Tapper A R, George A L
Department of Pharmacology and Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
J Gen Physiol. 2000 Sep;116(3):379-90. doi: 10.1085/jgp.116.3.379.
KvLQT1 is a voltage-gated potassium channel expressed in cardiac cells that is critical for myocardial repolarization. When expressed alone in heterologous expression systems, KvLQT1 channels exhibit a rapidly activating potassium current that slowly deactivates. MinK, a 129 amino acid protein containing one transmembrane-spanning domain modulates KvLQT1, greatly slowing activation, increasing current amplitude, and removing inactivation. Using deletion and chimeric analysis, we have examined the structural determinants of MinK effects on gating modulation and subunit association. Coexpression of KvLQT1 with a MinK COOH-terminus deletion mutant (MinK DeltaCterm) in Xenopus oocytes resulted in a rapidly activated potassium current closely resembling currents recorded from oocytes expressing KvLQT1 alone, indicating that this region is necessary for modulation. To determine whether MinK DeltaCterm was associated with KvLQT1, a functional tag (G55C) that confers susceptibility to partial block by external cadmium was engineered into the transmembrane domain of MinK DeltaCterm. Currents derived from coexpression of KvLQT1 with MinK DeltaCterm were cadmium sensitive, suggesting that MinK DeltaCterm does associate with KvLQT1, but does not modulate gating. To determine which MinK regions are sufficient for KvLQT1 association and modulation, chimeras were generated between MinK and the Na(+) channel beta1 subunit. Chimeras between MinK and beta1 could only modulate KvLQT1 if they contained both the MinK transmembrane domain and COOH terminus, suggesting that the MinK COOH terminus alone is not sufficient for KvLQT1 modulation, and requires an additional, possibly associative interaction between the MinK transmembrane domain and KvLQT1. To identify the MinK subdomains necessary for gating modulation, deletion mutants were designed and coexpressed with KvLQT1. A MinK construct with amino acid residues 94-129 deleted retained the ability to modulate KvLQT1 gating, identifying the COOH-terminal region critical for gating modulation. Finally, MinK/MiRP1 (MinK related protein-1) chimeras were generated to investigate the difference between these two closely related subunits in their ability to modulate KvLQT1. The results from this analysis indicate that MiRP1 cannot modulate KvLQT1 due to differences within the transmembrane domain. Our results allow us to identify the MinK subdomains that mediate KvLQT1 association and modulation.
KvLQT1是一种在心脏细胞中表达的电压门控钾通道，对心肌复极化至关重要。当在异源表达系统中单独表达时，KvLQT1通道表现出一种快速激活的钾电流，该电流会缓慢失活。MinK是一种含有一个跨膜结构域的129个氨基酸的蛋白质，可调节KvLQT1，极大地减缓激活速度，增加电流幅度，并消除失活。通过缺失和嵌合分析，我们研究了MinK对门控调节和亚基缔合作用的结构决定因素。在非洲爪蟾卵母细胞中，将KvLQT1与MinK的COOH末端缺失突变体（MinK DeltaCterm）共表达，产生了一种快速激活的钾电流，与单独表达KvLQT1的卵母细胞中记录到的电流非常相似，表明该区域对调节是必需的。为了确定MinK DeltaCterm是否与KvLQT1缔合，将一个赋予对外源镉部分阻断敏感性的功能标签（G55C）设计到MinK DeltaCterm的跨膜结构域中。KvLQT1与MinK DeltaCterm共表达产生的电流对镉敏感，表明MinK DeltaCterm确实与KvLQT1缔合，但不调节门控。为了确定MinK的哪些区域足以实现与KvLQT1的缔合和调节，在MinK和Na(+)通道β1亚基之间构建了嵌合体。MinK与β1之间的嵌合体只有在同时包含MinK跨膜结构域和COOH末端时才能调节KvLQT1，这表明仅MinK的COOH末端不足以调节KvLQT1，还需要MinK跨膜结构域与KvLQT1之间的额外的、可能是缔合性的相互作用。为了鉴定门控调节所需的MinK亚结构域，设计了缺失突变体并与KvLQT1共表达。一个缺失氨基酸残基94 - 129的MinK构建体保留了调节KvLQT1门控的能力，确定了COOH末端区域对门控调节至关重要。最后，构建了MinK/MiRP1（MinK相关蛋白 - 1）嵌合体，以研究这两个密切相关的亚基在调节KvLQT1能力上的差异。该分析结果表明，由于跨膜结构域内的差异，MiRP1不能调节KvLQT1。我们的结果使我们能够鉴定介导KvLQT1缔合和调节的MinK亚结构域。
