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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

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.

Ca 2+触发神经末梢释放递质和内分泌细胞释放激素。Ca2+ 信号由Ca 2+通过电压门控Ca 2+通道进入启动，并由Ca 2+结合到胞质Ca 2+缓冲液。通道已经被广泛研究，但对缓冲区知之甚少。这些蛋白质在进入时迅速结合97.5-99.5%的Ca 2+，并且与Ca 2+源和汇一起形成了一个高度规范但非常有活力的系统。传输和绑定之间复杂的相互作用对细胞内Ca ~（2+）信号的定量研究提出了严峻的挑战。缓冲剂限制了Ca 2+的上升，在进入位点周围设置陡峭的梯度，控制Ca 2+扩散，限制Ca 2+挤出的速率，螯合，并确定下游信号传导靶的Ca 2+的可用性。分子胞质Ca 2+缓冲液的结构是已知的，并且它们的Ca 2+结合特性已经在体外得到了充分的研究。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改变，并且它们在细胞内的锚定通常限制它们的移动性。本申请提出使用荧光在垂体后叶神经末梢中成像以原位探索细胞溶质Ca 2+缓冲液。早期Ca 2+成像工作提供的内源性缓冲能力的测量，表示为？e（总Ca ² +与游离Ca²+之比）。然而，原位结合特性很少被表征。很难从？e浓度和但我们需要这些信息，因为缓冲区饱和度可以降低？也就是一个或两个数量级。该应用程序将使用我们的创新新方法，结合膜片钳和Ca 2+荧光，随后原位滴定Ca 2+结合位点。这种方法远远超出了测量？e至表征多种内源性Ca 2+结合物质。在垂体终末，该方法鉴定了两个Ca 2 + 缓冲液，并测定其Kd和浓度。蛋白质印迹显示了众所周知的细胞质Ca 2 + 缓冲液calretinin和calbindin D28 K，它们的Kd值与我们的测量结果一致。完善我们的方法，并用它来检查不同神经末梢隔室的缓冲，原位，并调查每个物种的流动性，以评估其对Ca 2+扩散的影响。基因消融和计算机模拟将测试关于钙视网膜蛋白和钙结合蛋白D28 K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Ca 2+进入胞吐细胞， Ca 2+触发器。我们将检验缓冲液饱和促进释放的假设，并且缓冲液有助于促进两种垂体激素催产素和加压素的差异。我们将探索通过比较性别，缓冲剂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加压素的功能，通过评估水剥夺动物。这项工作将阐明细胞质的作用，内分泌功能中的Ca 2+缓冲剂，并澄清兴奋-分泌耦合领域中的长期问题。