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的选通行为有很大影响的区域，不仅与C型失活有关，而且在激活选通期间的通断转变方面也是如此。某些离子对发呆的影响已经被很好地记录下来了[1-4]。具有长占据态的离子(Rb+、Cs+、NH4+)倾向于通过栅极上的“踏门”效应来稳定开态，但通道离子相互作用的唯一区域出现在选择性滤光片上。 此外，在通向开放状态的过程中，通道似乎填充了副导通状态[5-7]，并且这些副导通状态显示出改变的选择性。针对内向整流K通道签名序列的非天然氨基酸突变揭示了快速门控转变的显著后果[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两侧的区域发生了微妙的构象变化，这些变化只有在有利于通道开放的条件下才会出现[9]。KCsA类似于其他生物上重要的K+通道[10，11]，经历C型失活。在向酸性pH转变后，位于内螺旋束的下闸门打开，并使选择性过滤器周围的构象发生变化。这种构象波导致C型失活。尽管如此，要证明选择性过滤器在影响激活门控中的作用，需要额外的结构方法。 Perozo实验室的三个最新进展打开了一个有趣的机会之窗，可以进一步分析通道的不同移动部分在门控中的作用。首先，如上所述，我们已经确定了KCSA中的C型失活机制，发现并鉴定了选择性过滤器周围的KCSA突变体。

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.