The format here is the same as that for (A, left). Right: native IKs recorded from guinea pig ventricular myocytes. Representative current traces are shown at the top, with a diagram of the voltage clamp protocol, and Boltzmann fit to the voltage dependence of channel activation is shown at the bottom, with the estimated parameter values in the inset. Data were pooled from nine myocytes of three hearts.

Article Snippet: Primary Abs used were: KCNE1 mouse Ab (H00003753-M01; AbNova, Taipei, Taiwan), HA mouse Ab (MMS-101P; Covance, Princeton, NJ, USA), dsRed rabbit Ab (catalog # 632496; Clontech, Mountain View, CA, USA), KCNQ1 rabbit Ab targeting the extracellular domain of KCNQ1 (APC-168; Alomone, Jerusalem, Israel), KCNQ1 goat Ab (C20; Santa Cruz Biotechnology, Santa Cruz, CA, USA), GFP goat Ab (ab5450; AbCam, Csambrudge,MA, USA), GFP rabbit Ab (ab290; AbCam), GFP mouse Ab (MA5-15256; Thermo Fisher) and EB1 rat Ab (ab53358; AbCam).

Techniques: Transduction, Control, Isolation, Cell Culture, Activation Assay, Immunofluorescence

Figure 2. Localized KCNQ1/KCNE1 assembly in cardiac myocytes by an in situ PLA Rat ventricular myocytes transduced with Q1-GFP and HA-E1-dsR were cultured for 24–36 h before experiments. A, diagram of PLA procedure. Primary antibodies (1st Abs) were GFP mouse Ab and dsRed rabbit Ab, binding to the FP tags in the cytoplasmic compartment. At sites where the 1st Abs were ≤40 nm apart, binding of nucleotide-conjugated PLA probes to the 1st Abs allowed nucleotide ligation and amplification. During the latter reaction, far red fluorophore was incorporated to mark these sites. Not shown in the diagram: after PLA procedure, myocytes were incubated with Alexa488 goat anti-mouse and Alexa568 goat anti-rabbit Abs to label total Q1-GFP and HA-E1-dsR. B, representative images of Q1-GFP, HA-E1-dsR and PLA (pseudo-coloured white) from a myo- cyte. To better appreciate the 3D distribution pattern of PLA signals in the myocyte, the PLA image is shown as z-projection of maxima. In total, 29 Z-slices were acquired and 20 Z-slices (excluding the bottom six and the top three slices to avoid interference by cell surface PLA signals) were used to create the z-projection. C, quantification of Q1-GFP, HA-E1-dsR and PLA signals in the cell periphery and cell interior. The cell periphery was defined as an area 2 μm wide within the cellular contour, whereas the cell interior was the area within the cell periphery. Shown are data points from individual myocytes (symbols, eight myocytes from two hearts) and mean ± SD (bar graphs). The three groups of data were subject to one-way ANOVA (P = 0.001), followed by Tukey’s pairwise tests. PLA signals have a higher percentage in the cell periphery, and a lower percentage in the cell interior, than Q1-GFP and HA-E1-dsR.

Journal: The Journal of Physiology

Article Title: Delayed KCNQ1/KCNE1 assembly on the cell surface helps I_Ks fulfil its function as a repolarization reserve in the heart

