Second coiled-coil domain of KCNQ channel controls current expression and subfamily specific heteromultimerization by salt bridge networks
Second coiled-coil domain of KCNQ channel controls current expression and subfamily specific heteromultimerization by salt bridge networks
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DOI：
10.1113/jphysiol.2007.148601
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发表时间：
2008-06-15
影响因子：
5.5
通讯作者：
Kubo, Yoshihiro
中科院分区：
文献类型：
--
作者：
Nakajo, Koichi;Kubo, Yoshihiro
KCNQ通道在可兴奋细胞如神经元中携带缓慢激活的电压依赖性M电流。虽然KCNQ 2同源多聚体可以形成功能性电压门控K+通道，但与KCNQ 3的异源多聚体化产生电流幅度增加> 10倍。所有KCNQ通道都含有双卷曲螺旋结构域（TCC 1和TCC 2，或A结构域头和尾），其中TCC 2（A结构域尾）被认为对亚基识别，通道组装和表面表达很重要。然而，TCC 2识别并与其伙伴联系的机制尚未完全了解。本研究的目的是通过检测TCC 2缺失突变体、TCC 2交换嵌合体和点突变体的表型来阐明其识别机制。使用爪蟾卵母细胞在双电极电压钳下的电生理分析显示，同源四聚体KCNQ 3 TCC 2是在没有KCNQ 2 TCC 2的情况下电流表达的负调节器。最近对KCNQ 4 TCC 2的结构分析揭示了螺旋间盐桥网络的存在。因此，我们交换了据报道参与盐桥形成的带电残基的符号，并在功能上证实了螺旋间盐桥网络负责KCNQ 2和KCNQ 3之间的亚基识别。最后，我们构建了TCC 2交换KCNQ 2/KCNQ 3突变体与KCNQ 1 TCC 2或GCN 4-pLI，卷曲螺旋结构域从一个无关的蛋白质，并发现TCC 2是可取代的，甚至GCN 4-pLI可以作为TCC 2的替代品。我们目前的数据提供了一些新的见解TCC 2在电流表达过程中所发挥的作用，也提供了功能证据的重要性的intercoil盐桥网络的亚基识别和卷曲螺旋的形成，最近的晶体学数据表明。
KCNQ channels carry the slowly activating, voltage-dependent M-current in excitable cells such as neurons. Although the KCNQ2 homomultimer can form a functional voltage-gated K+ channel, heteromultimerization with KCNQ3 produces a > 10-fold increase in current amplitude. All KCNQ channels contain double coiled-coil domains (TCC1 and TCC2, or A-domain Head and Tail), of which TCC2 (A-domain Tail) is thought to be important for subunit recognition, channel assembly and surface expression. The mechanism by which TCC2 recognizes and associates with its partner is not fully understood, however. Our aim in the present study was to elucidate the recognition mechanism by examining the phenotypes of TCC2-deletion mutants, TCC2-swapped chimeras and point mutants. Electrophysiological analysis using Xenopus oocytes under two-electrode voltage clamp revealed that homotetrameric KCNQ3 TCC2 is a negative regulator of current expression in the absence of KCNQ2 TCC2. Recent structural analysis of KCNQ4 TCC2 revealed the presence of intercoil salt bridge networks. We therefore swapped the sign of the charged residues reportedly involved in the salt bridge formation and functionally confirmed that the intercoil salt bridge network is responsible for the subunit recognition between KCNQ2 and KCNQ3. Finally, we constructed TCC2-swapped KCNQ2/KCNQ3 mutants with KCNQ1 TCC2 or GCN4-pLI, a coiled-coil domain from an unrelated protein, and found that TCC2 is substitutable and even GCN4-pLI can work as a substitute for TCC2. Our present data provide some new insights into the role played by TCC2 during current expression, and also provide functional evidence of the importance of the intercoil salt bridge network for subunit recognition and coiled-coil formation, as is suggested by recent crystallographic data.