PROPERTIES OF SINGLE SODIUM-CHANNELS TRANSLATED BY XENOPUS OOCYTES AFTER INJECTION WITH MESSENGER-RIBONUCLEIC-ACID
PROPERTIES OF SINGLE SODIUM-CHANNELS TRANSLATED BY XENOPUS OOCYTES AFTER INJECTION WITH MESSENGER-RIBONUCLEIC-ACID
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DOI：
10.1113/jphysiol.1987.sp016523
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发表时间：
1987-05-01
影响因子：
5.5
通讯作者：
SIGEL, E
中科院分区：
文献类型：
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作者：
SIGEL, E
1.用全细胞电压钳和膜片钳技术研究了非洲爪蟾卵母细胞质膜注入外源信使核糖核酸（mRNA）后所诱导的快瞬变钠通道的特性。2.表达的时间过程和代谢抑制剂放线菌素D和衣霉素的影响进行了研究。通道插入速率在注射mRNA后仅约3天达到最大值，对应于20个活性通道/s掺入单个卵母细胞。当应用于细胞内衣霉素几乎完全阻断了活跃通道的出现，而放线菌素D加入到培养基中没有效果。3.全细胞电流显示激活和失活特性，让人联想到骨骼肌Na+电流。最大峰值电流幅度为6 μ A。河豚毒素阻断所观察到的瞬时内向电流。在10 nM浓度下观察到50%抑制。 藜芦定抑制电流失活，导致尾电流延长。4.去除卵母细胞的周围层后，用膜片钳电极推到表面膜上，获得紧密的密封。可记录到卵母细胞内源性单通道电流和注射mRNA诱导的Na+通道电流。后者的单通道斜率电导为12-15 pS。两种不同类型的动力学行为是显而易见的单通道电流和合奏平均电流的分析。一种类型显示快速失活（τ）。< 1 ms）和短暂的通道开放（< 1 ms），而第二种类型的特征在于较慢的失活和爆发行为。5.当在移液管溶液中存在藜芦碱（75 μ M）时，单通道行为以复杂的方式改变。除了具有正常电导的沟道开口之外，还观察到具有3.5pS的斜率电导的第二开放状态。在返回到保持电位之后，仍然可以记录第二种类型的沟道打开。其最终闭合遵循指数时间过程，在-100 mV下的恒定时间常数为0.5 s。这些事件可能是全细胞结构中尾电流的基础。6.非洲爪蟾卵母细胞代表了一个有用的系统，用于研究外源mRNA诱导的通道表达，用于表征其单通道行为，并用于研究生物活性物质对这些通道的作用。这个系统可能被证明是有用的研究通道，无法访问膜片钳实验“原位”。
1. The properties of fast transient Na channels induced in the Xenopus laevis oocyte plasma membrane after injection of the oocyte with foreign messenger ribonucleic acid (mRNA) were investigated with the whole-cell voltage clamp and with the patch-clamp technique. 2. The time course of expression and the effect of the metabolic inhibitors actinomycin D and tunicamycin were studied. The rate of channel insertion reached a maximum only about 3 days after injection with mRNA and corresponded to the incorporation of 20 active channels/s, into a single oocyte. When applied intracellularly tunicamycin blocked the appearance of active channels nearly completely while actinomycin D added to the medium had no effect. 3. The whole-cell currents showed activation and inactivation properties reminiscent of skeletal muscle Na+ currents. The maximal peak current amplitude was 6 .mu.A. Tetrodotoxin blocked the observed transient inward current. 50% inhibition was observed at 10 nM concentration. Veratridine depressed inactivation of the current and led to prolonged tail currents. 4. After removal of the surrounding layers of the oocyte tight seals were obtained with a patch-clamp electrode pushed on the surface membrane. Single-channel currents endogenous to the oocyte and Na+-channel currents induced by injected mRNA could be recorded. The single-channel slope conductance of the latter was 12-15 pS. Two different types of kinetic behavior were evident from an analysis of single-channel currents and ensemble average currents. One type showed fast inactivation (.tau. < 1 ms) and brief channel opening (< 1 ms) whereas the second type was characterized by slower inactivation and a bursting behavior. 5. When veratridine (75 .mu.M) was present in the pipette solution the single-channel behavior was modified in a complex manner. In addition to the channel openings with normal conductance a second open state was observed with a slope conductance of 3.5 pS. This second type of channel opening could still be recorded after return to the holding potential. Its final closure followed an exponential time course with a constant time constant of 0.5 s at -100 mV. These events probably underlie the tail currents in the whole-cell configuration. 6. The Xenopus oocyte represents a useful system for the study of the expression of channels induced by foreign mRNA, for the characterization of their single-channel behavior and for the investigation of the action of pharmacologically active substances on these channels. This system may prove useful for the study of channels that are not accessible to patch-clamp experiments ''in situ''.