Naraghi M
Department of Membrane Biophysics, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany.
Cell Calcium. 1997 Oct;22(4):255-68. doi: 10.1016/s0143-4160(97)90064-6.
Recent experimental studies have investigated the kinetic competition between calcium chelators and the secretion apparatus at a fast central synapse. Simultaneously, mathematical modelling studies indicate the importance of a quantitative knowledge of the binding kinetics of the chelators in studying fast physiological processes operating on a millisecond time scale. Using the temperature-jump relaxation method, I have studied the in vitro kinetics of Bis-Fura-2, Furaptra, Fluo-3, Calcium-Green-1, Calcium-Green-5N, Calcium-Orange-5N as well as EGTA, BAPTA and H-EDTA in conditions which are identical to those implemented in our patch clamp recordings, i.e. 100-140 mM CsCl, 20-40 mM Cs-HEPES, 8 mM NaCl, pH = 7.2 at 22 degrees C. The results can be summarized as follows: all fluorescent indicators have on rates in the range of 10(8)-10(9) M-1s-1. They differ significantly with respect to their off-rates from each other according to their affinities, ranging from 100 s-1 up to 26,000 s-1. BAPTA is kinetically almost indistinguishable from Fura-2. EGTA and H-EDTA have small binding rate constants for calcium in the range of 3 x 10(6) M-1s-1 since, at pH 7.20, protons need to be dissociated from the chelators before they can bind calcium ions.
最近的实验研究探讨了钙螯合剂与快速中枢突触处分泌装置之间的动力学竞争。同时，数学建模研究表明，在研究以毫秒时间尺度运行的快速生理过程时，了解螯合剂结合动力学的定量知识非常重要。我使用温度跃变弛豫方法，研究了双呋喃-2、呋喃普特拉、Fluo-3、钙绿-1、钙绿-5N、钙橙-5N以及乙二醇双四乙酸（EGTA）、1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸（BAPTA）和氢乙二胺-N,N,N',N'-四乙酸（H-EDTA）在与我们膜片钳记录中所采用的条件相同的条件下的体外动力学，即100 - 140 mM氯化铯、20 - 40 mM Cs-HEPES、8 mM氯化钠，在22摄氏度时pH = 7.2。结果可总结如下：所有荧光指示剂的结合速率在10⁸ - 10⁹ M⁻¹s⁻¹范围内。根据它们的亲和力，它们的解离速率彼此有显著差异，范围从100 s⁻¹到26,000 s⁻¹。BAPTA在动力学上与呋喃-2几乎无法区分。EGTA和H-EDTA与钙的结合速率常数较小，在3×10⁶ M⁻¹s⁻¹范围内，因为在pH 7.20时，螯合剂需要先解离质子才能结合钙离子。
