Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche, 03202 Alicante, Spain.
Biochemistry. 2012 May 8;51(18):3891-900. doi: 10.1021/bi201497n. Epub 2012 Apr 27.
Ion permeation and selectivity, key features in ion channel function, are believed to arise from a complex ensemble of energetic and kinetic variables. Here we evaluate the contribution of pore cation binding to ion permeation and selectivity features of KcsA, a model potassium channel. For this, we used E71A and M96V KcsA mutants in which the equilibrium between conductive and nonconductive conformations of the channel is differently shifted. E71A KcsA is a noninactivating channel mutant. Binding of K(+) to this mutant reveals a single set of low-affinity K(+) binding sites, similar to that seen in the binding of K(+) to wild-type KcsA that produces a conductive, low-affinity complex. This seems consistent with the observed K(+) permeation in E71A. Nonetheless, the E71A mutant retains K(+) selectivity, which cannot be explained on the basis of just its low affinity for this ion. At variance, M96V KcsA is a rapidly inactivating mutant that has lost selectivity for K(+) and also conducts Na(+). Here, low-affinity binding and high-affinity binding of both cations are detected, seemingly in agreement with both being permeating species in this mutant channel. In conclusion, binding of the ion to the channel protein seemingly explains certain gating, ion selectivity, and permeation properties. Ion binding stabilizes greatly the channel and, depending upon ion type and concentration, leads to different conformations and ion binding affinities. High-affinity states guarantee binding of specific ions and mediate ion selectivity but are nonconductive. Conversely, low-affinity states would not discriminate well among different ions but allow permeation to occur.
离子渗透和选择性是离子通道功能的关键特征，据信它们源于复杂的能量和动力学变量组合。在这里，我们评估了孔阳离子结合对 KcsA（一种模型钾通道）离子渗透和选择性特征的贡献。为此，我们使用了 E71A 和 M96V KcsA 突变体，其中通道的导电和非导电构象之间的平衡以不同的方式移动。E71A KcsA 是一种非失活通道突变体。该突变体中 K(+)的结合揭示了一组低亲和力的 K(+)结合位点，类似于 K(+)与野生型 KcsA 的结合，产生了一种导电的、低亲和力的复合物。这似乎与在 E71A 中观察到的 K(+)渗透一致。尽管如此，E71A 突变体仍保留了 K(+)的选择性，这不能仅仅基于其对该离子的低亲和力来解释。相比之下，M96V KcsA 是一种快速失活的突变体，它已经失去了对 K(+)的选择性，并且还可以传导 Na(+)。在这里，检测到两种阳离子的低亲和力结合和高亲和力结合，似乎与这两种阳离子都是该突变体通道中的渗透物质一致。总之，离子与通道蛋白的结合似乎解释了某些门控、离子选择性和渗透特性。离子结合极大地稳定了通道，并且取决于离子类型和浓度，导致不同的构象和离子结合亲和力。高亲和力状态保证了特定离子的结合并介导离子选择性，但不导电。相反，低亲和力状态不能很好地区分不同的离子，但允许渗透发生。
