Recording ion channels in isolated, split-opened tubules.
Recording ion channels in isolated, split-opened tubules.
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DOI：
10.1007/978-1-62703-351-0_27
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发表时间：
2013-01-01
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影响因子：
--
通讯作者：
Stockand, James D
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文献类型：
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作者：
Mironova, Elena;Bugay, Vladislav;Stockand, James D
离子通道在生理学中发挥着关键作用。它们充当蛋白质传感器，能够将刺激和化学梯度转化为电信号。它们对于细胞信号传导也至关重要，并且在上皮运输中发挥着特别重要的作用，充当电解质跨上皮细胞膜移动的门户。然而，实验的局限性阻碍了许多类型组织中离子通道活动的记录。这减缓了理解这些通道的细胞和生理功能的进展，其中大多数功能是从体外系统和细胞培养模型推断的。在许多情况下，此类推论非但没有澄清事实，反而使情况变得更加模糊。在这里，我们描述了一种分离和膜片钳肾小管的当代方法，用于离体分析天然组织中的离子通道功能。重点是量化醛固酮敏感远端肾单位 (ASDN) 中上皮 Na(+) 通道 (ENaC) 的活性。这种分离的、裂开的小管制剂能够在天然细胞信号传导途径和受体的控制下记录接近天然环境中的肾离子通道。当与器官和系统功能的互补测量以及用于操纵功能和调节的当代分子遗传学和药理学相结合时，在分离的、裂开的肾小管中膜片钳肾通道能够了解这些关键蛋白质从分子到整个动物的生理功能。
Ion channels play key roles in physiology. They function as protein transducers able to transform stimuli and chemical gradients into electrical signals. They also are critical for cell signaling and play a particularly important role in epithelial transport acting as gateways for the movement of electrolytes across epithelial cell membranes. Experimental limitations, though, have hampered the recording of ion channel activity in many types of tissue. This has slowed progress in understanding the cellular and physiological function of these channels with most function inferred from in vitro systems and cell culture models. In many cases, such inferences have clouded rather than clarified the picture. Here, we describe a contemporary method for isolating and patch-clamping renal tubules for ex vivo analysis of ion channel function in native tissue. Focus is placed on quantifying the activity of the epithelial Na(+) channel (ENaC) in the aldosterone--sensitive distal nephron (ASDN). This isolated, split-open tubule preparation enables recording of renal ion channels in the close-to-native environment under the control of native cell signaling pathways and receptors. When combined with complementary measurements of organ and system function, and contemporary molecular genetics and pharmacology used to manipulate function and regulation, patch-clamping renal channels in the isolated, split-open tubule enables understanding to emerge about the physiological function of these key proteins from the molecule to the whole animal.