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: 7913618
• 负责人: Jennifer N. Greeson-Bernier
• 金额: $ 5.05万
• 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/7913618
• 关键词: Affinity; Aromatic Amino Acids; Binding; Calcium; Calmodulin; Calmodulin-Binding Proteins; Cells; Development; Disease; Drug Delivery Systems; EF Hand Motifs; Energy Transfer; Frequencies; Individual; Ion Channel; Ions; Lanthanoid Series Elements; Lobe; Measurement; Measures; Membrane; Methods; Monitor; Mutation; Neurons; Optics; SK potassium channel; Site; Specificity; Work; drug development; improved; luminescence; novel strategies; public health relevance; research study; tool development

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. PUBLIC HEALTH RELEVANCE: Ion channels, such as the small-conductance calcium-activated potassium (SK) channel, are important drug targets for a variety of diseases. The development of efficient and effective drugs that target SK channels would benefit greatly from a detailed understanding of their activation mechanism and function. The work proposed here will investigate the calcium activation of SK channels using a novel approach that can also be applied to a number of other ion channels that are modulated by calcium.

描述（由申请人提供）： 小电导钙 (Ca2+) 激活钾 (SK) 通道仅由细胞内 Ca2+ 激活，是中枢神经元和其他可兴奋细胞的后超极化 (AHP) 和放电频率的基础。由于其功能多样性，SK 通道是治疗多种不同疾病的药物开发的潜在靶标，通过更深入地了解其激活机制，这些努力将得到极大加强。 SK 通道依赖于普遍存在的 Ca2+ 结合蛋白钙调蛋白 (CaM)，它与通道组成型结合以感知细胞内 Ca2+ 的变化。了解 SK 通道 Ca2+ 激活的关键步骤取决于获得 Ca2+ 结合和通道开放的单独测量。通过这些测量，可以确定 Ca2+ 结合步骤对开放的贡献。在这里，我们建议使用光学和电生理学相结合的方法同时测量功能 SK 通道中的 Ca2+ 结合和通道激活。此外，我们建议通过测量 EF 手特异性亲和力和结合协同程度来充分表征 Ca2+ 与 CaM 的结合。发光的三价镧系元素离子 (Ln3+) 可以有效替代光谱上沉默的 Ca2+ 来激活 SK 通道。 Eu3+和Tb3+可以直接激发，或者Tb3+发光可以通过相邻芳香族氨基酸的能量转移来敏化。通过将能量转移残基引入 Ca2+ 结合域，后一种方法可以对 Tb3+ 结合进行位点特异性监测。使用直接 Ln3+ 激发，虽然失去了位点特异性，但通过 Ln3+ 发光衰变寿命的增加来区分结合，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 Ln3+ 的分数成正比，因此是结合等温线的直接测量。本提案中提出的实验是第一个直接测量完整 CaM 两个叶中单个 EF 手的位点特异性 Ca2+ 和 Ln3+ 结合亲和力的实验。此外，将使用显着降低 EF 手附近 Ca2+ 结合亲和力的突变来评估 Ca2+ 结合的协同性。 Ca2+ 亲和力的位点特异性测量和结合协同程度的直接测量相结合将有助于定量了解 Ca2+ 与 CaM 的结合。此外，直接测量 Ca2+ 与膜片中功能性 SK 通道的结合只是离子通道中的第二个此类方法。该工具的开发无疑对于提高我们对 SK 通道 Ca2+ 激活的理解具有重大意义，然而，所提出的方法也广泛适用于 CaM 调制的众多通道。 公共卫生相关性： 离子通道，例如小电导钙激活钾（SK）通道，是多种疾病的重要药物靶点。详细了解 SK 通道的激活机制和功能，开发高效且有效的针对 SK 通道的药物将大大受益。这里提出的工作将使用一种新方法研究 SK 通道的钙激活，该方法也可应用于由钙调节的许多其他离子通道。