PATCH CRAMMING - MONITORING INTRACELLULAR MESSENGERS IN INTACT-CELLS WITH MEMBRANE PATCHES CONTAINING DETECTOR ION CHANNELS
PATCH CRAMMING - MONITORING INTRACELLULAR MESSENGERS IN INTACT-CELLS WITH MEMBRANE PATCHES CONTAINING DETECTOR ION CHANNELS
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DOI：
10.1016/0896-6273(90)90046-i
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发表时间：
1990-03-01
期刊：
影响因子：
16.2
通讯作者：
KRAMER, RH
中科院分区：
文献类型：
--
作者：
KRAMER, RH
本文介绍了“补丁填鸭式”，一个新的程序，利用离子通道门控直接由细胞内信使分子作为探针，用于检测该分子在一个完整的细胞中的浓度变化。将含有膜贴片中的通道的贴片移液管插入受体细胞中，其中通道局部“感测”细胞内信使。在这项研究中，含有Caz+依赖性K+通道的贴片被插入到阻滞神经元中，在动作电位序列期间，它们被Ca*+内流激活。通道门直接由其他信使，包括环核苷酸和IP3，也已确定。因此，通过使用具有适当特异性的检测器通道，可以检测这些分子的局部细胞内波动。细胞内信使分子是细胞过程中普遍存在的调节因子。关于可扩散的细胞内信使如环AMP、环GMP、IP和Ca 2+的信号传导的生化事件已经了解了很多。然而，鲜为人知的是，这些分子的浓度变化的动力学和空间分布的完整细胞内。一个明显的例外是Ca2+。利用高灵敏度和特异性的Ca2+指示剂染料，在检测细胞内Ca2+方面取得了快速进展，其空间和时间分辨率不断提高，其中Ca2+选择性结合位点类似于众所周知的螯合剂ECXA。虽然这使得设计用于检测Ca2+的有用染料成为可能，但对于其他细胞内信使分子，如环核苷酸和IP3，还没有类似的指示剂。这些分子的细胞内浓度的变化仅通过破坏细胞并测定其内容物的生物化学方法来测量。
This paper introduces “patch cramming,” a new procedure that utilizes an ion channel gated directly by an intracellular messenger molecule as a probe for detecting changes in the concentration of that molecule in an intact cell. A patch pipette containing the channel in a membrane patch is inserted into a recipient cell where the channel locally “senses” the intracellular messenger. In this study patches containing Caz+ dependent K+ channels were inserted into Helix neurons, where they were activated by Ca*+ influx during trains of action potentials. Channels gated directly by other messengers, including cyclic nucleotides and IP3, have also been identified. Hence, by using detector channels with appropriate specificity, it may be possible to detect local intracellular fluctuations of these molecules. tntroductionIntracellular messenger molecules are ubiquitous regulators of cellular processes. Much has been learned about the biochemical events underlying signaling by diffusible intracellular messengers such as cyclic AMP,,: yclic GMP, IPs, and Ca2+. However, little is known about the dynamics and spatial distribution of concentration changes of these molecules inside intact cells. One notable exception is Ca2+. Rapid progress has been made in detecting intracellular Ca2+ with increasing spatial and temporal resolution with the use of highly sensitive and specific Ca2+ indicator dyes, in which the Ca2+-selective binding site resembles the well-known chelator, ECXA. While this has enabled the design of useful dyes for detecting Ca2+, there are no comparable indicators for other intracellular messenger molecules, such as cyclic nucleotides and IP3. Changes in the intracellular concentrations of these molecules have been measured only by disrupting cells and assaying their contents biochemic ally.