doi: 10.1113/jp281773

Figure Lengend Snippet: Figure 2. Localized KCNQ1/KCNE1 assembly in cardiac myocytes by an in situ PLA Rat ventricular myocytes transduced with Q1-GFP and HA-E1-dsR were cultured for 24–36 h before experiments. A, diagram of PLA procedure. Primary antibodies (1st Abs) were GFP mouse Ab and dsRed rabbit Ab, binding to the FP tags in the cytoplasmic compartment. At sites where the 1st Abs were ≤40 nm apart, binding of nucleotide-conjugated PLA probes to the 1st Abs allowed nucleotide ligation and amplification. During the latter reaction, far red fluorophore was incorporated to mark these sites. Not shown in the diagram: after PLA procedure, myocytes were incubated with Alexa488 goat anti-mouse and Alexa568 goat anti-rabbit Abs to label total Q1-GFP and HA-E1-dsR. B, representative images of Q1-GFP, HA-E1-dsR and PLA (pseudo-coloured white) from a myo- cyte. To better appreciate the 3D distribution pattern of PLA signals in the myocyte, the PLA image is shown as z-projection of maxima. In total, 29 Z-slices were acquired and 20 Z-slices (excluding the bottom six and the top three slices to avoid interference by cell surface PLA signals) were used to create the z-projection. C, quantification of Q1-GFP, HA-E1-dsR and PLA signals in the cell periphery and cell interior. The cell periphery was defined as an area 2 μm wide within the cellular contour, whereas the cell interior was the area within the cell periphery. Shown are data points from individual myocytes (symbols, eight myocytes from two hearts) and mean ± SD (bar graphs). The three groups of data were subject to one-way ANOVA (P = 0.001), followed by Tukey’s pairwise tests. PLA signals have a higher percentage in the cell periphery, and a lower percentage in the cell interior, than Q1-GFP and HA-E1-dsR.

Techniques: In Situ, Transduction, Cell Culture, Binding Assay, Ligation, Amplification, Incubation

Figure 3. Checking for non-specific signals from an in situ PLA used to detect KCNQ1/KCNE1 assembly Rat ventricular myocytes were incubated for the noted numbers of hours without or with adenovirus transduction of Q1-GFP and HA-E1-dsR. The PLA procedure and incubation with Alexa conjugated secondary Abs were the same as those described for Fig. 2. Asterisks mark non-specific PLA signals in nuclei of myocytes after 36 h of culture.

Journal: The Journal of Physiology

Article Title: Delayed KCNQ1/KCNE1 assembly on the cell surface helps I_Ks fulfil its function as a repolarization reserve in the heart

doi: 10.1113/jp281773

Figure Lengend Snippet: Figure 3. Checking for non-specific signals from an in situ PLA used to detect KCNQ1/KCNE1 assembly Rat ventricular myocytes were incubated for the noted numbers of hours without or with adenovirus transduction of Q1-GFP and HA-E1-dsR. The PLA procedure and incubation with Alexa conjugated secondary Abs were the same as those described for Fig. 2. Asterisks mark non-specific PLA signals in nuclei of myocytes after 36 h of culture.

Techniques: In Situ, Incubation, Transduction

Journal: The Journal of Physiology

Article Title: Delayed KCNQ1/KCNE1 assembly on the cell surface helps I_Ks fulfil its function as a repolarization reserve in the heart

doi: 10.1113/jp281773

Figure Lengend Snippet: Figure 5. Distinguish between cell surface and total KCNQ1 and KCNE1 in cardiac myocytes Adult rat ventricular myocytes transduced with Q1-GFP and HA-E1-dsR were fixed. Myocytes labeled as ‘Unpermeabilized’ were incubated with a Q1 rabbit Ab targeting aa 284 −297 in the extracellular domain of KCNQ1 (Q1 ext Ab), or HA mouse Ab (targeting HA epitope in the extracellular domain of HA-E1-dsR). This was followed by Alexa647-conjugated secondary Abs. Myocytes labeled as ‘Permeabilized’ were incubated with 0.1% Triton X-100 at room temperature for 10 min to permeabilize the cell membrane. This was followed by incubation with the same primary (Q1 ext Ab and HA Ab) and secondary Abs. A, Representative confocal images. Immuno- fluorescence (IF) signals from Q1 ext Ab and HA Ab report cell surface proteins (Unpermeabilized) or total proteins (Permeabilized), while fluorescence signals from GFP and dsRed (GFP FP and dsRed FP) report total Q1-GFP and HA-E1-dsR. B, Profiles of fluorescence signals across myocyte width. Black and magenta traces represent FP and IF signals, respectively. The fluorescence profiles and cell widths were normalized to values between 0 and 1.0. Gray shading denotes where cell periphery signals in (C) were quantified. C, Quantification of FP and IF signals in cell periphery of unpermeabilized myocytes. The percentage (%) of areas beneath the profile in the 0–0.2 and 0.8–1 cell width ranges was quantified for Q1-GFP and Q1-ext Ab IF (18 myocytes from 5 hearts), and for HA-E1-dsR and HA Ab IF (9 myocytes from 3 hearts). Shown are data points (circles) from individual myocytes and mean+SD (bar graphs), with p values by t-test. Aa: GFP FP and Q1 ext Ab IF signals in unpermeabilized and permeabilized myo- cytes (marked on top). Ab: dsRed FP and HA Ab IF signals in unpermeabilized and permeabilized myocytes (marked on top). Ba and Bb: Signal profiles (marked) in the same 4 myocytes; the profile is specified by the white dashed lines in A. Ca and Cb: % of specified fluorescence signals in the cell periphery of unpermeabilized myocytes.

