Conformational dynamics in the selectivity filter of KcsA in response to potassium ion concentration.
Conformational dynamics in the selectivity filter of KcsA in response to potassium ion concentration.
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DOI：
10.1016/j.jmb.2010.06.031
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发表时间：
2010-08-13
影响因子：
5.6
通讯作者：
McDermott AE
中科院分区：
文献类型：
--
作者：
Bhate MP;Wylie BJ;Tian L;McDermott AE
利用固体核磁共振研究了KcsA选择性滤光片的构象变化随环境钾浓度的变化。这个高度保守的蛋白质区域被认为是选择性地螯合钾离子。我们报告了两种不同构象的选择性滤波器的固态核磁共振化学位移指纹；当缓冲钾离子浓度从50 mM变化到1µM时，过滤器中关键残留物的化学位移发生了显著变化。钾离子滴定研究表明，关键孔残基Val 76上K+结合的位点特异性Kd约为~ 7µM，观察低K+构象需要相对高的样品水化。在低环境钾浓度下同时检测两种构象表明，高K+态和低K+态在核磁共振时间尺度上（kex < 500 s−1）处于缓慢交换状态。同时清除两个内部位点的缓慢速率和紧密结合，与先前在溶液中的洗涤剂中观察到的不同，但与电生理学的测量结果很一致，并且似乎是由于我们使用了水合双层环境。这些特征排除了低K+态参与离子传输的时间尺度，这被认为涉及到一个内部结合位点总是被占用的状态交换。另一方面，内部位置的这些动力学和热力学特征当然可以与低离子浓度下参与控制机制相兼容，例如c型失活，这是一个与激活相耦合的过程，涉及关闭通道的外口。
The conformational change in the selectivity filter of KcsA as a function of ambient potassium concentration is studied with solid state NMR. This highly conserved region of the protein is known to chelate potassium ions selectively. We report solid-state NMR chemical shift fingerprints of two distinct conformations of the selectivity filter; significant changes are observed in the chemical shifts of key residues in the filter as the buffer potassium ion concentration is changed from 50 mM to 1 µM. Potassium ion titration studies reveal that the site-specific Kd for K+ binding at the key pore residue Val 76, is on the order of ∼7 µM and that relatively high sample hydration is necessary to observe the low K+ conformer. Simultaneous detection of both conformers at low ambient potassium concentration suggests that the high K+ and low K+ states are in slow exchange on the NMR timescale (kex < 500 s−1). The slow rate and tight binding for evacuating both inner sites simultaneously, differ from prior observations in detergents in solution, but agree well with measurements by electrophysiology, and appear to result from our use of a hydrated bilayer environment. These characteristics rule out participation of the low K+ state on the timescale of ion transmission, which has been assumed to involve interchange of states where one of the inner binding sites is always occupied. On the other hand, these kinetic and thermodynamic characteristics of evacuation of the inner sites certainly could be compatible with participation in a control mechanism at low ion concentration, such as C-type inactivation, a process that is coupled to activation and involves closing of the outer mouth of the channel.
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影响因子：
56.9
作者：
Doyle, DA;Cabral, JM;MacKinnon, R
通讯作者：
MacKinnon, R
DOI：
10.1085/jgp.200709844
发表时间：
2007-11
期刊：
The Journal of general physiology
影响因子：
--
作者：
Chakrapani S;Cordero-Morales JF;Perozo E
通讯作者：
Perozo E
影响因子：
64.8
作者：
Åqvist, J;Luzhkov, V
通讯作者：
Luzhkov, V
影响因子：
2.7
作者：
DELAGLIO, F;GRZESIEK, S;BAX, A
通讯作者：
BAX, A
影响因子：
5.6
作者：
Domene, Carmen;Furini, Simone
通讯作者：
Furini, Simone