Modulation of the conductance-voltage relationship of the BKCa channel by mutations at the putative flexible interface between two RCK domains
Modulation of the conductance-voltage relationship of the BKCa channel by mutations at the putative flexible interface between two RCK domains
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DOI：
10.1529/biophysj.107.108738
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发表时间：
2008-01-15
影响因子：
3.4
通讯作者：
Park, Chul-Seung
中科院分区：
文献类型：
--
作者：
Kim, Hyun-Ju;Lim, Hyun-Ho;Park, Chul-Seung
大电导Ca 2+激活的K+（BKCa）通道的钙依赖性门控由含有K+电导调节因子（RCK）的两个结构域和高亲和力Ca 2+结合位点（Ca 2+碗）的大胞质羧基末端赋予。在我们之前的研究中，我们定位了假定的第二个RCK结构域（RCK 2），并证明它通过疏水的“组装界面”与RCK 1直接相互作用。在这项研究中，我们测试了结构模型的其他接口，“灵活的接口”，通过战略定位电荷对跨假定的接口。RCK 2上的几个电荷突变影响了通道的电压依赖性激活。特别是，在位置803处的Gly至Asp取代通过稳定通道的开放构象而对通道的Ca 2+亲和力和电压敏感性影响最小，从而深刻地影响通道激活。Gly-803的各种突变使通道的电导-电压曲线在145 mV范围内向左或向右移动。由于预测该残基位于RCK 2的第一个环中，因此这些结果强烈表明该环在确定通道开放的内在平衡常数方面起着关键作用，并且它们支持该环是介导RCK 1和RCK 2之间构象偶联的界面的一部分的假设。
Calcium-dependent gating of the large-conductance Ca2+-activated K+ (BKCa) channel is conferred by the large cytosolic carboxyl terminus containing two domains of the regulator of K+ conductance (RCK) and the high-affinity Ca2+-binding site (the Ca2+-bowl). In our previous study, we located the putative second RCK domain (RCK2) and demonstrated that it interacts directly with RCK1 via a hydrophobic '' assembly interface ''. In this study, we tested the structural model of the other interface, the '' flexible interface '', by strategically positioning charge pairs across the putative interface. Several charge mutations on RCK2 affected the voltage-dependent activation of the channel. In particular, the Gly-to-Asp substitution at position 803 profoundly affected channel activation by stabilizing the open conformation of the channel with minimal effects on its Ca2+ affinity and voltage sensitivity. Various mutations at Gly-803 shifted the channel's conductance-voltage curve either left or right over a 145-mV range. Since this residue is predicted to be in the first loop of RCK2 these results strongly suggest that this loop plays a critical role in determining the intrinsic equilibrium constant for channel opening, and they support the hypothesis that this loop is part of an interface that mediates conformational coupling between RCK1 and RCK2.