Techniques: Transduction, Labeling, Incubation, Membrane, Fluorescence

Figure 6. Tracking KCNE1 and KCNQ1 movements after their ER exit by the RUSH strategy A, RUSH procedure. Protein-of-interest (POI) was fused with a streptavidin-binding-peptide (POI-SBP) and sub- cloned into a bi-cistronic plasmid (with internal ribosome entry ‘IRE’) that had an upstream ER-resident protein (an isoform of the human invariant chain of the major histocompatibility complex, Ii, retained in the ER membrane by a double arginine motif in its cytoplasmic domain) (Boncompain et al. 2012) fused with ‘streptavidin core domain’ (ER-hook-SCD). POI-SBP and ER-hook-SCD were translated as two independent proteins in the same cells. COS-7 cells were transfected with RUSH plasmid and cultured under control conditions, during which the POI-SBP was retained at the ER by ER-hook-SCD. Biotin (membrane permeable) added to the culture medium would compete off POI-SBP from the ER-hook-SCD, allowing POI-SBP to exit the ER and travel down its secretory path. B, fluorescence images of ER-hooked Q1-GFP, HA-E1-dsR and junctophilin-2 (JPH2, an ER marker) in COS-7 cells fixed before (0 min, left) or 30 min (right) after biotin application. Q1-GFP, HA-E1-dsR and JPH2 were detected by GFP mouse Ab/Alexa488 donkey anti-mouse, dsR rabbit Ab/Alexa568 donkey anti-rabbit and JPH2 goat Ab/Alexa647 chicken anti-goat (pseudo-coloured white).

Journal: The Journal of Physiology

Article Title: Delayed KCNQ1/KCNE1 assembly on the cell surface helps I_Ks fulfil its function as a repolarization reserve in the heart

doi: 10.1113/jp281773

Figure Lengend Snippet: Figure 6. Tracking KCNE1 and KCNQ1 movements after their ER exit by the RUSH strategy A, RUSH procedure. Protein-of-interest (POI) was fused with a streptavidin-binding-peptide (POI-SBP) and sub- cloned into a bi-cistronic plasmid (with internal ribosome entry ‘IRE’) that had an upstream ER-resident protein (an isoform of the human invariant chain of the major histocompatibility complex, Ii, retained in the ER membrane by a double arginine motif in its cytoplasmic domain) (Boncompain et al. 2012) fused with ‘streptavidin core domain’ (ER-hook-SCD). POI-SBP and ER-hook-SCD were translated as two independent proteins in the same cells. COS-7 cells were transfected with RUSH plasmid and cultured under control conditions, during which the POI-SBP was retained at the ER by ER-hook-SCD. Biotin (membrane permeable) added to the culture medium would compete off POI-SBP from the ER-hook-SCD, allowing POI-SBP to exit the ER and travel down its secretory path. B, fluorescence images of ER-hooked Q1-GFP, HA-E1-dsR and junctophilin-2 (JPH2, an ER marker) in COS-7 cells fixed before (0 min, left) or 30 min (right) after biotin application. Q1-GFP, HA-E1-dsR and JPH2 were detected by GFP mouse Ab/Alexa488 donkey anti-mouse, dsR rabbit Ab/Alexa568 donkey anti-rabbit and JPH2 goat Ab/Alexa647 chicken anti-goat (pseudo-coloured white).

