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首页> 外文学位 >A kinetic model of calcium binding to calretinin: Experimental measurements and predicted effects on calcium signaling at neuronal synapses.
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A kinetic model of calcium binding to calretinin: Experimental measurements and predicted effects on calcium signaling at neuronal synapses.

机译：钙结合钙调蛋白的动力学模型：实验测量和神经突触对钙信号传导的预测影响。

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摘要

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.
机译：钙调蛋白（CR）是一种钙结合蛋白（CaBP），在某些神经元中以高浓度表达，在其中它可以调节突触传递和其他依赖钙作为细胞内信使的细胞过程。本论文涉及三项研究以确定CR的钙结合特性并预测CR和其他CABP在突触中的作用。首次研究涉及开发一种方法来进行Ca2 +与CR的体外结合的平衡和动力学测量。我首先确定了该技术所需的几种Ca 2+指示剂染料和笼状螯合剂的结合速率常数。然后，我设计并测试了光裂解过程和光解产物的详细模型。我用两个已知动力学值的合成Ca2 +缓冲液确认了模型和参数值。第二次研究确定了Ca2 +与CR结合的平衡和动力学性质。发现每个CR分子以约1.1μM的表观亲和力（Kd，o）和正协同性（希尔系数nH约1.7）结合6 Ca2 +。还有一个Kd〜190μM的低亲和力结合位点。我使用快速光解法进行了一系列详尽的测量，以增加游离Ca2 +和钙指示剂染料的弛豫动力学。我开发了Ca2 +与CR结合的模型，该模型可准确说明这些平衡和动力学数据，包括协同作用。第三次调查使用数值模拟来探索CR在突触中的可能作用。我开发了一种非常快速且强大的计算算法，该算法可在简单的几何结构中模拟Ca2 +缓冲液的扩散和化学结合反应特性。我还为具有不同反应方案的反应扩散方程式开发了近似解析解，从而产生了协同的Ca2 +结合。我得出结论，CR可以通过结合靠近Ca2 +进入位点的游离Ca2 +并带走它来迅速终止突触传递。预测多个结合位点的协同行为将显着影响CR中Ca2 +的释放，这种释放发生在距Ca2 +进入位点一定距离的地方，可能对较慢的Ca2 +调节过程具有生理学意义。

著录项

  • 作者

    Alp, Murat.;

  • 作者单位

    University of Oregon.;

  • 授予单位 University of Oregon.;
  • 学科 Biology Neuroscience.; Biophysics General.
  • 学位 Ph.D.
  • 年度 2005
  • 页码 269 p.
  • 总页数 269
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 神经科学;生物物理学;
  • 关键词

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