<資料>新西蘭,加奈陀,印度の中央銀行設立計畫

<p><b>Panels A–C,</b> representative images obtained from confocal microscopy of transiently transfected HEK cells. R835Q mutant channels do not appear differently distributed in comparison to WT KCNH2. <b>D</b>, Immunoblots using anti-erg1 (2, 5 µg/mL) of crude membrane extracts from heterologous expression in HEK cells, indicating equal protein expression level. Illustrated below are endoplasmic reticulum and plasma membrane fraction with respective markers of equal protein loading (calnexin for endoplasmic reticulum, spectrin for plasma membranes). Exemplary Western blots of preparations at physiological temperature (37°C) and 40°C (to simulate febrile illness of the index patient’s brother) are shown. No differences were observed in Kv11.1-WT or Kv11.1-R835Q plasma membrane representation of the two proteins under the two conditions. ER: endoplasmic reticulum fraction; PM: plasma-membrane fraction; WT: wild type; NT: non-transfected cells.</p

mRNA expression levels.

<p>Mean±SEM results of quantitative real-time PCR for mRNA expression of KCNE1 (<b>A</b>) and Kv7.1 (<b>B</b>) from HEK cells transfected with Kv7.1 and the various KCNE1 constructs (n = 5 transfections for each panel). Data were normalized to ß-actin expression. There were no differences in mRNA transcription that could account for changes in membrane currents.</p

Immunofluorescent studies of membrane representation.

<p>Confocal images of HEK cells transfected with Kv7.1 and flag-tagged KCNE1 constructs. The top row localizes Kv7.1 (red) within cells. The middle row shows the distribution of KCNE1 (green), bottom panels illustrate the plasma-membrane marker pan-cadherin (blue). Bars represent 10 µm. The respective bar graphs below represent the mean±SEM data of Kv7.1 and KCNE1 correlation with plasma-membrane marker pan-cadherin. White bars represent Kv7.1/pan-cadherin ratio, hatched bars are KCNE1/pan-cadherin ratio. Results are from 4-9 experiments each. Cad – cadherin, other abbreviations as above.</p

Electrophysiological properties.

<p><b>A–F</b>, representative ionic currents elicited by whole-cell patch-clamp with the protocol shown in inset A. <b>G</b>, current-voltage relations of mean±SEM depolarization induced activating step-current densities from cells transfected with Kv7.1 plus various KCNE1 constructs. <b>H,</b> mean±SEM current-voltage relationships of repolarization induced tail-current densities. P-values are shown for currents recorded from cells transfected with Kv7.1+’38S’ vs. all other constructs. The remainder of constructs did not lead to significantly different current sizes compared with ‘38G’ (<i>P</i> = n.s.). TP – test potential.</p

Biophysical characteristics.

<p><b>A</b>, illustrates half-activation voltages (V₅₀) of currents resulting from expression of Kv7.1 with respective constructs. Mean±SEM V₅₀ values were similar between constructs ‘38S’: 5.8±3.7 mV, ‘38G’: −2.7±4.1 mV, ‘Δ1-38’: 6.0±4.1 mV, ‘linker’: 1.0±2.5 mV, ‘38S-3xA’: 3.4±8.6 mV, ‘38G-3xA’: 8.7±6.8 mV; <i>P</i> = n.s vs. ‘38S’. Lines shown are Boltzmann fits to mean data (obtained with the formula: A = A₀/(1+exp[(V₅₀-V)/S])). <b>B</b> shows results of mono-exponential fits (y = A^(−t/τ)+C) to activating currents with time-constants plotted over a test potential of 0 mV. <b>C</b> shows results of mono-exponential fits to deactivating ionic currents. Currents obtained from co-transfection of Kv7.1 with ‘linker’ and ‘38S-3xA’ deactivated more slowly than currents obtained with the remainder of the constructs (<i>P</i><0.05 vs. ‘38S’). TP – test potential.</p

Primers for KCNE1 mutagenesis.

<p>Mutation containing sequences in italic, underlined.</p

Primers for quantitation of Kv7.1 and KCNE1 mRNA.

<p>F – forward primer, R – reverse primer.</p

Schematic of KCNE1 and constructs.

<p>This figure schematically illustrates KCNE1 structure and mutants used in the present study. <b>A, left</b>, alignment of KCNE1 sequences from various mammalian species. Grey underlines conserved residues. Glycine at position 38 is not strongly conserved among species providing no first-glance evidence for evolutionary importance. <b>A, right</b>, schematic of KCNE1 at the membrane with the N-terminal part oriented towards the cell exterior and C-terminus towards the cytosol. <b>B</b>, schematic of KCNE1 N-terminal constructs and mutations created for the present study. Ten N-terminal amino-acids (AA) illustrate differences between KCNE1 constructs. Position 38 carries a glycine in the wild-type (common allele) and is associated with atrial fibrillation. Position 38 carries a serine in the prevalent single nucleotide polymorphism. One of the constructs contained an N-terminal truncation (‘Δ1-38’), another one (‘linker’) replaced position 38 by 5 alanines. Additionally, three positively-charged arginines at positions 32, 33 and 36 have been exchanged for alanines in order to probe the role of these AA in KCNE1 function.</p

Immunodetection of heterologously expressed constructs.

<p>Immunodetection of flag-tagged KCNE1 constructs. <b>A</b>, crude membrane preparations from HEK cells transiently transfected with respective KCNE1 constructs. Actin (∼42 kD) is shown as loading control. <b>B</b>, effective co-immunoprecipitation (IP) occurred for Kv7.1 with all flag-tagged KCNE1 constructs. The upper blot shows protein samples from HEK cells precipitated by anti-flag and bands detected by anti-Kv7.1. The lower blot shows samples precipitated by anti-Kv7.1 and bands detected by anti-flag antibody (n = 2 experiments each). <b>C</b> shows respective confocal images of KCNE1 subunits expressed without Kv7.1. Bars represent 5 µm. Images are representative of at least 5 different experiments. NT – non-transfected control, IP – immunoprecipitation.</p

Membrane representation of Kv7.1 and KCNE1.

<p>This figure illustrates results from cell compartment fractionation experiments yielding plasma-membrane (PM) and endoplasmic reticulum (ER) fractions. <b>A, B</b> show representative blots of cells transfected with Kv7.1 and KCNE1 constructs. The upper part of each panel shows a blot of plasma-membrane proteins, the lower part of each panel illustrates the endoplasmic reticulum-fraction. Spectrin was used as loading control for plasma-membrane, calnexin for endoplasmic reticulum. Freedom from contamination of plasma-membrane and endoplasmic reticulum fractions is illustrated by representation of spectrin and calnexin, respectively. <b>A</b>, mean±SEM optical densities (OD) quantified from immunoblots of Kv7.1 illustrate an increase in Kv7.1 protein at the plasma-membrane and <b>B</b> shows a similarly greater amount of KCNE1-38S protein at the plasma-membrane. The respective bar graphs below represent the mean±SEM data. White bars represent Kv7.1 or KCNE1 protein content normalized to spectrin as loading control for plasma-membrane fractions, black bars represent protein content normalized to calnexin as loading control for endoplasmic reticulum fractions. Data of plasma-membrane protein content were thereafter normalized against endoplasmic reticulum (n = 4).</p