Techniques: Binding Assay, Clone Assay, Plasmid Preparation, Immunopeptidomics, Membrane, Transfection, Cell Culture, Control, Fluorescence, Marker

$Figure 9. Testing the preference of KCNQ1 and KCNE1 assembly inside cells or on the cell surface A, experimental design. COS-7 cells transfected with Q1-Q147C and E1-G40C were cultured under the control conditions or in the presence of BFA (25 μg mL–1, preventing proteins from reaching the cell surface). The two Cys side chains (147C on Q1 and 40C on E1) could come very close to each other to form a disulphide bond in the open state of a fully-assembled KCNQ1/KCNE1 channel. Therefore, the abundance of Q1-S-S-E1 reported the degree of KCNQ1/KCNE1 assembly. Two scenarios are depicted. Left: Q1 and E1 preferentially assemble inside cells. Forcing Q1-Q147C and E1-G40C to stay inside cells by BFA would not prevent, or even encourage, Q1-S-S-E1 formation. Right: Q1 and E1 preferentially assemble on the cell surface. BFA would prevent Q1-S-S-E1 formation. B, testing the effectiveness of BFA in preventing proteins from reaching the cell surface. Cell surface proteins were biotinylated with a membrane-impermeable, amine-reactive biotin derivative, and the biotinylated fraction was purified from whole cell lysate using neutravidin bead pull-down. Top: representative immunoblot image of W(CL) and b(iotinylated) fraction probed by Ab targeting Q1. Bottom: immunoblot of a cytoplasmic protein, actin, confirming no contamination by cytoplasmic proteins in biotinylated fraction. D, lower: densitometry quantification of percentage cell surface Q1. Under the control conditions, Q1-Q147C reached the same level of cell surface expression as Q1-WT. Incubation with BFA markedly reduced surface level of Q1-Q147C, indicating that BFA treatment effectively prevented Q1-Q147C (and E1-G40C, by inference) from reaching the cell surface. C, quantification of the degree of Q1-S-S-E1 formation by non-reducing SDS-PAGE. Shown is a representative immunoblot image. WCL from cells expressing Q1-Q147C and E1-G40C cultured under the control conditions or in the presence of BFA (–BFA and +BFA, respectively), with or without reducing agent pretreatment (DTT, breaking disulphide bonds) were analyzed by immunoblotting with Ab targeting Q1. The 80 kDa band represented disulphide-linked Q1-Q147C/E1-G40C, confirmed by DTT treatment, which collapsed the 80 kDa band into the 60 kDa band. D, upper: quantification of KCNQ1/KCNE1 assembly (80 kDa divided by 60 kDa band intensity). Incubation with BFA significantly reduced KCNQ1/KCNE1 assembly, supporting the scenario shown on right in (A). In (D), symbols of different styles and colours represent data from individual experiments. For illustration purpose, the symbols are connected by lines of the same colours. t test, ‘+BFA’ vs. ‘-BFA’, P < 0.05.$

Journal: The Journal of Physiology

Article Title: Delayed KCNQ1/KCNE1 assembly on the cell surface helps I_Ks fulfil its function as a repolarization reserve in the heart

