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...