Ca regulation of Ca channels

描述（由申请人提供）：钙调蛋白（Calmodulin, CaM）对CaV1-2通道的调节-被称为钙调-已被证明是丰富的，无论是生物学上还是作为具有区分Ca2+解码能力的一般调节原型。在早期的周期中，发现无Ca2+ CaM （apoCaM）与通道胞内羧基端上的IQ结构域预先相关。Ca2+结合到这个常驻CaM诱导了尚不清楚的构象变化，以某种方式促进（CDF）或灭活（CDI）通道开放，将钙调作为Ca2+的正反馈或负反馈控制系统。有趣的是，Ca2+结合到CaM的C端和n端叶各自可以诱导不同形式的通道调节，这与早期在草履虫中CaM“功能双分区”的发现相呼应。更值得注意的是，C-lobe响应由相关通道驱动的~100毫米Ca2+脉冲（局部Ca2+选择性），而N-lobe在某种程度上能够感知来自远处Ca2+源的远弱信号（全局选择性）。在当前周期中，我们粗略地推导了CaM的c端瓣（慢CaM方案）和n端瓣（SQS方案）的钙调的功能机制。这些机制解释了这两个调节子系统显著的空间Ca2+解码特性。然而，由于通常驱动钙调的Ca2+通道内流的复杂性，明显缺乏这些低分辨率机制草图的Ca2+依赖性的定量意义。更大的空白涉及由慢速CaM和SQS方案所假定的通道/CaM接口。如果我们遵循初步数据的引导，并放弃流行的“以智商为中心”的观点，即apoam和Ca2+/CaM都与智商结构域相互作用以触发通道调节，那么我们将知之甚少。这种不确定性模糊了我们有希望的功能机制与分子现实的联系，并混淆了对刚刚发现的通过RNA编辑CaV通道的CaM调节的生理调节的理解。这些突出的挑战将通过三个具体目标来解决。1)通过动态控制Ca2+输入通道来量化钙调的Ca2+依赖性，如UV Ca2+ uncaging所提供的。2)利用一种称为单独转化朗缪尔（iTL）分析的方法，结合CaM/通道构型的计算机预测，确定CaM调控CaV通道的分子状态。3)利用Aim 2的洞察力，推断RNA编辑CaV1.3通道改变其CaM调控的机制。

DESCRIPTION (provided by applicant): Calmodulin (CaM) regulation of CaV1-2 channels-termed calmodulation has proven rich, both biologically and as a general modulatory prototype with discriminating Ca2+ decoding capabilities. In earlier cycles, Ca2+-free CaM (apoCaM) was found to be pre-associated to an IQ domain on the intracellular carboxy terminus of channels. Ca2+-binding to this resident CaM induces as-yet-unclear conformational changes that somehow facilitate (CDF) or inactivate (CDI) channel opening, casting calmodulation as a positive or negative feedback control system for Ca2+. Intriguingly, Ca2+-binding to the C- and N-terminal lobes of CaM can each induce distinct forms of channel regulation, echoing earlier findings of CaM 'functional bipartition' in Paramecium. More remarkably, the C-lobe responds to the ~100-mM Ca2+ pulses driven by the associated channel (local Ca2+ selectivity), whereas the N-lobe is somehow capable of sensing far weaker signals from distant Ca2+ sources (global selectivity). In the current cycle, we deduced in coarse outline the functional mechanisms of calmodulation by the C-terminal lobe of CaM (slow CaM scheme), and by the N-terminal lobe (SQS scheme). These mechanisms explain the notable spatial Ca2+ decoding properties of these two calmodulatory subsystems. Prominently absent, however, is a quantitative sense of the Ca2+ dependence of these low-resolution mechanistic sketches, owing to the complexity of Ca2+ channel influx that normally drives calmodulation. A greater void concerns the channel/CaM interfaces postulated by slow CaM and SQS schemes. If we follow the lead of preliminary data, and abandon a prevailing 'IQ-centric' view, where apoCaM and Ca2+/CaM both interact with the IQ domain to trigger channel regulation, little would be known. This indeterminacy obscures the linkage of our promising functional mechanisms to molecular reality, and confounds understanding of just discovered physiological tuning of CaM regulation via RNA editing of CaV channels. These prominent challenges will be addressed by three specific aims. 1) To quantify the Ca2+ dependence of calmodulation via dynamic control of Ca2+ inputs to channels, as afforded by UV Ca2+ uncaging. 2) To identify the molecular states underlying CaM regulation of CaV channels, using an approach termed individually Transformed Langmuir (iTL) analysis, combined with in silico prediction of CaM/channel configurations. 3) To deduce, using Aim 2 insight, the mechanism whereby RNA editing of CaV1.3 channels alters their CaM regulation.