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

钙离子触发神经末梢的递质释放和内分泌细胞的激素释放。Ca2+ 信号是由钙离子通过电压门控钙离子通道进入启动的，并由钙离子结合到 胞内钙离子缓冲液。人们对这些通道进行了广泛的研究，但对缓冲区的了解要少得多。 这些蛋白质在进入时迅速结合97.5-99.5%的钙离子，并与钙源和钙汇合在一起 形成一个高度监管但非常有活力的体系。运输和结合之间复杂的相互作用 这对细胞内钙信号的定量研究提出了严峻的挑战。缓冲液限制了钙离子的上升， 在入口点周围设置陡峭的梯度，控制钙离子的扩散，限制钙离子的挤出速度和 隔离，并确定下游信号转导靶标的钙离子可用性。分子 细胞内钙缓冲液的结构已为人所知，其与钙离子的结合特性已在体外得到了很好的研究。 然而，它们在细胞中的浓度很难测量，它们的结合特性可能会在细胞质中发生变化， 而它们在细胞内的锚定往往会限制它们的流动性。此应用程序建议使用荧光 在垂体后神经末梢进行成像，以原位探测胞浆中的钙缓冲。早期的钙离子成像工作 提供了内源性缓冲能力的测量，表示为？E(总钙与游离钙的比率)。 然而，原位结合的性质很少被表征。很难从精神集中到集中精神。 Kd，但我们需要这个信息，因为缓冲区饱和可以减少一个或两个数量级。 这一应用将使用我们创新的新方法，即结合膜片钳和钙离子荧光来 进行钙离子结合部位的原位滴定。这种方法远远超出了？e到？的测量范围。 表征多种内源性钙离子结合物种。在脑垂体末梢，该方法鉴定出两种钙离子 缓冲液，并测定它们的解离常数和浓度。Western blotting揭示了众所周知的胞内钙离子 缓冲液Calretinin和Calbindin D28K，它们的Kd与我们的测量结果一致。我们会改进的 我们的方法并用它来检查不同神经末梢隔间的缓冲，表征了 并研究每个物种的迁移率，以评估其对钙离子扩散的影响。基因消融和 计算机模拟将检验有关calretinin和calbindin D28K生物学功能的假说。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钙离子进入胞吐 CA2+触发器。我们将测试缓冲区饱和有助于释放的假设，以及缓冲区有助于 催产素和加压素这两种垂体激素促进作用的差异。我们将探索其潜力 通过性别比较，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 通过评估缺水动物的加压素功能。这项工作将阐明细胞质的作用 钙离子在内分泌功能中的缓冲作用，并阐明了兴奋-分泌耦合领域中长期存在的问题。

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.