Dual roles of the C2B domain of synaptotagmin I in synchronizing Ca2+-dependent neurotransmitter release
Dual roles of the C2B domain of synaptotagmin I in synchronizing Ca2+-dependent neurotransmitter release
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DOI：
10.1523/jneurosci.2545-04.2004
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发表时间：
2004-09-29
影响因子：
5.3
通讯作者：
Augustine, GJ
中科院分区：
文献类型：
--
作者：
Nishiki, T;Augustine, GJ
虽然囊泡蛋白synaptotagmin I含有两个Ca 2+结合结构域（C（2）A和C2B），但Ca 2+与C2B结构域的结合对于触发同步神经递质释放更为重要。我们已经使用点突变，以确定5个带负电荷的天冬氨酸（Asp）残基的功能贡献，构成了突触结合蛋白I的C2B结构域中的Ca 2+结合位点。将野生型synaptotagmin I DNA转染到来自synaptotagmin I基因敲除小鼠的培养海马神经元中，挽救了Ca 2+依赖性同步递质释放，并减少了释放的较慢的异步成分，表明synaptotagmin I抑制异步释放。突变C2B结构域的第二或第三个天冬氨酸残基有效地抑制了突触结合蛋白I拯救同步释放的能力，但没有改变其抑制异步释放的能力。在C2B结构域的第一个或第四个Asp残基突变的Synaptotagmin I部分拯救同步释放和部分抑制异步释放，而中和第五个Asp残基的能力synaptotagmin I救援发射释放没有影响。因此，我们得出结论，突触结合蛋白I的C2B结构域调节神经递质释放至少有两种方式。同步释放绝对需要结合的Ca 2+的第二个和第三个天冬氨酸残基在这个域。对于异步释放的抑制，Ca 2+结合到突触结合蛋白I的C2B结构域显然是不必要的，因为第二个Asp残基的突变抑制Ca 2+结合，但仍然允许这种蛋白质抑制异步释放。
Although the vesicular protein synaptotagmin I contains two Ca2+-binding domains (C(2)A and C2B), Ca2+ binding to the C2B domain is more important for triggering synchronous neurotransmitter release. We have used point mutagenesis to determine the functional contributions of the five negatively charged aspartate ( Asp) residues that constitute the Ca2+-binding sites in the C2B domain of synaptotagmin I. Transfecting wild-type synaptotagmin I DNA into cultured hippocampal neurons from synaptotagmin I knock-out mice rescued Ca2+-dependent synchronous transmitter release and reduced a slower, asynchronous component of release, indicating that synaptotagmin I suppresses asynchronous release. Mutating either the second or third Asp residues of the C2B domain potently inhibited the ability of synaptotagmin I to rescue synchronous release but did not change its ability to suppress asynchronous release. Synaptotagmin I with mutations in the first or fourth Asp residues of the C2B domain partially rescued synchronous release and partially suppressed asynchronous release, whereas neutralizing the fifth Asp residue had no effect on the ability of synaptotagmin I to rescue transmitter release. Thus, we conclude that the C2B domain of synaptotagmin I regulates neurotransmitter release in at least two ways. Synchronous release absolutely requires binding of Ca2+ to the second and third Asp residues in this domain. For the suppression of asynchronous release, Ca2+ binding to the C2B domain of synaptotagmin I apparently is not necessary because mutation of the second Asp residue inhibits Ca2+ binding, yet still allows this protein to suppress asynchronous release.