Stable expression of the human large-conductance Ca2+-activated K+ channel alpha- and beta-subunits in HEK293 cells
Stable expression of the human large-conductance Ca2+-activated K+ channel alpha- and beta-subunits in HEK293 cells
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DOI：
10.1016/s0014-5793(97)01096-x
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发表时间：
1997-09-22
期刊：
影响因子：
3.5
通讯作者：
Johansen, TE
中科院分区：
文献类型：
--
作者：
Ahring, PK;Strobaek, D;Johansen, TE
我们已经生成了高水平表达人BK通道α -亚基或BK通道α -亚基和β -亚基的HEK293细胞系。为了共表达，构建了带有三个表达盒的质粒。膜片钳在转染细胞的内外膜片上记录，产生反映每个膜片200-800个BK通道表达的宏观电流，在生长超过50代的细胞中未检测到通道表达的减少，α -亚基单独表达时在生理范围内传导对细胞内Ca2+敏感的电流。BK通道开启剂NS1608 （10 μ M）使α -亚基和α - β -亚基的稳态激活曲线移动了-40 ~ -50 mV。(C) 1997年欧洲生化学会联合会。
We have generated HEK293 cell lines stably expressing high levels of either the human BK channel alpha-subunit alone or the BK channel alpha-subunit and beta-subunit together. For co-expression a plasmid with three expression cassettes was constructed. Patch-clamp recordings on inside-out patches from the transfected cells resulted in macroscopic currents reflecting the expression of 200-800 BK channels per patch, No decrease in channel expression could be detected in cells grown for more than 50 passages, The alpha-subunit when expressed alone conducted currents which were sensitive to intracellular Ca2+ in the physiological range. In the presence of the beta-subunit the steady-state activation curves were shifted by -20 to -30 mV and channel deactivation kinetics were slowed, The BK channel opener NS1608 (10 mu M) shifted the steady-state activation curves for the alpha-subunit as well as for the alpha beta-subunits by -40 to -50 mV. (C) 1997 Federation of European Biochemical Societies.