Molecular microfluorometry: converting arbitrary fluorescence units into absolute molecular concentrations to study binding kinetics and stoichiometry in transporters.
Molecular microfluorometry: converting arbitrary fluorescence units into absolute molecular concentrations to study binding kinetics and stoichiometry in transporters.
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分子微荧光测定：将任意荧光单位转换为绝对分子浓度，以研究转运蛋白中的结合动力学和化学计量。
DOI：
10.1007/3-540-29784-7_2
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发表时间：
2006
影响因子：
--
通讯作者：
DeFelice,LJ
中科院分区：
文献类型：
--
作者：
Schwartz,JW;Piston,D;DeFelice,LJ
协同转运蛋白利用储存在Na+或H+梯度中的能量逆其自身梯度转运神经递质或其他底物。协同转运是快速和有效的，在突触上它有助于终止信号传导。去甲肾上腺素（NET）、肾上腺素（EpiT）、多巴胺（DAT）和5-羟色胺（SERT）转运蛋白中的协同转运将下行Na+通量与上行递质通量偶联。例如，NETs通过有效地从突触间隙清除NE来减弱肾上腺素能突触处的信号传导，从而为下一个信号准备突触。三环类抗抑郁药的转运抑制作用可减少突触间隙中神经递质的存在，从而可能减轻抑郁症状。可卡因或安非他明分别阻断或替代正常转运，从而抑制转运，可能导致多动症。底物或药物与转运蛋白的动力学相互作用知之甚少，主要是因为已经成功发现转运蛋白激动剂和拮抗剂的技术不能产生详细的动力学信息。机制数据在很大程度上局限于全局参数，如KmandVmax，从数千个细胞平均的大量转运分子中测量。三个相对较新的技术用于转运蛋白的研究是电生理学，电流分析法，和显微荧光。这篇评论的重点是荧光为基础的方法，它不同于任何其他技术，允许同时测量的结合和运输。显微荧光法提供了独特的见解结合动力学和运输机制从荧光数据的定量分析。在这里，我们演示了如何量化结合底物分子的数量，运输底物分子的数量，以及底物与单个转运蛋白结合的动力学。虽然我们描述了一个特定的神经递质转运实验，这些方法也适用于其他膜蛋白。
Cotransporters use energy stored in Na+or H+gradients to transport neurotransmitters or other substrates against their own gradient. Cotransport is rapid and efficient, and at synapses it helps terminate signaling. Cotransport in norepinephrine (NET), epinephrine (EpiT), dopamine (DAT), and serotonin (SERT) transporters couples downhill Na+flux to uphill transmitter flux. NETs, for example, attenuate signaling at adrenergic synapses by efficiently clearing NE from the synaptic cleft, thus preparing the synapse for the next signal. Transport inhibition with tricyclic antidepressants prolongs neurotransmitter presence in the synaptic cleft, potentially alleviating symptoms of depression. Transport inhibition with cocaine or amphetamine, which respectively block or replace normal transport, may result in hyperactivity. Little is known about the kinetic interactions of substrates or drugs with transporters, largely because the techniques that have been successful in discovering transporter agonists and antagonists do not yield detailed kinetic information. Mechanistic data are for the most part restricted to global parameters, such asKmandVmax, measured from large populations of transporter molecules averaged over thousands of cells. Three relatively new techniques used in transporter research are electrophysiology, amperometry, and microfluorometry. This review focuses on fluorescence-based methodologies, which—unlike any other technique—permit the simultaneous measurement of binding and transport. Microfluorometry provides unique insights into binding kinetics and transport mechanisms from a quantitative analysis of fluorescence data. Here we demonstrate how to quantify the number of bound substrate molecules, the number of transported substrate molecules, and the kinetics of substrate binding to individual transporters. Although we describe experiments on a specific neurotransmitter transporter, these methods are applicable to other membrane proteins.