Functional interactions between residues in the S1, S4, and S5 domains of Kv2.1
Functional interactions between residues in the S1, S4, and S5 domains of Kv2.1
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DOI：
10.1007/s00249-011-0694-3
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发表时间：
2011-06-01
影响因子：
2
通讯作者：
Snyders, D. J.
中科院分区：
文献类型：
--
作者：
Bocksteins, E.;Ottschytsch, N.;Snyders, D. J.
电压门控钾通道亚基Kv2.1与沉默亚基Kv6.4形成异四聚体通道。含有来自Kv6.4的单个跨膜区段的嵌合Kv2.1通道已显示出功能性。然而，含有来自Kv6.4的S1和S5的Kv2.1嵌合体没有功能。该嵌合体中单个残基的回复突变（相对于Kv2.1对应物）确定了对功能性至关重要的四个位置：S1中的A200 V和A203 T，以及S5中的T343 M和P347 S。为了测试Kv2.1中可能的相互作用，我们使用带电残基和色氨酸取代最外对203/347。在T203和S347处具有相反电荷的取代的组合是耐受的，但导致具有改变的门控动力学的通道，带负电荷的天冬氨酸取代的组合也是如此。这些突变体的双突变体循环分析表明，这两个残基是积极耦合。相反，用带正电荷的赖氨酸一起替换两个残基（T203 K + S347 K）是不耐受的，并且导致折叠或运输缺陷。通过在电压传感器的S4片段中引入R300 E突变，可以恢复T203 K + S347 K突变的非功能性。这些结果表明，这些特定的S1、S4和S5残基在功能通道中非常接近并相互作用，但也是Kv2.1通道成熟的重要决定因素。这些数据支持S1和S5之间锚定相互作用的观点，但表明这种相互作用表面比以前提出的更广泛。
The voltage-gated potassium channel subunit Kv2.1 forms heterotetrameric channels with the silent subunit Kv6.4. Chimeric Kv2.1 channels containing a single transmembrane segment from Kv6.4 have been shown to be functional. However, a Kv2.1 chimera containing both S1 and S5 from Kv6.4 was not functional. Back mutation of individual residues in this chimera (to the Kv2.1 counterpart) identified four positions that were critical for functionality: A200V and A203T in S1, and T343M and P347S in S5. To test for possible interactions in Kv2.1, we used substitutions with charged residues and tryptophan for the outermost pair 203/347. Combinations of substitutions with opposite charges at both T203 and S347 were tolerated but resulted in channels with altered gating kinetics, as did the combination of negatively charged aspartate substitutions. Double mutant cycle analysis with these mutants indicated that both residues are energetically coupled. In contrast, replacing both residues with a positively charged lysine together (T203K + S347K) was not tolerated and resulted in a folding or trafficking deficiency. The nonfunctionality of the T203K + S347K mutation could be restored by introducing the R300E mutation in the S4 segment of the voltage sensor. These results indicate that these specific S1, S4, and S5 residues are in close proximity and interact with each other in the functional channel, but are also important determinants for Kv2.1 channel maturation. These data support the view of an anchoring interaction between S1 and S5, but indicate that this interaction surface is more extensive than previously proposed.