Atluri Pradeep P, Ryan Timothy A
Department of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021, USA.
J Neurosci. 2006 Feb 22;26(8):2313-20. doi: 10.1523/JNEUROSCI.4425-05.2006.
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 (pKa approximately 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 approximately 4-5 s) and that their rate of reacidification is subject to slowing by increased external buffer.
胞吐作用之后，突触小泡在突触终末进行局部循环利用，并通过囊泡转运体重新填充神经递质。重新填充的生物物理机制尚不清楚，但很明显，跨囊泡膜产生质子梯度至关重要。为了更好地理解囊泡重新填充的决定因素，我们开发了一种新方法，能够明确测量单个突触终末处突触小泡重新酸化的动力学。用突触pH荧光蛋白（SpH）转染的海马神经元，SpH是一种靶向突触小泡的腔内绿色荧光蛋白（GFP），其荧光在质子化时淬灭（pKa约为7.1），在重复电刺激序列后立即进行快速表面淬灭。最近内吞的SpH碱性池受到这种表面淬灭的保护，其荧光衰减反映了重新酸化动力学。这些测量结果表明，在代偿性内吞作用之后，突触小泡以一级动力学重新酸化（时间常数约为4 - 5秒），并且它们的重新酸化速率会因外部缓冲液增加而减慢。
