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

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

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

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

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

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