Control of ion channel expression for patch clamp recordings using an inducible expression system in mammalian cell lines.
Control of ion channel expression for patch clamp recordings using an inducible expression system in mammalian cell lines.
复制标题
使用哺乳动物细胞系中的诱导表达系统控制斑块夹记录的离子通道表达。
DOI：
10.1186/1471-2202-4-15
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发表时间：
2003-07-02
期刊：
影响因子：
2.4
通讯作者：
Korn SJ
中科院分区：
文献类型：
--
作者：
Trapani JG;Korn SJ
许多离子通道功能的分子研究依赖于使用膜片钳技术获得高质量电压钳记录的能力。对于在哺乳动物细胞异源表达系统中研究的多种通道类型，缺乏实验者对表达水平的控制严重阻碍了获得具有适合于高质量记录的表达水平的高百分比细胞的能力。此外，几乎不可能在哺乳动物细胞中获得非常适合单通道记录的表达水平。我们在这里描述的诱导型启动子系统在稳定转染的哺乳动物细胞系，产生近100%的成功，在获得离子通道的表达水平，无论是全细胞或单离子通道研究合适的使用。我们使用四环素调控的表达系统来控制CHO（中国仓鼠卵巢）细胞系中K+通道的表达。对于宏观或单通道记录，可以容易且可靠地获得合理窄范围内的电流幅度。在几乎100%的测试细胞中可以获得1 - 2nA的宏观电流。仅需2 ~ 3小时即可获得所需的表达水平，并在室温下保持稳定。也可以获得非常低表达水平的转染通道，这导致从贴片记录单通道电流的能力的成功率>70%。此外，在这些低表达水平下，似乎内源性通道产生很少或没有污染。这种控制离子通道表达的方法相对简单，极大地提高了收集高质量宏观电流数据的速度和效率，并且使得有可能容易且可靠地记录哺乳动物细胞异源表达系统中的单通道电流。尽管我们证明了该系统控制电压门控K+通道表达水平的能力，但它应该适用于在哺乳动物表达系统中表达良好的所有其他通道类型。
Many molecular studies of ion channel function rely on the ability to obtain high quality voltage clamp recordings using the patch clamp technique. For a variety of channel types studied in mammalian cell heterologous expression systems, the lack of experimenter control over expression levels severely hinders the ability to obtain a high percentage of cells with an expression level suitable for high quality recordings. Moreover, it has been nearly impossible to obtain expression levels in mammalian cells well suited for single channel recordings. We describe here the use of an inducible promoter system in a stably transfected mammalian cell line that produces nearly 100% success in obtaining ion channel expression levels suitable for either whole cell or single ion channel studies. We used a tetracycline-regulated expression system to control K+ channel expression in a CHO (Chinese hamster ovary) cell line. Current magnitudes within a reasonably narrow range could be easily and reliably obtained for either macroscopic or single channel recordings. Macroscopic currents of 1 – 2 nA could be obtained in nearly 100% of cells tested. The desired expression level could be obtained within just 2 to 3 hours, and remained stable at room temperature. Very low expression levels of transfected channels could also be obtained, which resulted in a >70% success rate in the ability to record single channel currents from a patch. Moreover, at these low expression levels, it appeared that endogenous channels produced little or no contamination. This approach to controlling ion channel expression is relatively simple, greatly enhances the speed and efficiency with which high quality macroscopic current data can be collected, and makes it possible to easily and reliably record single channel currents in a mammalian cell heterologous expression system. Whereas we demonstrate the ability of this system to control expression levels of voltage-gated K+ channels, it should be applicable to all other channel types that express well in mammalian expression systems.