doi: 10.1113/jp281773

Figure Lengend Snippet: Figure 9. Testing the preference of KCNQ1 and KCNE1 assembly inside cells or on the cell surface A, experimental design. COS-7 cells transfected with Q1-Q147C and E1-G40C were cultured under the control conditions or in the presence of BFA (25 μg mL–1, preventing proteins from reaching the cell surface). The two Cys side chains (147C on Q1 and 40C on E1) could come very close to each other to form a disulphide bond in the open state of a fully-assembled KCNQ1/KCNE1 channel. Therefore, the abundance of Q1-S-S-E1 reported the degree of KCNQ1/KCNE1 assembly. Two scenarios are depicted. Left: Q1 and E1 preferentially assemble inside cells. Forcing Q1-Q147C and E1-G40C to stay inside cells by BFA would not prevent, or even encourage, Q1-S-S-E1 formation. Right: Q1 and E1 preferentially assemble on the cell surface. BFA would prevent Q1-S-S-E1 formation. B, testing the effectiveness of BFA in preventing proteins from reaching the cell surface. Cell surface proteins were biotinylated with a membrane-impermeable, amine-reactive biotin derivative, and the biotinylated fraction was purified from whole cell lysate using neutravidin bead pull-down. Top: representative immunoblot image of W(CL) and b(iotinylated) fraction probed by Ab targeting Q1. Bottom: immunoblot of a cytoplasmic protein, actin, confirming no contamination by cytoplasmic proteins in biotinylated fraction. D, lower: densitometry quantification of percentage cell surface Q1. Under the control conditions, Q1-Q147C reached the same level of cell surface expression as Q1-WT. Incubation with BFA markedly reduced surface level of Q1-Q147C, indicating that BFA treatment effectively prevented Q1-Q147C (and E1-G40C, by inference) from reaching the cell surface. C, quantification of the degree of Q1-S-S-E1 formation by non-reducing SDS-PAGE. Shown is a representative immunoblot image. WCL from cells expressing Q1-Q147C and E1-G40C cultured under the control conditions or in the presence of BFA (–BFA and +BFA, respectively), with or without reducing agent pretreatment (DTT, breaking disulphide bonds) were analyzed by immunoblotting with Ab targeting Q1. The 80 kDa band represented disulphide-linked Q1-Q147C/E1-G40C, confirmed by DTT treatment, which collapsed the 80 kDa band into the 60 kDa band. D, upper: quantification of KCNQ1/KCNE1 assembly (80 kDa divided by 60 kDa band intensity). Incubation with BFA significantly reduced KCNQ1/KCNE1 assembly, supporting the scenario shown on right in (A). In (D), symbols of different styles and colours represent data from individual experiments. For illustration purpose, the symbols are connected by lines of the same colours. t test, ‘+BFA’ vs. ‘-BFA’, P < 0.05.

Techniques: Transfection, Cell Culture, Control, Membrane, Purification, Western Blot, Expressing, Incubation, SDS Page

Figure 10. KCNQ1 and KCNE1 are closer to each other on the cell surface than inside cells A, experimental design. Live COS-7 cells coexpressing Q1-GFP and HA-E1-dsR were incubated with Q1 ext rabbit Ab and HA mouse Ab, followed by Alexa647 goat anti-rabbit and Alexa405 goat anti-mouse. Therefore, far red and blue fluorescence signals reported cell surface Q1-GFP and HA-E1-dsR. Green and red fluorescence signals reported total Q1-GFP and HA-E1-dsR. B, representative raw images of far red and blue channels (cell surface Q1-GFP and HA-E1-dsR), and green/red channels (cytoplasmic counterparts). The raw images were deconvoluted, chromatic aberration corrected and segmented to identify fluorescence foci. C, segmented images of the same cells, showing fluorescence foci. The co-ordinates of fluorescence foci were used to calculate the nearest neighbour distances. D, distribution of distances between nearest Q1-GFP neighbours to HA-E1-dsR on the cell surface (top, far red foci to blue foci) and in cytoplasm (bottom, green foci to red foci) based on 22,648 and 47,119 nearest neighbour distances, respectively, from 10 cells. The median distances were 0.44 and 1.76 μm, respectively.

