Ca Regulation of Ca Channels

描述（由申请人提供）：在过去的资助周期中，发现了钙通道家族钙调素（CaM）调节的一般规则。CaM有两个叶，每个叶有两个Ca 2+结合位点。第一个一般方面是每个叶可以自主触发一种形式的通道调节。其次，每当CaM的C端叶（C叶）触发通道调节时，对局部Ca 2+信号有优先反应。相反，无论N端叶（N叶）启动调节，都对全局Ca 2+信号具有选择性。第三，有理由预期，这一规则的运作推广到钙通道以外，许多复合物中，钙调素是preassociated与目标分子。由于钙通道（和其他信号分子）的钙调素调节是至关重要的正常神经加工，并可能重要的治疗有关的疼痛，精神病和心脏血管生成，回答这些一般规则如何发生是下一个研究周期的首要推力。三个目标将涉及这一总的主题。1.开发并进行CaM/Ca通道复合物的动力学Ca 2+解码机制的初步测试。这一目标制定了一个“动力学Ca 2+解码”的CaM解码如何发生的理论，并设计了新的电压块的实验，使这一理论的初步测试。2.设计CaM/Ca通道调节的局部/全局Ca 2+偏好，作为动力学Ca 2+解码理论的高阶测试，并作为收集开发新型通道调节剂的设计原则的手段。动力学Ca 2+解码理论的一个主要预测是，局部/全球的Ca 2+偏好的通道调节反映了竞争之间的通道亲和力的Ca 2+结合和Ca 2 +-自由形式的一叶钙调素。目标2将改变这些亲和力，并检查预测的Ca 2+偏好变化。目标2还将探索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.通过实验确定Ca通道纳米内的Ca 2+浓度和扩散。下一阶段进展的关键是在实际通道“纳米域”环境中直接测量局部和全局Ca 2+浓度信号以及Ca 2+扩散。基因编码的Ca 2+传感器（TNL-15）与通道的融合，结合TIRF显微镜，有望揭示这些长期寻求的实体。这些目标有望取得大胆的进展，并产生基本和实际的影响。

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.