Bridging Project 8: Dynamics of ion permeation & conformational coupling in Kcsa

11.2.背景和意义 许多独立的证据表明，选择性过滤器是一个对通道门控行为有很大影响的区域，不仅在C型失活方面，而且在激活门控期间的开关转换方面。某些渗透性离子对gafing的影响已得到很好的证明[1-4]。具有长占据时间的离子（Rb+、Cs+、NH 4+）倾向于通过对门的“脚在门中”效应来稳定开放状态，然而通道-离子相互作用的唯一区域发生在选择性过滤器处。 此外，通道似乎在通往开放状态的途中填充子导电状态[5-7]，并且这些子导电状态显示出改变的选择性。针对内向整流钾通道特征序列的非天然氨基酸诱变显示了快速门控转换的显著后果[8]，再次指出选择性过滤器是门控过程的贡献者。在结构上，有早期证据表明选择性过滤器侧翼区域的细微构象变化，并且这些变化仅在有利于通道开放的条件下出现[9]。KcsA经历C型失活，类似于其他生物学上重要的K+通道[10，11]。在过渡到酸性pH值后，内螺旋束的下门打开，并赋予选择性过滤器周围的构象变化。这种构象波导致C型失活。尽管如此，选择性过滤器在影响激活门控中的作用的演示需要额外的结构方法。 Perozo实验室最近的三项进展为进一步分析通道的不同运动部件对门控的作用打开了一扇有趣的机会之窗。首先，如上所述，我们已经确定了在KcsA的C型失活机制，发现和表征KcsA突变体周围的选择性过滤器。

11.2. BACKGROUND AND SIGNIFICANCE A number of independent pieces of evidence point to the selectivity filter as a region with a great deal of influence over the gating behavior of a channel, not only in regards to C-type inactivation but also in terms of on-off transitions during activation gating. The effect of certain permeant ions on gafing is have been well documented [1-4]. Ions with long occupancy fimes (Rb+, Cs+, NH4+) tend to stabilize the open state through a "foot in the door" effect on the gate, yet the only region of channel-ion interacfion occurs at the selecfivity filter. Additionally, channels seem to populate sub-conducting states on the way to the open state [5-7], and these sub-conducting states show altered selectivity. Unnatural amino acid mutagenesis targeted to the signature sequence of an inward rectifier K channel revealed dramatic consequences upon rapid gating transitions [8], again, pointing to the selectivity filter as a contributor to the gating process. Structurally, there is eariy evidence of subtle conformational changes in regions flanking the selectivity filter, and these changes appear only under conditions that favor channel opening [9]. KcsA undergoes C-type inacfivation similar to other biologically important K+ channels [10, 11]. After a transition to acidic pH, the lower gate at the inner-helical bundle opens and imparts conformational changes around the selectivity filter. This conformational wave leads to C-type inactivation. Nonetheless, a demonstration of the role of the selectivity filter in influencing activation gating requires additional structural approaches. Three recent developments in the Perozo lab have opened an interesting window of opportunity to further analyze the role of the different moving parts of a channel on gating. First, as stated above, we have identified a C-type inactivation mechanism in KcsA, found and characterized KcsA mutants around the selectivity filter.