Journal: The Journal of Physiology

Article Title: Delayed KCNQ1/KCNE1 assembly on the cell surface helps I_Ks fulfil its function as a repolarization reserve in the heart

doi: 10.1113/jp281773

Figure Lengend Snippet: Figure 10. KCNQ1 and KCNE1 are closer to each other on the cell surface than inside cells A, experimental design. Live COS-7 cells coexpressing Q1-GFP and HA-E1-dsR were incubated with Q1 ext rabbit Ab and HA mouse Ab, followed by Alexa647 goat anti-rabbit and Alexa405 goat anti-mouse. Therefore, far red and blue fluorescence signals reported cell surface Q1-GFP and HA-E1-dsR. Green and red fluorescence signals reported total Q1-GFP and HA-E1-dsR. B, representative raw images of far red and blue channels (cell surface Q1-GFP and HA-E1-dsR), and green/red channels (cytoplasmic counterparts). The raw images were deconvoluted, chromatic aberration corrected and segmented to identify fluorescence foci. C, segmented images of the same cells, showing fluorescence foci. The co-ordinates of fluorescence foci were used to calculate the nearest neighbour distances. D, distribution of distances between nearest Q1-GFP neighbours to HA-E1-dsR on the cell surface (top, far red foci to blue foci) and in cytoplasm (bottom, green foci to red foci) based on 22,648 and 47,119 nearest neighbour distances, respectively, from 10 cells. The median distances were 0.44 and 1.76 μm, respectively.

Techniques: Incubation, Fluorescence

$Figure 12. Microtubule plus-end binding protein 1 (EB1) binds KCNQ1 in rat ventricular myocytes and is colocalized with myocyte surface KCNQ1 Rat ventricular myocytes transduced with Q1-GFP and HA-E1-dsR were cultured for 36 h and used for two types of experiments. A, coimmunoprecipitation: three equal aliquots of myocyte whole cell lysate (WCL, in 1% Triton X-100) were incubated with immunoprecipitating (IP) antibodies: GFP rabbit Ab, HA mouse Ab and EB1 rat Ab, in the presence of protein A/G beads. WCL, negative control ([–], WCL incubated with protein A/G beads without IP Ab) and positive immunoprecipitate ([+]) were fractionated by SDS-PAGE. Proteins were transferred to PVDF membrane, and the membrane was cut below the 75 kDa size marker. The upper portion was probed with GFP goat Ab and the lower portion was probed with dsR rabbit Abs (left). After ECL, the lower portion was stripped and reprobed with EB1 rat Ab. For illustration purposes, irrelevant bands were removed. EB1 monomer and dimer bands are noted as 1× and 2×. Q1-GFP and native EB1 dimer co-immunoprecipitated reciprocally (indicated by ‘$’). HA-E1-dsR and Q1-GFP co-immunoprecipitated reciprocally (indicated by ‘∗’ and ‘#’). However, there was no sign of co-immunoprecipitation between HA-E1-dsR and EB1. ‘IgG’: GFP rabbit Ab heavy chain detected by the secondary goat anti-rabbit Ab. B, immunofluorescence/confocal microscopy: fixed myocytes without membrane permeabilization were incubated with Q1 ext rabbit Ab to label cell surface Q1. After membrane permeabilization, myocytes were incubated with GFP mouse Ab (binding total pool of Q1-GFP) and EB1 rat Ab (binding native EB1), followed by Alexa568-, Alexa488- and Alexa647-conjugated secondary Abs targeting rabbit, mouse and rat Abs, respectively. Shown are representative images from a myocyte at the cell centre and close to the top surface, acquired with a Zeiss 880 microscope in the Airyscan mode.$

Journal: The Journal of Physiology

Article Title: Delayed KCNQ1/KCNE1 assembly on the cell surface helps I_Ks fulfil its function as a repolarization reserve in the heart

