Div. of Cardiology, Section of Electrophysiology, Goethe-Universität, Frankfurt, Germany.
Heart Rhythm. 2009 Dec;6(12):1792-801. doi: 10.1016/j.hrthm.2009.08.009. Epub 2009 Aug 13.
KCNQ1-T587M is a trafficking-deficient long QT syndrome (LQTS) missense mutation. Affected patients exhibit severe clinical phenotypes that are not explained by the mutant's effects on I(Ks). Previous work showed a KCNH2 and KCNQ1 alpha-subunit interaction that increases KCNH2 membrane localization and function.
We hypothesized that failure of trafficking-deficient KCNQ1-T587M to enhance KCNH2 membrane expression could reduce KCNH2 current versus wild-type KCNQ1 (KCNQ1-WT), contributing to the LQTS phenotype of KCNQ1-T587M carriers.
Patch-clamp, protein biochemical studies, confocal imaging, and in vivo transfection of guinea pig cardiomyocytes were performed.
KCNQ1-T587M failed to generate functional current when coexpressed with KCNE1 and caused haploinsufficiency when coexpressed with KCNQ1-WT/KCNE1. Coexpression of KCNQ1-WT with KCNH2 increased I(KCNH2) versus KCNH2 alone (P <.05). Immunoblots and confocal microscopy indicated increased plasma membrane localization of KCNH2 alpha-subunits in cells cotransfected with KCNQ1-WT plasmid, while total KCNH2 protein synthesis and KCNH2 glycosylation remained unaffected, which suggests a chaperone effect of KCNQ1-WT to enhance the membrane localization of KCNH2. KCNH2 also coimmunoprecipitated with KCNQ1-WT. Although KCNQ1-T587M coprecipitated with KCNH2, the mutant was retained intracellularly and failed to increase KCNH2 membrane localization, abolishing the KCNQ1-WT chaperone function and reducing I(KCNH2) upon coexpression substantially compared with coexpression with KCNQ1-WT (P <.05). In vivo transfection of KCNQ1-T587M in guinea pigs suppressed I(Kr) in isolated cardiomyocytes.
The trafficking-deficient LQTS mutation KCNQ1-T587M fails to show the chaperoning function that enhances KCNH2 membrane localization with KCNQ1-WT. This novel mechanism results in reduced I(KCNH2), which would be expected to decrease repolarization reserve and synergize with reduced I(KCNQ1) caused directly by the mutation, potentially explaining the malignant clinical phenotype in affected patients.
KCNQ1-T587M 是一种转运缺陷型长 QT 综合征（LQTS）错义突变。受影响的患者表现出严重的临床表型，这不能用突变对 I(Ks) 的影响来解释。先前的工作表明 KCNH2 和 KCNQ1 亚基之间存在相互作用，这种相互作用增加了 KCNH2 的膜定位和功能。
我们假设转运缺陷型 KCNQ1-T587M 不能增强 KCNH2 的膜表达，可能会降低 KCNH2 电流相对于野生型 KCNQ1（KCNQ1-WT），从而导致 KCNQ1-T587M 携带者的 LQTS 表型。
进行了膜片钳、蛋白生化研究、共聚焦成像和豚鼠心肌细胞的体内转染。
KCNQ1-T587M 与 KCNE1 共表达时不能产生功能性电流，与 KCNQ1-WT/KCNE1 共表达时表现为半不足。KCNQ1-WT 与 KCNH2 共表达可增加 I(KCNH2)，而与 KCNH2 单独表达相比（P<0.05）。免疫印迹和共聚焦显微镜表明，与 KCNQ1-WT 质粒共转染的细胞中 KCNH2 亚基的质膜定位增加，而 KCNH2 总蛋白合成和 KCNH2 糖基化保持不变，这表明 KCNQ1-WT 具有伴侣蛋白作用，可增强 KCNH2 的膜定位。KCNH2 也与 KCNQ1-WT 共免疫沉淀。虽然 KCNQ1-T587M 与 KCNH2 共沉淀，但突变体被保留在细胞内，无法增加 KCNH2 的膜定位，从而消除了 KCNQ1-WT 的伴侣蛋白功能，并与 KCNQ1-WT 共表达相比，大大降低了共表达时的 I(KCNH2)（P<0.05）。豚鼠体内转染 KCNQ1-T587M 可抑制分离心肌细胞中的 I(Kr)。
转运缺陷型 LQTS 突变 KCNQ1-T587M 未能显示出伴侣蛋白功能，该功能可增强 KCNQ1-WT 与 KCNH2 的膜定位。这种新的机制导致 I(KCNH2)减少，这可能会降低复极化储备，并与突变直接导致的 I(KCNQ1)减少协同作用，从而解释了受影响患者的恶性临床表型。
