Thermodynamic and kinetic properties of amino-terminal and S4-S5 loop HERG channel mutants under steady-state conditions
Thermodynamic and kinetic properties of amino-terminal and S4-S5 loop HERG channel mutants under steady-state conditions
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DOI：
10.1529/biophysj.107.116731
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发表时间：
2008-05-15
影响因子：
3.4
通讯作者：
Barros, Francisco
中科院分区：
文献类型：
--
作者：
Alonso-Ron, Carlos;de la Pena, Pilar;Barros, Francisco
门控动力学和基本的热力学性质的人ether-a-go-go-related基因（HERG）的K+通道在非洲爪蟾卵母细胞中表达的研究使用协议能够产生真正的稳态动力学参数。在保守的eag/PAS区域之前缺乏氨基末端的最初16个残基的通道突变体显示出与z（g）值的降低和较小的负Δ G（o）相关的激活电压依赖性的显著正位移，表明平衡向闭合状态的缺失诱导的位移。相反，在缺乏氨基末端近端结构域的通道中观察到负位移和增加的Δ G（o），表明闭合状态不稳定。此外，加速激活和失活动力学时，在这些结构中观察到的驱动力的差异被认为是，这表明远端和近端氨基末端片段的存在有助于在野生型通道的特定的化学相互作用，提高激活的能量屏障。一些单点突变体在细胞内环连接S4和S5螺旋的稳态特性揭示了这些突变的影响和氨基末端modi的影响之间的惊人的平行性。阳离子。我们的数据表明，除了公认的影响HERG失活的初始氨基末端区域，这个胞质区域也影响激活行为。数据还表明，不仅电压传感器本身的缓慢移动，而且可能作用于S4-S5环的一些细胞质结构延迟其与激活门的功能性偶联，可能有助于HERG的缓慢门控。
Gating kinetics and underlying thermodynamic properties of human ether-a-go-go-related gene (HERG) K+ channels expressed in Xenopus oocytes were studied using protocols able to yield true steady-state kinetic parameters. Channel mutants lacking the initial 16 residues of the amino terminus before the conserved eag/PAS region showed significant positive shifts in activation voltage dependence associated with a reduction of z(g) values and a less negative Delta G(o), indicating a deletion-induced displacement of the equilibrium toward the closed state. Conversely, a negative shift and an increased Delta G(o), indicative of closed-state destabilization, were observed in channels lacking the amino-terminal proximal domain. Furthermore, accelerated activation and deactivation kinetics were observed in these constructs when differences in driving force were considered, suggesting that the presence of distal and proximal amino-terminal segments contributes in wild-type channels to specific chemical interactions that raise the energy barrier for activation. Steady-state characteristics of some single point mutants in the intracellular loop linking S4 and S5 helices revealed a striking parallelism between the effects of these mutations and those of the amino-terminal modi. cations. Our data indicate that in addition to the recognized influence of the initial amino-terminus region on HERG deactivation, this cytoplasmic region also affects activation behavior. The data also suggest that not only a slow movement of the voltage sensor itself but also delaying its functional coupling to the activation gate by some cytoplasmic structures possibly acting on the S4-S5 loop may contribute to the atypically slow gating of HERG.