Ca2+ buffering in the regulation of secretion from peptidergic nerve terminals

肽能神经末梢分泌调节中的 Ca2 缓冲

• 批准号: 10000213
• 负责人: MEYER B. JACKSON
• 金额: $ 32.91万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10000213
• 关键词: Ablation; Action Potentials; Address; Affinity; Alzheimer' s Disease; Animals; Axon; Binding; Binding Proteins; Binding Sites; Biological Process; Buffers; Cell membrane; Cells; Complex; Computer Simulation; Coupled; Coupling; Cytoplasm; Data; Defect; Development; Diffuse; Diffusion; Disease; Dyes; Electric Capacitance; Endocrine; Environment; Epilepsy; Equilibrium; Evoked Potentials; Exocytosis; Failure; Female; Fluid Balance; Fluorescence; Frequencies; Genetic; Hormones; Image; Immunohistochemistry; In Situ; In Vitro; Ions; Knock-out; Knowledge; Link; Measurement; Measures; Methods; Modeling; Molecular; Molecular Structure; Mus; Nerve; Nervous system structure; Neurons; Oxytocin; Physiologic pulse; Pituitary Gland; Pituitary Hormones; Posterior Pituitary Gland; Process; Property; Proteins; Regulation; Role; Sex Differences; Shapes; Signal Transduction; Site; Source; Swelling; Synapses; Synaptic Transmission; Testing; Time; Titrations; Vasopressins; Water; Western Blotting; Work; calbindin-D28K; calretinin; dynamic system; fluorescence imaging; improved; innovation; insight; knockout animal; male; neurotransmitter release; novel; patch clamp; protein function; reproductive function; sensor; sex; simulation; spatiotemporal; voltage

Ca2+ triggers the release of transmitters from nerve terminals and hormones from endocrine cells. Ca2+ signals are initiated by Ca2+ entry through voltage-gated Ca2+ channels, and shaped by Ca2+ binding to cytosolic Ca2+ buffers. The channels have been extensively studied, but much less is known about the buffers. These proteins rapidly bind 97.5-99.5% of the Ca2+ upon entry, and together with the Ca2+ sources and sinks form a highly regulated but very dynamic system. The complex interplay between transport and binding presents a formidable challenge to the quantitative study of cellular Ca2+ signaling. Buffers limit the rise in Ca2+, set up steep gradients around sites of entry, control Ca2+ diffusion, limit the rate of Ca2+ extrusion and sequestration, and determine the availability of Ca2+ for downstream signaling targets. The molecular structures of cytosolic Ca2+ buffers are known and their Ca2+ binding properties have been well studied in vitro. However, their concentrations in cells are hard to measure, their binding properties can change in cytoplasm, and their anchoring within cells often restricts their mobility. This application proposes to use fluorescence imaging in posterior pituitary nerve terminals to explore cytosolic Ca2+ buffers in situ. Early Ca2+ imaging work provided measurements of the endogenous buffering capacity, denoted as ?e (the ratio of total to free Ca²+). However, the in situ binding properties are rarely characterized. It is difficult to go from ?e to concentration and Kd, but we need this information because buffer saturation can reduce ?e by one or two orders of magnitude. This application will use our innovative new method that combines patch clamping and Ca2+ fluorescence to follow the titration of Ca2+ binding sites in situ. This method goes well beyond measurements of ?e to characterize multiple endogenous Ca2+ binding species. In pituitary terminals this method identified two Ca2+ buffers, and determined their Kd and concentration. Western blots revealed the well-known cytosolic Ca2+ buffers calretinin and calbindin D28K, and their Kd’s are consistent with our measurements. We will improve our approach and use it to examine buffering in different nerve terminal compartments, characterize diffusion in situ, and investigate the mobility of each species to assess its influence on Ca2+ diffusion. Genetic ablation and computer simulation will test hypotheses about the biological functions of calretinin and calbindin D28K. We will explore the role of these proteins in secretion and determine how they control Ca2+ access to the exocytotic Ca2+ trigger. We will test the hypothesis that buffer saturation facilitates release, and that buffers contribute to differences in facilitation of the two pituitary hormones, oxytocin and vasopressin. We will explore the potential roles of buffers in reproductive functions of oxytocin by comparing sexes, and potential roles in fluid balance functions of vasopressin by evaluating water-deprived animals. This work will illuminate the role of cytosolic Ca2+ buffers in endocrine function and clarify longstanding issues in the field of excitation-secretion coupling.

Ca2+ 触发神经末梢释放递质和内分泌细胞释放激素。钙离子 信号由 Ca2+ 通过电压门控 Ca2+ 通道进入启动，并由 Ca2+ 结合形成 胞质 Ca2+ 缓冲液。这些通道已被广泛研究，但对缓冲区的了解却少之又少。 这些蛋白质在进入后迅速结合 97.5-99.5% 的 Ca2+，并与 Ca2+ 源和汇结合在一起 形成一个高度监管但又非常动态的系统。运输和结合之间复杂的相互作用 对细胞 Ca2+ 信号传导的定量研究提出了巨大的挑战。缓冲剂限制 Ca2+ 的上升， 在入口点周围设置陡峭的梯度，控制 Ca2+ 扩散，限制 Ca2+ 挤出速率和 封存，并确定 Ca2+ 对下游信号传导目标的可用性。分子 胞质 Ca2+ 缓冲液的结构是已知的，并且它们的 Ca2+ 结合特性已在体外得到充分研究。 然而，它们在细胞中的浓度很难测量，它们的结合特性可以在细胞质中发生变化， 它们在细胞内的锚定通常限制了它们的移动性。该应用建议使用荧光 垂体后神经末梢成像以原位探索胞质 Ca2+ 缓冲液。早期Ca2+成像工作 提供了内源性缓冲能力的测量值，表示为 ?e（总 Ca2+ 与游离 Ca2+ 的比率）。 然而，原位结合特性很少被表征。从专注到专注是很困难的 Kd，但我们需要此信息，因为缓冲区饱和可以将 εe 降低一两个数量级。 该应用将使用我们创新的新方法，结合膜片钳和 Ca2+ 荧光来 遵循原位 Ca2+ 结合位点的滴定。该方法远远超出了 ?e 的测量范围 表征多种内源性 Ca2+ 结合物种。在垂体末梢中，该方法识别出两种 Ca2+ 缓冲液，并测定其 Kd 和浓度。蛋白质印迹揭示了众所周知的胞质 Ca2+ 缓冲液 calretinin 和 calbindin D28K，其 Kd 与我们的测量结果一致。我们会改进 我们的方法并用它来检查不同神经末梢室的缓冲，表征扩散 原位，并研究每个物种的迁移率以评估其对 Ca2+ 扩散的影响。基因消融和 计算机模拟将检验有关钙结合蛋白 D28K 和钙结合蛋白生物功能的假设。我们 将探索这些蛋白质在分泌中的作用并确定它们如何控制 Ca2+ 进入胞吐 Ca2+ 触发。我们将测试缓冲液饱和促进释放以及缓冲液有助于释放的假设 催产素和加压素这两种垂体激素的促进作用存在差异。我们将探索潜力 通过比较性别来了解缓冲液在催产素生殖功能中的作用，以及在液体平衡中的潜在作用 通过评估缺水动物来了解加压素的功能。这项工作将阐明细胞质的作用 Ca2+ 缓冲内分泌功能，并澄清兴奋-分泌耦合领域长期存在的问题。