The kinetics of synaptic vesicle reacidification at hippocampal nerve terminals
The kinetics of synaptic vesicle reacidification at hippocampal nerve terminals
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DOI：
10.1523/jneurosci.4425-05.2006
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发表时间：
2006-02-22
影响因子：
5.3
通讯作者：
Ryan, TA
中科院分区：
文献类型：
--
作者：
Atluri, PP;Ryan, TA
胞吐作用后，突触囊泡在突触终末局部再循环，并通过囊泡转运蛋白重新填充神经递质。再填充的生物物理机制知之甚少，但很明显，跨囊泡膜产生质子梯度是至关重要的。为了更好地了解囊泡再填充的决定因素，我们开发了一种新的方法来明确地测量单个突触末端突触囊泡再固定的动力学。用突触-pHluorin（SpH）转染的海马神经元在一连串重复电刺激后立即快速表面淬灭，所述突触-pHluorin（SpH）是一种突触囊泡靶向的内腔GFP（绿色荧光蛋白），其荧光在质子化时淬灭（pK（a）类似于7.1）。最近内吞的碱性池的SPH的保护，从这样的表面淬灭，其荧光衰减反映reacidification动力学。这些测量结果表明，在补偿性内吞作用后，突触囊泡以一级动力学（τ类似于4 - 5 s）再活化，并且它们的再活化速率受到增加的外部缓冲液的减慢。
After exocytosis, synaptic vesicles are recycled locally in the synaptic terminal and are refilled with neurotransmitter via vesicular transporters. The biophysical mechanisms of refilling are poorly understood, but it is clear that the generation of a proton gradient across the vesicle membrane is crucial. To better understand the determinants of vesicle refilling, we developed a novel method to measure unambiguously the kinetics of synaptic vesicle reacidification at individual synaptic terminals. Hippocampal neurons transfected with synapto-pHluorin (SpH), a synaptic vesicle-targeted lumenal GFP ( green fluorescent protein), whose fluorescence is quenched when protonated (pK(a) similar to 7.1), were rapidly surface-quenched immediately after trains of repetitive electrical stimulation. The recently endocytosed alkaline pool of SpH is protected from such surface quenching, and its fluorescence decay reflects reacidification kinetics. These measurements indicate that, after compensatory endocytosis, synaptic vesicles reacidify with first-order kinetics (tau similar to 4 - 5 s) and that their rate of reacidification is subject to slowing by increased external buffer.