Journal: eLife

Article Title: The angiopoietin-like protein ANGPTL4 catalyzes unfolding of the hydrolase domain in lipoprotein lipase and the endothelial membrane protein GPIHBP1 counteracts this unfolding

doi: 10.7554/eLife.20958

Figure Lengend Snippet: Panel A shows a sequence alignment of the first 55 residues of fully processed human ANGPTL3, ANGPTL4, and ANGPTL8. Identical sequences are highlighted by blue letters. The filled red box highlights the two-residue acidic motif at the start of the N-terminal α-helix, followed by a conserved α-helical region (open box). The two cysteine residues unique to ANGPTL4 are shown in yellow boxes. Panel B shows the time-dependent unfolding of 10 µM LPL by 1 µM ANGPTL3 ( green circles ) as defined by the appearance of bimodality in the isotope envelopes for peptide 131–165. For comparison the unfolding of LPL by 1 µM ANGPTL4 1–159 is shown as solid (wild-type) and hatched (E15K) gray lines. Spontaneous unfolding is shown by black triangles. Panel C shows the residual lipolytic activity of 10 µM LPL incubated for 10 min alone or in the presence of 1 µM ANGPTL3, 1 µM ANGPTL3 and 30 µM GPIHBP1, or 1 µM ANGPTL4 1–159 . DOI: http://dx.doi.org/10.7554/eLife.20958.012

Article Snippet: Full-length ANGPTL3 produced in HEK293 cells was purchased from ProSpec (Ness-Ziona, Israel).

Techniques: Sequencing, Residue, Comparison, Activity Assay, Incubation

Journal: Physiological Reports

Article Title: Multiple mechanisms underlie reduced potassium conductance in the p.T1019PfsX38 variant of hERG

doi: 10.14814/phy2.15341

Figure Lengend Snippet: Step‐ramp voltage clamp protocol on cells expressing WT‐ or T1019PfsX38‐hERG. (ai and bi) Example current recordings from HEK293 cells that were transfected with WT‐ (ai) or T1019PfsX38‐hERG (bi) channels, as indicated. The currents were elicited using the protocol shown above A (V hold = –80 mV). The grey arrows mark the level where V C was 50 mV. (aii and bii) Current recordings from (ai) and (bi) presented as functions of membrane voltage. The ramp durations in (aii) and (bii) were colour‐coded as indicated in the box. The dotted lines represent ramp slope lines between 50 and –80 mV. (c) Average densities of peak current amplitudes plotted as functions of ramp durations. (d) Average values of area under each curve of current densities (I integral ) at different ramp durations. (e) Average values of membrane voltages at the time of occurance of peak current amplitudes. Data are mean ± SEM, * p < 0.05; WT‐hERG, n = 12, grey circles; T1019PfsX38‐hERG, n = 11 (except in E where n = 7 at 10 ms ramp duration), blue circles. At 20 ms in d and 40 ms in c and d the individual data points obtained from T1019PfsX38‐hERG did not meet normal statistical distribution criteria (Shapiro–Wilk test), in which case Mann–Whitney rank sum test was used for statistical comparison only at these three data points. Other details as in Materials and Methods and Results sections.

Article Snippet: For patch‐clamp recording, we used human embryonic kidney 293 (HEK‐293) cell line (passage <40) (ECACC, MERCK, Catalog # 85120602) grown in Dulbecco's Modified Eagle Medium (DMEM) high glucose (Sigma‐Aldrich, St. Louis, MO, USA).

Techniques: Expressing, Transfection, MANN-WHITNEY

Journal: Physiological Reports

Article Title: Multiple mechanisms underlie reduced potassium conductance in the p.T1019PfsX38 variant of hERG

doi: 10.14814/phy2.15341

Figure Lengend Snippet: The current densities and the voltage dependence of activation of WT‐ and T1019PfsX38‐hERG channels. (a, b) Representative whole‐cell currents recorded from WT‐ (a) and T1019PfsX38‐hERG (b) channels expressed in HEK293 cells. The currents were elicited using the protocol shown above A (V hold = −80 mV). The grey arrows mark the level where V C was −80 mV. (c) Quantification of step currents measured as average current densities in the last 50 ms of the depolarizing test potentials. (d) Quantification of peak tail currents measured after stepping to −40 mV. The continuous lines in (d) are best fits to the average data using the Boltzmann equation. (e, f) Whisker plots of the V mid and k values predicted by fitting the data in (d). Data in (c) and (d) are mean ± SEM; WT‐hERG, n = 5, grey circles and triangles; T1019PfsX38‐hERG, n = 6, blue circles and triangles. Other details as in Materials and Methods and Results sections.

