Analysis of the interactions between the C-terminal cytoplasmic domains of KCNQ1 and KCNE1 channel subunits.
Analysis of the interactions between the C-terminal cytoplasmic domains of KCNQ1 and KCNE1 channel subunits.
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DOI：
10.1042/bj20090977
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发表时间：
2010-04-28
期刊：
影响因子：
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通讯作者：
McDonald TV
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文献类型：
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作者：
Zheng R;Thompson K;Obeng-Gyimah E;Alessi D;Chen J;Cheng H;McDonald TV
KCNQ1 和 KCNE1 编码的离子通道亚基产生缓慢激活的 K+ 电流 (IK)，在心肌复极中发挥核心作用。 KCNQ1α 亚基和 KCNE1 β 亚基通过其跨膜片段进行组装，相互作用，导致通道激活动力学发生转变。我们最近报道了涉及两个亚基 C 端部分的功能相互作用，以及随后的通道失活调节。在这里，我们提供了表征 KCNQ1 和 KCNE1 的 C 末端之间物理相互作用的证据。当在培养细胞中表达时，KCNE1 (KCNE1-CT) 的 C 末端与 KCNQ1 共定位，与 KCNQ1 共免疫沉淀，并扰乱 KCNQ1 电流的失活动力学。纯化的 KCNQ1 C 末端 (KCNQ1-CT) 和 KCNE1-CT 在下拉实验中发生物理相互作用，表明存在直接关联。 KCNQ1-CT 的删除分析表明，KCNE1-CT 与最后一个跨膜片段之后但四聚化结构域 N 端的 KCNQ1 区域结合。表面等离子共振 (SPR) 证实了下拉结果，表明最近端区域 (KCNQ1 a.a.349–438) 对双分子相互作用的贡献最大，解离常数约为 4μM。 KCNE1-CT、D76N 和 W87F 的 LQT 突变体以相当的亲和力保留与 KCNQ1-CT 的结合，表明这些致病突变不会通过破坏关联来改变通道行为。然而，涉及 KCNQ1 C 末端的几种 LQT 突变对 KCNQ1/KCNE1 关联显示出不同的影响。我们的结果表明，KCNQ1 和 KCNE1 的 C 末端包含一个独立的相互作用结构域，该结构域可能在 IK 通道调节中发挥作用，而 IK 通道调节可能在某些长 QT 综合征突变中受到影响。
Ion channel subunits encoded by KCNQ1 and KCNE1 produce the slowly activating K+ current (IKs) that plays a central role in myocardial repolarization. The KCNQ1α-subunit and the KCNE1 β-subunit assemble with their membrane-spanning segments interacting resulting in transformation of channel activation kinetics. We recently reported a functional interaction involving C-terminal portions of the two subunits with ensuing regulation of channel deactivation. Here we provide evidence characterizing a physical interaction between the C-termini of KCNQ1 and KCNE1. When expressed in cultured cells the C-terminus of KCNE1 (KCNE1-CT) co-localized with KCNQ1, co-immunoprecipitated with KCNQ1 and perturbed deactivation kinetics of the KCNQ1 currents. Purified KCNQ1 C-terminus (KCNQ1-CT) and KCNE1-CT physically interacted in pull-down experiments indicating a direct association. Deletion-analysis of KCNQ1-CT indicated that KCNE1-CT binds to a KCNQ1 region just after the last transmembrane segment but N-terminal to the tetramerization domain. Surface plasmon resonance (SPR) corroborated the pull-down results showing that the most proximal region (KCNQ1 a.a.349–438) contributed most to the bimolecular interaction with dissociation constant of ~4μM. LQT mutants of KCNE1-CT, D76N and W87F retained binding to KCNQ1-CT with comparable affinity, indicating that these disease-causing mutations do not alter channel behavior by disruption of the association. Several LQT mutations involving the C-terminus of KCNQ1 however, showed varying effects on KCNQ1/KCNE1 association. Our results indicate that the C-termini of KCNQ1 and KCNE1 comprise an independent interaction domain that may play a role in IKs channel regulation that is potentially affected in some Long QT Syndrome mutations.