A kinetic model of calcium binding to calretinin: Experimental measurements and predicted effects on calcium signaling at neuronal synapses

Calretinin (CR) is a calcium binding protein (CaBP) that is expressed at high concentrations in some neurons, where it may regulate synaptic transmission and other cellular processes that rely on calcium as an intracellular messenger. This dissertation involves three investigations to determine the calcium-binding properties of CR and to predict the effects of CR and other CABPs at synapses.; The first investigation involved development of a method to perform equilibrium and kinetic measurements of Ca2+ binding to CR in vitro . I first determined the binding rate constants of several Ca 2+ indicator dyes and caged chelators that were needed for this technique. I then devised and tested a detailed model of the photo-cleavage process and the photolysis products. I confirmed the model and parameter values by two synthetic Ca2+ buffers with known kinetic values.; The second investigation determined the equilibrium and kinetic properties of Ca2+ binding to CR. Each CR molecule was found to bind 6 Ca2+ with an apparent affinity (Kd,o) of ∼1.1 muM and positive cooperativity (Hill coefficient nH ∼ 1.7). There is an additional low affinity binding site with Kd ∼ 190 muM. I performed an exhaustive series of measurements using flash photolysis to increase free Ca2+ and calcium indicator dyes to follow the relaxation kinetics. I developed a model for Ca2+ binding to CR that accurately accounts for these equilibrium and kinetic data, including cooperativity.; The third investigation used numerical simulation to explore possible roles of CR at synapses. I developed a very fast and robust computational algorithm which simulates the diffusion and chemical binding reaction properties of Ca2+ buffers in a simple geometry. I also developed approximate analytical solutions to reaction-diffusion equations with different reaction schemes giving rise to cooperative Ca2+ binding. I conclude that CR can serve to rapidly terminate synaptic transmission by binding free Ca2+ close to the site of Ca2+ entry and carrying it away. The cooperative behavior of multiple binding sites is predicted to significantly influence the release of Ca2+ from CR, which takes place at a distance from Ca2+ entry sites and may have physiological implications for slower Ca2+-regulated processes.