Article Snippet: For patch‐clamp recording, we used human embryonic kidney 293 (HEK‐293) cell line (passage <40) (ECACC, MERCK, Catalog # 85120602) grown in Dulbecco's Modified Eagle Medium (DMEM) high glucose (Sigma‐Aldrich, St. Louis, MO, USA).

Techniques: Activation Assay, Whisker Assay

Journal: Physiological Reports

Article Title: Multiple mechanisms underlie reduced potassium conductance in the p.T1019PfsX38 variant of hERG

doi: 10.14814/phy2.15341

Figure Lengend Snippet: Kinetics of activation of WT‐ and T1019PfsX38‐hERG channels. (a, b) Example whole cell currents recorded from WT‐ (a) and T1019PfsX38‐hERG (b) channels expressed in HEK293 cells. The currents were elicited using the protocol shown above (a). (V hold = −100 mV). The example recordings in (a) and (b) were from cells activated at 80 mV. For better visualization, the x‐axes in (a) and (b) were presented in logarithmic scales. The grey and black arrows mark the levels where V C was −100 mV and 80 mV, respectively. (c–e) Quantification of the negative peak tail currents obtained after activating the channels at 80 mV (c), 40 mV (d) or 0 mV (e) and presented as fractions of the peak inward tail currents after depolarization to 80 mV for 5.12 s in the same cell. The continuous lines in (c–e) are best fits to the average data using an exponential function. (f) Whisker plots and individual data points of the time constants of activation of WT‐ and T1019PfsX38‐hERG channels. Data are mean ± SEM; WT‐hERG, grey circles/lines, n = 7–8 (c), n = 5–6 (d), n = 5 (e) and n = 4–7 (f); T1019PfsX38‐hERG, blue circles / lines, n = 7–9 (c), n = 6–7 (d), n = 6 (e) and n = 4–8 (f). Other details as in Materials and Methods, and Results sections.

Article Snippet: For patch‐clamp recording, we used human embryonic kidney 293 (HEK‐293) cell line (passage <40) (ECACC, MERCK, Catalog # 85120602) grown in Dulbecco's Modified Eagle Medium (DMEM) high glucose (Sigma‐Aldrich, St. Louis, MO, USA).

Techniques: Activation Assay, Whisker Assay

Journal: Physiological Reports

Article Title: Multiple mechanisms underlie reduced potassium conductance in the p.T1019PfsX38 variant of hERG

doi: 10.14814/phy2.15341

Figure Lengend Snippet: The voltage dependence of recovery from inactivation of WT‐ and T1019PfsX38‐hERG channels. (a, b) Representative whole cell currents recorded from WT‐ (a) and T1019PfsX38‐hERG (b) channels expressed in HEK293 cells. The currents were elicited using the protocol shown above A (V hold = −80 mV). The grey arrows mark the level where V C was 50 mV. For better representation, isolated segments from (a) and (b) at different test potentials are shown separately. (c) Quantification of peak current amplitudes measured after stepping to the test potential and normalized to the maximum inward current amplitudes. Data are mean ± SEM, * p < 0.05; WT‐hERG, n = 6, grey; T1019PfsX38‐hERG, n = 7, blue. Other details as in Materials and Methods and Results sections.

Article Snippet: For patch‐clamp recording, we used human embryonic kidney 293 (HEK‐293) cell line (passage <40) (ECACC, MERCK, Catalog # 85120602) grown in Dulbecco's Modified Eagle Medium (DMEM) high glucose (Sigma‐Aldrich, St. Louis, MO, USA).

