Lotan I, Volterra A, Dash P, Siegelbaum S A, Goelet P
Department of Pharmacology, Howard Hughes Medical Institute, Columbia University, New York, New York 10032.
Neuron. 1988 Dec;1(10):963-71. doi: 10.1016/0896-6273(88)90153-5.
Ionic currents were recorded from Xenopus oocytes injected with RNA isolated from chick or mouse brain. Three currents were studied: a rapid tetrodotoxin-sensitive Na+ current (Ina), an early outward K+ current sensitive to 4-aminopyridine (IA), and an inward current activated by the excitatory amino acid receptor agonist kainate. Oligonucleotides (60-80 bases long) complementary to rat brain Na+ channel sequences were prehybridized to chick brain RNA. These DNA sequences, upon injection into oocytes, specifically inhibited expression of INa relative to IA and the kainate-induced current in a dose-dependent manner. By contrast, prehybridization of oligonucleotides complementary to sequences either from the Drosophila Shaker locus (which codes for an early K+ current in Drosophila muscle) or from a homologous clone from mouse brain did not block the expression of the early outward K+ current induced in the oocytes by mRNA from chick or mouse brain. This method provides a convenient means for testing the functional role of cloned DNA species.
从注射了从鸡或小鼠大脑中分离出的RNA的非洲爪蟾卵母细胞中记录离子电流。研究了三种电流：一种对河豚毒素敏感的快速Na⁺电流（Ina）、一种对4-氨基吡啶敏感的早期外向K⁺电流（IA）以及一种由兴奋性氨基酸受体激动剂海人酸激活的内向电流。与大鼠脑Na⁺通道序列互补的寡核苷酸（60 - 80个碱基长）与鸡脑RNA进行预杂交。将这些DNA序列注射到卵母细胞中后，相对于IA和海人酸诱导的电流，它们以剂量依赖的方式特异性抑制Ina的表达。相比之下，与果蝇Shaker基因座（其编码果蝇肌肉中的早期K⁺电流）的序列或来自小鼠脑的同源克隆序列互补的寡核苷酸预杂交，并不会阻断由鸡或小鼠脑mRNA在卵母细胞中诱导产生的早期外向K⁺电流的表达。这种方法为测试克隆DNA物种的功能作用提供了一种便捷手段。