doi: 10.1113/jp281773

Figure Lengend Snippet: Figure 12. Microtubule plus-end binding protein 1 (EB1) binds KCNQ1 in rat ventricular myocytes and is colocalized with myocyte surface KCNQ1 Rat ventricular myocytes transduced with Q1-GFP and HA-E1-dsR were cultured for 36 h and used for two types of experiments. A, coimmunoprecipitation: three equal aliquots of myocyte whole cell lysate (WCL, in 1% Triton X-100) were incubated with immunoprecipitating (IP) antibodies: GFP rabbit Ab, HA mouse Ab and EB1 rat Ab, in the presence of protein A/G beads. WCL, negative control ([–], WCL incubated with protein A/G beads without IP Ab) and positive immunoprecipitate ([+]) were fractionated by SDS-PAGE. Proteins were transferred to PVDF membrane, and the membrane was cut below the 75 kDa size marker. The upper portion was probed with GFP goat Ab and the lower portion was probed with dsR rabbit Abs (left). After ECL, the lower portion was stripped and reprobed with EB1 rat Ab. For illustration purposes, irrelevant bands were removed. EB1 monomer and dimer bands are noted as 1× and 2×. Q1-GFP and native EB1 dimer co-immunoprecipitated reciprocally (indicated by ‘$’). HA-E1-dsR and Q1-GFP co-immunoprecipitated reciprocally (indicated by ‘∗’ and ‘#’). However, there was no sign of co-immunoprecipitation between HA-E1-dsR and EB1. ‘IgG’: GFP rabbit Ab heavy chain detected by the secondary goat anti-rabbit Ab. B, immunofluorescence/confocal microscopy: fixed myocytes without membrane permeabilization were incubated with Q1 ext rabbit Ab to label cell surface Q1. After membrane permeabilization, myocytes were incubated with GFP mouse Ab (binding total pool of Q1-GFP) and EB1 rat Ab (binding native EB1), followed by Alexa568-, Alexa488- and Alexa647-conjugated secondary Abs targeting rabbit, mouse and rat Abs, respectively. Shown are representative images from a myocyte at the cell centre and close to the top surface, acquired with a Zeiss 880 microscope in the Airyscan mode.

Techniques: Binding Assay, Transduction, Cell Culture, Incubation, Negative Control, SDS Page, Membrane, Marker, Immunoprecipitation, Immunofluorescence, Confocal Microscopy, Microscopy

$Figure 13. EB1 promotes Q1-GFP to reach the cell surface and, although wild-type Q1-GFP preferentially binds EB1 dimer, an LQT1-associated, trafficking defective, mutation (Y111C) preferentially binds EB1 monomer A, fluorescence images of COS-7 cells co-transfected with Q1-GFP and EB1-tdTomato (tdTomato stands for ‘tandem dimer of Tomato proteins’, which behaves as a monomer). Top: in the same field, Q1-GFP in cells coexpressing both constructs had Q1-GFP in the cell periphery (∗), whereas those expressing Q1-GFP without EB1-tdTomato had Q1-GFP in the ER (#). Bottom: Q1-GFP in the cell coexpressing both constructs had more Q1-GFP on the cell surface (detected by Q1 ext Ab IF under unpermeabilized conditions as described for Fig. 4A) (∗) than the cell expressing Q1-GFP alone (#). B, top: KCNQ1 N-terminal amino acid sequence (from amino acids 1 to 123, ending at the beginning of S1), highlighting two putative EB1-binding sites and position 111. Bottom: fluorescence images of Q1-GFP (wild-type, ‘WT’, or Y111C), dsRed-ER and their merged image. Both WT and Y111C resided in the ER. C, immunoblot images of Q1-GFP WT and Y111C in whole cell lysate (W) and on the cell surface (b, biotinylated fraction, as described for Fig. 9B). Immunoblotting of actin confirmed lack of cytosolic proteins in the biotinylated fraction. Y111C had much lower total and cell surface protein levels than WT. D, Comparison of EB1 co-immunoprecipitation with Q1-GFP between WT and Y111C. The experimental procedure and presentation format are similar to those in Fig. 11B. Cells expressing Q1-GFP, WT or Y111C, were lysed in 1% Triton, and whole cell lysates were subject to immunoprecipitation with GFP rabbit Ab. WCL, super (WCL after immunoprecipitation), [–] IP (incubation with protein A/B beads without Ab) and [+] IP were analyzed by immunoblotting with Q1 (C-20 goat) Ab and EB1 rat Ab. EB1 monomer and dimer bands are marked (1× and 2×). Experiments in (A), (B) and (C) were conducted via COS-7 expression. The experiment in (D) was conducted in HEK293 cells.$

