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    Detection of transmembrane potentials by optical methods
    5.
    发明授权
    Detection of transmembrane potentials by optical methods 有权
    通过光学方法检测跨膜电位

    公开(公告)号：US07173130B2

    公开(公告)日：2007-02-06

    申请号：US09967772

    申请日：2001-09-28

    申请人： Roger Y. Tsien ,  Jesus E. Gonzalez, III

    发明人： Roger Y. Tsien ,  Jesus E. Gonzalez, III

    IPC分类号： C07D403/06

    CPC分类号： G01N33/502 ,  C07D209/08 ,  C07D215/227 ,  C07D239/60 ,  C07D239/62 ,  C07D239/66 ,  C07D239/80 ,  C07D241/44 ,  C07D265/36 ,  C07D279/16 ,  C07D311/16 ,  C07D487/04 ,  C07F5/003 ,  C07F5/02 ,  C07F5/027 ,  C07F9/10 ,  C07F9/5728 ,  C07K2319/033 ,  C09B23/02 ,  G01N33/5005 ,  G01N33/5008 ,  G01N33/5044 ,  G01N33/5058 ,  G01N33/5061 ,  G01N33/5076 ,  G01N33/52 ,  G01N33/542 ,  G01N33/582 ,  G01N33/6872 ,  G01N33/92 ,  G01N2405/04 ,  G01N2500/10 ,  Y10S436/80 ,  Y10S436/805

    摘要： Methods and compositions are provided for detecting changes in membrane potential in membranes biological systems. In one aspect, the method comprises; a) providing a living cell with a first reagent comprising a charged hydrophobic molecule which is typically a fluorescence resonance energy transfer (FRET) acceptor or donor, or is a quencher and is capable of redistributing within the membrane of a biological membrane in response to changes in the potential across the membrane; b) providing the cell with a second reagent that can label the first face or the second face of a biological membrane within the cell; c) detecting light emission from the first reagent or the second reagent. One aspect of this method involves monitoring membrane potential changes in subcellular organelle membranes in a living cells.Another aspect of the invention is the use of certain embodiments of the method for the screening of test chemicals for activity to modulate the activity of a target ion channel.Another aspect of the present invention is a transgenic organism comprising a first reagent that comprises a charged hydrophobic fluorescent molecule, and a second reagent comprising a bioluminescent or naturally fluorescent protein.

    摘要翻译： 提供了用于检测膜生物系统中膜电位变化的方法和组合物。 一方面，该方法包括： a）向活细胞提供包含通常为荧光共振能量转移（FRET）受体或供体的带电荷的疏水性分子的第一试剂，或者是猝灭剂，并且能够响应于变化而重新分布在生物膜的膜内 在膜的潜力; b）向细胞提供可以标记细胞内生物膜的第一面或第二面的第二试剂; c）检测来自第一试剂或第二试剂的发光。 该方法的一个方面涉及监测活细胞中亚细胞细胞器膜中的膜电位变化。 本发明的另一方面是该方法的某些实施方案用于筛选测试化学品以调节靶离子通道的活性的活性。 本发明的另一方面是转基因生物，其包含含有带电荷的疏水荧光分子的第一试剂和包含生物发光或天然荧光蛋白的第二试剂。

    Detection of transmembrane potentials by optical methods
    10.
    发明授权
    Detection of transmembrane potentials by optical methods 有权
    通过光学方法检测跨膜电位

    公开(公告)号：US06596522B2

    公开(公告)日：2003-07-22

    申请号：US09378534

    申请日：1999-08-20

    申请人： Roger Y. Tsien ,  Jesus E. Gonzalez, III

    发明人： Roger Y. Tsien ,  Jesus E. Gonzalez, III

    IPC分类号： C12N1300

    CPC分类号： G01N33/5076 ,  C07D209/08 ,  C07D239/60 ,  C07D239/62 ,  C07D239/66 ,  C07D311/16 ,  C07F5/003 ,  C07F5/02 ,  C07F5/027 ,  C07F9/10 ,  C07F9/5728 ,  C09B23/02 ,  G01N33/5005 ,  G01N33/5008 ,  G01N33/502 ,  G01N33/5044 ,  G01N33/5058 ,  G01N33/5061 ,  G01N33/52 ,  G01N33/542 ,  G01N33/582 ,  G01N33/6872 ,  G01N2500/10 ,  Y10S436/80 ,  Y10S436/805

    摘要： Methods and compositions are provided for determining the potential of a membrane. In one aspect, the method comprises: (a) introducing a first reagent comprising a hydrophobic fluorescent ion capable of redistributing from a first face of the membrane to a second face of the membrane in response to changes in the potential of the membrane, as described by the Nernst equation, (b) introducing a second reagent which labels the first face or the second face of the membrane, which second reagent comprises a chromophore capable of undergoing energy transfer by either (i) donating excited state energy to the fluorescent ion, or (ii) accepting excited state energy from the fluorescent ion, (c) exposing the membrane to radiation; (d) measuring energy transfer between the fluorescent ion and the second reagent, and (e) relating the energy transfer to the membrane potential. Energy transfer is typically measured by fluorescence resonance energy transfer. In some embodiments the first and second reagents are bound together by a suitable linker. In one aspect the method is used to identify compounds which modulate membrane potentials in biological membranes.

    摘要翻译： 提供了用于确定膜的电位的方法和组合物。 一方面，该方法包括：（a）如所描述的那样，引入包含能够从膜的第一面重新分布到膜的第二面的疏水性荧光离子的第一试剂，以响应膜的电位变化 通过能斯特方程，（b）引入标记膜的第一面或第二面的第二试剂，该第二试剂包含能够通过（i）向荧光离子提供激发态能量而进行能量转移的发色团， 或（ii）从荧光离子接收激发态能量，（c）将膜暴露于辐射;（d）测量荧光离子和第二试剂之间的能量传递，和（e）将能量转移与膜电位相关联。 能量转移通常通过荧光共振能量转移来测量。 在一些实施方案中，第一和第二试剂通过合适的接头结合在一起。在一个方面，该方法用于鉴定调节生物膜中膜电位的化合物。