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

基本信息

批准号：
10240521
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
项目类别：
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/10240521
关键词：
Ablation Action Potentials Address Affinity Alzheimer&apos 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

Ablation Action Potentials Address Affinity Alzheimer&apos 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

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项目摘要

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+触发了从内分泌细胞中神经末端和激素的发射器释放。 Ca2+ 信号是通过CA2+通过电压门控Ca2+通道启动的，并由Ca2+结合到胞质CA2+缓冲液。这些通道已经进行了广泛的研究，但是对缓冲区的了解少得多。这些蛋白质在进入时迅速结合97.5-99.5％的Ca2+，并与CA2+源一起结合形成一个高度调节但非常动态的系统。运输和结合之间的复杂相互作用对细胞Ca2+信号传导的定量研究提出了巨大的挑战。缓冲区限制Ca2+的上升，设置进入入口部位，控制Ca2+扩散，限制CA2+扩展速率的蒸汽梯度和隔离，并确定CA2+对于下游信号通转目标的可用性。分子胞质Ca2+缓冲液的结构是已知的，其Ca2+结合特性在体外已经很好地研究了。但是，它们在细胞中的浓度很难测量，它们的结合特性可能会改变细胞质，它们在细胞内的锚定通常会限制其活动能力。此应用程序提出使用荧光的建议在垂体后神经末端进行成像，以探索原位的胞质Ca2+缓冲液。早期CA2+成像工作提供了内源缓冲能力的测量值，表示为？E（总CA²+的比率）。但是，原位结合特性很少是表征的。从“ e”到集中度很难 KD，但是我们需要此信息，因为缓冲满意度可以减少一个或两个数量级。该应用程序将使用我们的创新新方法，该方法将贴片夹紧和Ca2+荧光结合到遵循原位Ca2+结合位点的滴定。此方法远远超出了“ 表征多个内源性Ca2+结合物种。在垂体终端中，此方法确定了两个Ca2+ 缓冲区，并确定其KD和浓度。 Western印迹揭示了众所周知的胞质CA2+ 缓冲仪calletin和calbindin D28K及其KD与我们的测量值一致。我们将有所改善我们的方法并使用它来检查不同神经终端隔室中的缓冲原位，并研究每个物种评估其对Ca2+扩散的影响的迁移率。遗传消融和计算机模拟将测试有关Calletin和Calbindin D28K的生物学功能的假设。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+进入胞吐的访问 CA2+触发器。我们将检验以下假设：缓冲满意度准备释放，并且缓冲区有助于两种垂体激素，氧气和加压素的促进差异。我们将探索潜力通过比较性别，缓冲液在氧气生殖功能中的作用，并在流体平衡中的潜在作用加压素的功能通过评估水分剥夺动物。这项工作将阐明胞质的作用内分泌功能中的Ca2+缓冲液，并阐明了兴奋 - 分泌耦合领域的长期问题。