Journal: The Journal of Physiology

Article Title: Delayed KCNQ1/KCNE1 assembly on the cell surface helps I_Ks fulfil its function as a repolarization reserve in the heart

doi: 10.1113/jp281773

Figure Lengend Snippet: Figure 13. EB1 promotes Q1-GFP to reach the cell surface and, although wild-type Q1-GFP preferentially binds EB1 dimer, an LQT1-associated, trafficking defective, mutation (Y111C) preferentially binds EB1 monomer A, fluorescence images of COS-7 cells co-transfected with Q1-GFP and EB1-tdTomato (tdTomato stands for ‘tandem dimer of Tomato proteins’, which behaves as a monomer). Top: in the same field, Q1-GFP in cells coexpressing both constructs had Q1-GFP in the cell periphery (∗), whereas those expressing Q1-GFP without EB1-tdTomato had Q1-GFP in the ER (#). Bottom: Q1-GFP in the cell coexpressing both constructs had more Q1-GFP on the cell surface (detected by Q1 ext Ab IF under unpermeabilized conditions as described for Fig. 4A) (∗) than the cell expressing Q1-GFP alone (#). B, top: KCNQ1 N-terminal amino acid sequence (from amino acids 1 to 123, ending at the beginning of S1), highlighting two putative EB1-binding sites and position 111. Bottom: fluorescence images of Q1-GFP (wild-type, ‘WT’, or Y111C), dsRed-ER and their merged image. Both WT and Y111C resided in the ER. C, immunoblot images of Q1-GFP WT and Y111C in whole cell lysate (W) and on the cell surface (b, biotinylated fraction, as described for Fig. 9B). Immunoblotting of actin confirmed lack of cytosolic proteins in the biotinylated fraction. Y111C had much lower total and cell surface protein levels than WT. D, Comparison of EB1 co-immunoprecipitation with Q1-GFP between WT and Y111C. The experimental procedure and presentation format are similar to those in Fig. 11B. Cells expressing Q1-GFP, WT or Y111C, were lysed in 1% Triton, and whole cell lysates were subject to immunoprecipitation with GFP rabbit Ab. WCL, super (WCL after immunoprecipitation), [–] IP (incubation with protein A/B beads without Ab) and [+] IP were analyzed by immunoblotting with Q1 (C-20 goat) Ab and EB1 rat Ab. EB1 monomer and dimer bands are marked (1× and 2×). Experiments in (A), (B) and (C) were conducted via COS-7 expression. The experiment in (D) was conducted in HEK293 cells.

Techniques: Mutagenesis, Fluorescence, Transfection, Construct, Expressing, Sequencing, Binding Assay, Western Blot, Comparison, Immunoprecipitation, Incubation

Figure 14. Searching for KCNQ1 and KCNE1 interactors in their trafficking paths A, Venn diagram of proteins found in KCNQ1-GFP immunoprecipitate (109 proteins) and in HA-KCNE1-dsR immunoprecipitate (109 proteins). Sixty eight are shared. B, cartoon of KCNQ1 and KCNE1 forward trafficking, assembly and endocytosis.

Journal: The Journal of Physiology

Article Title: Delayed KCNQ1/KCNE1 assembly on the cell surface helps I_Ks fulfil its function as a repolarization reserve in the heart

doi: 10.1113/jp281773

Figure Lengend Snippet: Figure 14. Searching for KCNQ1 and KCNE1 interactors in their trafficking paths A, Venn diagram of proteins found in KCNQ1-GFP immunoprecipitate (109 proteins) and in HA-KCNE1-dsR immunoprecipitate (109 proteins). Sixty eight are shared. B, cartoon of KCNQ1 and KCNE1 forward trafficking, assembly and endocytosis.

Techniques:

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