Ca Regulation of Ca Channels

描述（由申请人提供）：在过去的资助周期中，钙调蛋白（CaM）调节钙通道家族的一般规则被识别出来。CaM有两个裂片，每个裂片有两个Ca2+结合位点。第一个普遍的方面是，每个叶可以自主地触发一种形式的通道调节。其次，当CaM的c端瓣（C-lobe）触发通道调节时，对局部Ca2+信号有优先响应。相反，只要n端叶（N-lobe）启动调控，就存在对全局Ca2+信号的选择性。第三，有理由期望这一操作规则可以推广到Ca通道以外的许多配合物中，其中CaM与靶分子预结合。因为CaM对Ca通道（和其他信号分子）的调节对正常的神经处理至关重要，并且可能对与疼痛、精神病和心律失常有关的治疗很重要，回答这些一般规则是如何发生的是下一个研究周期的首要任务。三个目标将解决这一总体主题。1. 开发和执行动力学Ca2+解码机制的CaM/Ca通道复合物的基本测试。这一目标制定了一个“动态Ca2+解码”理论，CaM解码是如何发生的，并设计了新的电压块实验，使这一理论的基本测试。2. 设计CaM/Ca通道调节的局部/全局Ca2+偏好，作为动态Ca2+解码理论的高阶测试，并作为收集开发新型通道调制器的设计原则的手段。动力学Ca2+解码理论的一个主要预测是，通道调节的局部/全局Ca2+偏好反映了Ca2+结合和Ca2+自由形式的通道亲和性之间的竞争。Aim 2将改变这些亲和力，并检查Ca2+偏好的预测变化。目的2还将探讨这些修饰是否可以为以新方式调节通道调节的药物化合物的设计提供信息。3. 实验确定Ca2+浓度和扩散内的纳米钙通道。下一阶段进展的关键是直接测量局部和全局Ca2+浓度信号，以及Ca2+在实际通道“纳米域”环境中的扩散。基因编码的Ca2+传感器（TNL-15）与通道的融合，结合TIRF显微镜有望揭示这些长期受欢迎的实体。这些目标有望取得大胆的进展，并带来基本的和实际的影响。

DESCRIPTION (provided by applicant): Over the past grant cycle, general rules for calmodulin (CaM) regulation of the family of Ca channels were discerned. CaM has two lobes, each with two Ca2+ binding sites. A first general aspect was that each lobe can autonomously trigger a form of channel regulation. Secondly, whenever the C-terminal lobe of CaM (C-lobe) triggers channel regulation, there is preferential responsiveness to local Ca2+ signals. Conversely, wherever the N-terminal lobe (N-lobe) initiates regulation, there is selectivity for global Ca2+ signals. Thirdly, there is reason to expect that this rule of operation generalizes beyond Ca channels, to many complexes in which CaM is preassociated with target molecules. Because CaM regulation of Ca channels (and other signaling molecules) is crucial for normal neuroprocessing, and likely important for therapeutics relating to pain, psychosis, and cardiac arrhythmogenesis, answering how these general rules occur is the overarching thrust for the next cycle of research. Three aims will address this overall theme. 1. To develop and perform elementary tests of a kinetic Ca2+ decoding mechanism for the CaM/Ca channel complex. This aim formulates a 'kinetic Ca2+ decoding' theory of how the CaM decoding occurs, and devises novel Voltage-block' experiments to enable elementary tests of this theory. 2. To engineer the local/global Ca2+ preference of CaM/Ca channel regulation, as a higher-order test of the kinetic Ca2+ decoding theory, and as means to glean design principles for developing novel channel modulators. A principal prediction of the kinetic Ca2+ decoding theory is that the local/global Ca2+ preference of channel regulation reflects competition between channel affinities for the Ca2+-bound and Ca2+-free forms of a lobe of CaM. Aim 2 will alter these affinities and check for the predicted changes in Ca2+ preference. Aim 2 will also explore whether these modifications can inform the design of drug compounds that modulate channel regulation in new ways. 3. To experimentally determine Ca2+ concentrations and diffusion within the nanometers of the Ca channel. Crucial to the next phase of progress is the direct measurements of local and global Ca2+ concentration signals, and Ca2+ diffusion, in the actual channel 'nanodomain' environment. Fusions of a genetically-encoded Ca2+ sensor (TNL-15) to channels, combined with TIRF microscopy promise to reveal these long sought-after entities. These aims promise bold progress, with basic and applied ramifications.