Ca2+ Buffering and Dynamics in a Large, Peptidergic Nerve Terminal

The large, peptidergic nerve terminals of the posterior pituitary provide a useful model system for the study of presynaptic physiology. Details of Ca2+ buffering and dynamics were investigated in a population of large diameter (≥ 5mum) nerve terminals from acute pituitary slices using two-photon fluorescence imaging. Nerve terminals were loaded with 50-100 muM of the Ca2+ indicator fluo-8 by whole-cell patch clamp recording. Ca2+ imaging has provided significant insights into the dynamics of cellular Ca2+ signaling. The spatiotemporal evolution of intracellular [Ca2+]free is shaped by binding and unbinding to cytoplasmic Ca2+ buffers, as well as the fluorescent indicator used for imaging, sequestration into intracellular compartments and extrusion. These factors must be properly accounted for when interpreting Ca2+ imaging data, and can be employed to study endogenous Ca2+ signaling mechanisms. We improved the use of Ca2+ fluorometry for characterization of cytosolic Ca2+ binding molecules, extending previous methods of in situ titration of cytosolic Ca2+ binding sites with measured amounts of Ca2+ entering through voltage-gated Ca2+ channels. We developed a systematic procedure for fitting fluorescence data acquired during a series of voltage steps to models with multiple Ca 2+ binding sites. The method accounts for the measurement errors providing robust estimation of buffering parameters, even in the presence of noisy data. The method was validated with simulated data, and then applied to 2-photon fluorescence imaging data from rat posterior pituitary nerve terminals patch clamp-loaded with the Ca2+ indicator fluo-8. The Ca 2+ buffering capacity of the cytosol (K) was found to decrease as a function of [Ca2+]Free in a manner consistent with one or two saturable endogenous buffers. The nerve terminals in the posterior pituitary were found to express a combination of mobile, high affinity (Kd 400 nM) buffers and immobile, low affinity (K d ∼ 4 muM) buffers. These experiments provide detailed information regarding endogenous Ca2+ buffers which we incorporated into a computational model for Ca2+ removal. This analysis implicated mitochondria as a significant participant in Ca2+ signaling, accounting for ∼45% of the initial phase of Ca2+ removal following a train of depolarizing pulses.