Optical microwell assay of membrane transport kinetics
Optical microwell assay of membrane transport kinetics
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DOI：
10.1016/s0006-3495(03)74655-4
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发表时间：
2003-10-01
影响因子：
3.4
通讯作者：
Peters, R
中科院分区：
文献类型：
--
作者：
Kiskin, NI;Siebrasse, JP;Peters, R
在光学单转运体记录中，膜牢固地附着在含有小孔或测试室（TC）的平坦固体基板上。荧光分子通过tc跨越膜斑块的运输是由溶液变化引起的，并通过共聚焦显微镜记录。以前，轨迹蚀刻膜过滤器被用来制造含有随机分布的tc种群的固体基质。在这项研究中，探索了由激光微钻创建的有序TC阵列提供的可能性。考虑到膜传输、溶液变化和扩散的卷积，建立了一个理论框架。研究了有序TC阵列的光学性质，并测量了溶液变化动力学。分析了爪蟾卵母细胞核孔复合物（NPC）在离体包膜中的输出和输入。根据先前的报道，直接与NPC蛋白结合的核转运受体NTF2被发现比类似大小的“惰性”分子转运快得多。出乎意料的是，据报道，NTF2的同源物NXT1不能直接与NPC蛋白结合，它的易位速度与NTF2一样快。因此，微结构TC阵列为光学单转运子记录提供了新的基础。
In optical single transporter recording, membranes are firmly attached to flat solid substrates containing small wells or test compartments (TC). Transport of fluorescent molecules through TC-spanning membrane patches is induced by solution change and recorded by confocal microscopy. Previously, track-etched membrane filters were used to create solid substrates containing populations of randomly distributed TCs. In this study the possibilities offered by orderly TC arrays as created by laser microdrilling were explored. A theoretical framework was developed taking the convolution of membrane transport, solution change, and diffusion into account. The optical properties of orderly TC arrays were studied and the kinetics of solution change measured. Export and import through the nuclear pore complex (NPC) was analyzed in isolated envelopes of Xenopus oocyte nuclei. In accordance with previous reports nuclear transport receptor NTF2, which binds directly to NPC proteins, was found to be translocated much faster than "inert" molecules of similar size. Unexpectedly, NXT1, a homolog of NTF2 reportedly unable to bind to NPC proteins directly, was translocated as fast as NTF2. Thus, microstructured TC arrays were shown to provide optical single transporter recording with a new basis.