Techniques: Isolation

Journal: Physiological Reports

Article Title: Multiple mechanisms underlie reduced potassium conductance in the p.T1019PfsX38 variant of hERG

doi: 10.14814/phy2.15341

Figure Lengend Snippet: The time dependence of WT‐ and T1019PfsX38‐hERG recovery from inactivation and deactivation. (a, b) Representative whole‐cell currents recorded from WT‐ (a) and T1019PfsX38‐hERG (b) channels expressed in HEK293 cells. The currents were elicited using the protocol shown above A (V hold = −80 mV). The example recordings in (a) and (b) were from cells activated at 80 mV. The grey arrows mark the level where V C was 80 mV. (c–e) Quantification of the negative peak tail currents obtained after 80 mV (c) 40 mV (d) and 0 mV (e) test potentials. The values in (c) to (e) are shown after normalization, the normalization range was from the lowest peak current amplitudes (at 0 mV) to the highest peak current amplitudes (at 80 mV) in the same cell. The continuous lines in (c–e) are best fits to the average data using a 2‐component exponential function, τ 1 is the time constant for recovery from inactivation and τ 2 is the time constant for deactivation. Data are mean ± SEM, WT‐hERG, n = 4, grey circles; T1019PfsX38‐hERG, n = 4, blue circles. Other details as in Materials and Methods and Results sections.

Article Snippet: For patch‐clamp recording, we used human embryonic kidney 293 (HEK‐293) cell line (passage <40) (ECACC, MERCK, Catalog # 85120602) grown in Dulbecco's Modified Eagle Medium (DMEM) high glucose (Sigma‐Aldrich, St. Louis, MO, USA).

Techniques:

Journal: Physiological Reports

Article Title: Multiple mechanisms underlie reduced potassium conductance in the p.T1019PfsX38 variant of hERG

doi: 10.14814/phy2.15341

Figure Lengend Snippet: The voltage dependence of inactivation of WT‐ and T1019PfsX38‐hERG channels. (a, b) Representative whole‐cell currents recorded from WT‐ (a) and T1019PfsX38‐hERG (b) channels expressed in HEK293 cells. The currents were elicited using the protocol shown above A (V hold = −80 mV). The grey arrows mark the level where V C was 40 mV. (c) Quantification of currents measured at 1 ms before the end of the test potential and normalized to the maximal inward peak current amplitude. (d) Quantification of normalized tail currents measured at 5 ms after stepping to 20 mV. The grey and blue continuous lines in d are best fits to the average data after correction for deactivation. (e, f) Whisker plots of the V mid and the k values as predicted by fitting the raw (open boxes) and the corrected (dashed boxes) data in d. Data are mean ± SEM; WT‐hERG, n = 5, grey boxes, squares, circles, and lines; T1019PfsX38‐hERG, n = 5–6, blue boxes, squares, circles, and lines. Other details as in Materials and Methods and Results sections.

Article Snippet: For patch‐clamp recording, we used human embryonic kidney 293 (HEK‐293) cell line (passage <40) (ECACC, MERCK, Catalog # 85120602) grown in Dulbecco's Modified Eagle Medium (DMEM) high glucose (Sigma‐Aldrich, St. Louis, MO, USA).

Techniques: Whisker Assay

Journal: Physiological Reports

Article Title: Multiple mechanisms underlie reduced potassium conductance in the p.T1019PfsX38 variant of hERG

doi: 10.14814/phy2.15341

Figure Lengend Snippet: The inactivation rates of WT‐ and T1019PfsX38‐hERG channels. (a, b) Representative whole‐cell currents recorded from WT‐ (a) and T1019PfsX38‐hERG (b) channels expressed in HEK293 cells. The currents were elicited using the protocol shown above A (V hold = −80 mV). The grey arrows mark the level where V C was 50 mV. (c) Quantification of the rate constants of channel inactivation measured by fitting the tail currents at the different tested potentials using a standard exponential function. Data are mean ± SEM, * p < 0.05; WT‐hERG, n = 7 except at −10 mV ( n = 6), grey circles; T1019PfsX38‐hERG, n = 5, blue circles. Other details as in Materials and Methods and Results sections.

Article Snippet: For patch‐clamp recording, we used human embryonic kidney 293 (HEK‐293) cell line (passage <40) (ECACC, MERCK, Catalog # 85120602) grown in Dulbecco's Modified Eagle Medium (DMEM) high glucose (Sigma‐Aldrich, St. Louis, MO, USA).

Techniques: