Molecular features of an alcohol binding site in a neuronal potassium channel.
Molecular features of an alcohol binding site in a neuronal potassium channel.
复制标题
神经元钾通道中酒精结合位点的分子特征。
DOI：
10.1021/bi034738f
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发表时间：
2003
期刊：
影响因子：
2.9
通讯作者：
Covarrubias,Manuel
中科院分区：
文献类型：
--
作者：
Shahidullah,Mohammad;Harris,Thanawath;Germann,MarkusW;Covarrubias,Manuel
脂肪醇（1-链烷醇）选择性地抑制神经元Shaw 2 K+通道的内部结合位点。这种抑制作用是由构成孔形成亚基中S4−S5环的13个残基序列赋予的。在这里，我们结合了功能和结构的方法，以深入了解这种相互作用的分子基础。为了推断所涉及的力，我们采用快速浓度钳方法（10 - 90%交换时间= 800 μs）来检查1-链烷醇同系列的三个成员（乙醇、1-丁醇和1-己醇）与异种卵母细胞由内而外斑块中Shaw 2 K+通道的相互作用动力学。正如预期的二阶机制，涉及一个受体网站，只有观察到的缔合速率常数线性依赖于1-烷醇浓度。虽然烷基链长对解离速率常数的影响不大（在乙醇和1-己醇之间仅降低了0.02倍），但二级缔合速率常数增加了1倍。因此，疏水相互作用控制在1-烷醇结合位点产生碰撞的可能性，并且短程极性相互作用有助于稳定复合物。我们还研究了1-烷醇结合的能量学与S4−S5环的结构性质之间的关系。将圆二色光谱应用于对应于各种K+通道的S4−S5环的肽，揭示了1-烷醇结合位点的表观结合亲和力与S4−S5环的α-螺旋倾向之间的相关性。这些数据表明，在肖2 1-烷醇结合位点的两亲性相互作用取决于特定的结构限制的通道的成孔亚基。
Aliphatic alcohols (1-alkanols) selectively inhibit the neuronal Shaw2 K+channel at an internal binding site. This inhibition is conferred by a sequence of 13 residues that constitutes the S4−S5 loop in the pore-forming subunit. Here, we combined functional and structural approaches to gain insights into the molecular basis of this interaction. To infer the forces that are involved, we employed a fast concentration-clamp method (10−90% exchange time = 800 μs) to examine the kinetics of the interaction of three members of the homologous series of 1-alkanols (ethanol, 1-butanol, and 1-hexanol) with Shaw2 K+channels inXenopusoocyte inside-out patches. As expected for a second-order mechanism involving a receptor site, only the observed association rate constants were linearly dependent on the 1-alkanol concentration. While the alkyl chain length modestly influenced the dissociation rate constants (decreasing only ∼2-fold between ethanol and 1-hexanol), the second-order association rate constants increasede-fold per carbon atom. Thus, hydrophobic interactions govern the probability of productive collisions at the 1-alkanol binding site, and short-range polar interactions help to stabilize the complex. We also examined the relationship between the energetics of 1-alkanol binding and the structural properties of the S4−S5 loop. Circular dichroism spectroscopy applied to peptides corresponding to the S4−S5 loop of various K+channels revealed a correlation between the apparent binding affinity of the 1-alkanol binding site and the α-helical propensity of the S4−S5 loop. The data suggest that amphiphilic interactions at the Shaw2 1-alkanol binding site depend on specific structural constraints in the pore-forming subunit of the channel.