MinK subdomains that mediate modulation of and association with KvLQT1.
MinK subdomains that mediate modulation of and association with KvLQT1.
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DOI：
10.1085/jgp.116.3.379
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发表时间：
2000-09
期刊：
影响因子：
--
通讯作者：
George AL Jr
中科院分区：
文献类型：
--
作者：
Tapper AR;George AL Jr
KvLQT 1是一种在心肌细胞中表达的电压门控钾通道，对心肌复极至关重要。当在异源表达系统中单独表达时，KvLQT 1通道表现出快速激活的钾电流，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KvLQT 1，大大减缓激活，增加电流幅度，并消除失活。使用删除和嵌合分析，我们已经研究了MinK效应的结构决定因素的门控调制和亚基协会。KvLQT 1与MinK COOH末端缺失突变体（MinK ΔCterm）在非洲爪蟾卵母细胞中的共表达导致快速激活的钾电流，其与单独表达KvLQT 1的卵母细胞记录的电流非常相似，表明该区域是调节所必需的。为了确定MinK ΔCterm是否与KvLQT 1相关，将赋予对外部镉的部分阻断敏感性的功能标签（G55 C）工程化到MinK ΔCterm的跨膜结构域中。KvLQT 1与MinK ΔCterm共表达产生的电流对镉敏感，表明MinK ΔCterm确实与KvLQT 1相关，但不调节门控。为了确定哪些MinK区域足以用于KvLQT 1缔合和调节，在MinK和Na+通道β1亚基之间产生嵌合体。MinK和β1之间的嵌合体只有在同时含有MinK跨膜结构域和COOH末端的情况下才能调节KvLQT 1，这表明MinK COOH末端单独不足以调节KvLQT 1，并且需要MinK跨膜结构域和KvLQT 1之间的额外的、可能的缔合相互作用。为了鉴定门控调节所必需的MinK亚结构域，设计缺失突变体并与KvLQT 1共表达。氨基酸残基94-129缺失的MinK构建体保留了调节KvLQT 1门控的能力，确定了对门控调节至关重要的COOH末端区域。最后，MinK/MiRP 1（MinK相关蛋白-1）嵌合体的产生，以研究这两个密切相关的亚基之间的差异，他们的能力，以调节KvLQT 1。该分析的结果表明，由于跨膜结构域内的差异，MiRP 1不能调节KvLQT 1。我们的研究结果使我们能够确定介导KvLQT 1关联和调制的MinK子域。
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 ΔCterm) 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 ΔCterm 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 ΔCterm. Currents derived from coexpression of KvLQT1 with MinK ΔCterm were cadmium sensitive, suggesting that MinK ΔCterm 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 β1 subunit. Chimeras between MinK and β1 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.
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影响因子：
3.4
作者：
Pusch, M;Magrassi, R;Conti, F
通讯作者：
Conti, F
DOI：
10.1073/pnas.94.8.4017
发表时间：
1997-04-15
影响因子：
11.1
作者：
Yang, WP;Levesque, PC;Blanar, MA
通讯作者：
Blanar, MA
影响因子：
30.8
作者：
Wang, Q;Curran, ME;Keating, MT
通讯作者：
Keating, MT
影响因子：
64.8
作者：
Sanguinetti, MC;Curran, ME;Keating, MT
通讯作者：
Keating, MT
影响因子：
4.8
作者：
Romey, G;Attali, B;Lazdunski, M
通讯作者：
Lazdunski, M