Division of Cardiology, Section of Electrophysiology, JW Goethe-University, Frankfurt, Germany.
PLoS One. 2011;6(11):e26967. doi: 10.1371/journal.pone.0026967. Epub 2011 Nov 4.
The slow delayed rectifier current (I(Ks)) is important for cardiac action potential termination. The underlying channel is composed of Kv7.1 α-subunits and KCNE1 β-subunits. While most evidence suggests a role of KCNE1 transmembrane domain and C-terminus for the interaction, the N-terminal KCNE1 polymorphism 38G is associated with reduced I(Ks) and atrial fibrillation (a human arrhythmia). Structure-function relationship of the KCNE1 N-terminus for I(Ks) modulation is poorly understood and was subject of this study.
We studied N-terminal KCNE1 constructs disrupting structurally important positively charged amino-acids (arginines) at positions 32, 33, 36 as well as KCNE1 constructs that modify position 38 including an N-terminal truncation mutation. Experimental procedures included molecular cloning, patch-clamp recording, protein biochemistry, real-time-PCR and confocal microscopy.
All KCNE1 constructs physically interacted with Kv7.1. I(Ks) resulting from co-expression of Kv7.1 with non-atrial fibrillation '38S' was greater than with any other construct. Ionic currents resulting from co-transfection of a KCNE1 mutant with arginine substitutions ('38G-3xA') were comparable to currents evoked from cells transfected with an N-terminally truncated KCNE1-construct ('Δ1-38'). Western-blots from plasma-membrane preparations and confocal images consistently showed a greater amount of Kv7.1 protein at the plasma-membrane in cells co-transfected with the non-atrial fibrillation KCNE1-38S than with any other construct.
The results of our study indicate that N-terminal arginines in positions 32, 33, 36 of KCNE1 are important for reconstitution of I(Ks). Furthermore, our results hint towards a role of these N-terminal amino-acids in membrane representation of the delayed rectifier channel complex.
缓慢延迟整流电流（I(Ks)）对于心脏动作电位的终止非常重要。该通道由 Kv7.1α 亚基和 KCNE1β 亚基组成。虽然大多数证据表明 KCNE1 跨膜域和 C 末端对于相互作用很重要，但 KCNE1N 末端的 38G 多态性与 I(Ks)减少和心房颤动（人类心律失常）有关。KCNE1N 末端对 I(Ks)调节的结构-功能关系尚不清楚，这也是本研究的主题。
我们研究了破坏结构上重要的带正电荷的氨基酸（精氨酸）的 KCNE1N 末端结构，这些氨基酸位于 32、33、36 位，以及修饰 38 位的 KCNE1 结构，包括 N 末端截短突变。实验程序包括分子克隆、膜片钳记录、蛋白质生化、实时 PCR 和共聚焦显微镜。
所有 KCNE1 结构都与 Kv7.1 相互作用。与任何其他构建体相比，Kv7.1 与非心房颤动“38S”共同表达产生的 I(Ks)更大。与转染 N 末端截断 KCNE1 构建体（“Δ1-38”）的细胞相比，转染具有精氨酸取代的 KCNE1 突变体（“38G-3xA”）的离子电流相当。质膜制剂的 Western 印迹和共聚焦图像一致显示，与任何其他构建体相比，与非心房颤动 KCNE1-38S 共转染的细胞质膜上 Kv7.1 蛋白的量更大。
我们的研究结果表明，KCNE1 第 32、33、36 位的 N 末端精氨酸对于 I(Ks)的重建很重要。此外，我们的结果暗示这些 N 末端氨基酸在延迟整流通道复合物的膜表达中起作用。
