Lanthanide Ions as Spectroscopic Probes of Calmodulin and SK Channel Activation

镧系离子作为钙调蛋白和 SK 通道激活的光谱探针

• 批准号: 8069574
• 负责人: Jennifer N. Greeson-Bernier
• 金额: $ 5.3万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8069574
• 关键词: Affinity; Agonist; Aromatic Amino Acids; Binding; Biological Assay; Calcium; Calmodulin; Calmodulin-Binding Proteins; Cells; Complex; Coupled; Detection; Development; Disease; Drug Delivery Systems; EF Hand Motifs; Energy Transfer; Europium; Frequencies; Goals; Individual; Ion Channel; Ions; Knowledge; Lanthanoid Series Elements; Ligand Binding; Lobe; Measurement; Measures; Membrane; Mental disorders; Methodology; Methods; Modeling; Monitor; Mutation; Neurons; Optics; Play; Property; Reaction; Role; SK potassium channel; Sampling; Shapes; Site; Solutions; Specificity; Synapses; Techniques; Terbium; Time; Work; cell type; drug development; improved; luminescence; nervous system disorder; novel strategies; research study; response; tool development; voltage

DESCRIPTION (provided by applicant): Small-conductance calcium (Ca2+)-activated potassium (SK) channels are activated solely by intracellular Ca2+ and underlie the after hyperpolarization (AHP) and firing frequency in central neurons and other excitable cells. Due to their functional diversity, SK channels are potential targets for drug development in the treatment of a number of different diseases, and these efforts would be greatly enhanced by a deeper understanding of their activation mechanism. SK channels rely on the ubiquitous Ca2+ binding protein, calmodulin (CaM), which associates with the channel constitutively to sense changes in intracellular Ca2+. A critical step to understanding the Ca2+-activation of SK channels hinges on obtaining separate measurements of Ca2+ binding and channel opening. Through these measurements, the contribution of Ca2+ binding steps to opening can be determined. Here we propose to measure Ca2+ binding and channel activation simultaneously in functioning SK channels using a combined optical and electrophysiological approach. Additionally, we propose to fully characterize Ca2+ binding to CaM through measures of EF hand-specific affinities and the degree of binding cooperativity. Luminescent, trivalent lanthanide ions (Ln3+) can effectively substitute for spectroscopically silent Ca2+ in activating SK channels. Eu3+ and Tb3+ can be directly excited, or Tb3+ luminescence can be sensitized through energy transfer from an adjacent aromatic amino acid. By introducing an energy-transferring residue into the Ca2+ binding domain, the latter method allows for site-specific monitoring of Tb3+ binding. Using direct Ln3+ excitation, though site specificity is lost, binding is distinguished through an increase in the Ln3+ luminescence decay lifetime, and measurements can utilize wild-type CaM. In either case, the measured luminescence intensity is directly proportional to the fraction of bound Ln3+ and is therefore a direct measurement of the binding isotherm. The experiments presented in this proposal are the first to directly measure the site-specific Ca2+ and Ln3+ binding affinities of individual EF hands in both lobes of intact CaM. Additionally, the cooperativity of Ca2+ binding will be assessed using mutations that significantly reduce Ca2+ binding affinity adjacent EF hands. The combination of site-specific measurements of Ca2+ affinities and direct measurements of the degree of binding cooperativity will aid in the development of a quantitative understanding of Ca2+ binding to CaM. Additionally, the direct measurement of Ca2+ binding to functioning SK channels in membrane patches is only the second of its kind in an ion channel. The development of this tool certainly has large implications for improving our understanding of the Ca2+-activation of the SK channel, however, the proposed method is also widely applicable to numerous channels that are modulated by CaM.

描述（由申请人提供）： 小电导钙激活钾通道（SK）仅由细胞内Ca ~（2+）激活，是中枢神经元和其他可兴奋细胞后超极化（AHP）和放电频率的基础。由于它们的功能多样性，SK通道是治疗许多不同疾病的药物开发的潜在靶点，并且这些努力将通过对其激活机制的更深入理解而大大增强。SK通道依赖于普遍存在的Ca 2+结合蛋白，钙调蛋白（CaM），其与通道组成性地结合以感知细胞内Ca 2+的变化。理解SK通道的Ca 2+激活的关键步骤在于获得Ca 2+结合和通道开放的单独测量。通过这些测量，可以确定Ca 2+结合步骤对开放的贡献。在这里，我们建议测量Ca 2+结合和通道激活同时在功能SK通道使用相结合的光学和电生理方法。此外，我们建议通过EF手特异性亲和力和结合协同性的程度的措施，充分表征钙离子结合钙调素。发光的三价镧系离子（Ln 3+）可以有效地取代光谱上沉默的Ca 2+激活SK通道。Eu 3+和Tb 3+可以被直接激发，或者Tb 3+发光可以通过来自相邻芳香族氨基酸的能量转移而被敏化。通过将能量转移残基引入Ca 2+结合结构域，后一种方法允许对Tb 3+结合进行位点特异性监测。使用直接的Ln 3+激发，虽然失去了位点特异性，但通过增加Ln 3+发光衰减寿命来区分结合，并且测量可以利用野生型CaM。在任一情况下，测量的发光强度与结合的Ln 3+的分数成正比，因此是结合等温线的直接测量。在这个建议中提出的实验是第一个直接测量的站点特异性的Ca 2+和Ln 3+结合亲和力的个人EF手在两个完整的钙调素叶。此外，将使用显著降低邻近EF手的Ca 2+结合亲和力的突变来评估Ca 2+结合的协同性。结合位点特异性测量的Ca 2+的亲和力和直接测量的结合协同性的程度将有助于发展的定量理解的Ca 2+结合钙调素。此外，直接测量Ca 2+结合到膜补丁中的功能SK通道，这只是离子通道中的第二种。这个工具的开发当然有很大的影响，提高我们的理解钙激活的SK通道，然而，所提出的方法也广泛适用于许多通道，由钙调素。