Properties of BKCa channels formed by bicistronic expression of hSloα and β1-4 subunits in HEK293 cells
Properties of BKCa channels formed by bicistronic expression of hSloα and β1-4 subunits in HEK293 cells
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DOI：
10.1007/s00232-002-1070-0
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发表时间：
2003-03-15
影响因子：
2.4
通讯作者：
Davies, NW
中科院分区：
文献类型：
--
作者：
Lippiat, JD;Standen, NB;Davies, NW
大电导Ca 2+激活的K+（BKCa）通道对电压和内部[Ca 2 +]均敏感，并且在许多组织中发现。它们的生理作用范围从引起平滑肌松弛到调节动作电位放电的频率。不同组织的钙离子依赖性和电压依赖性有相当大的差异。这种变化的大部分是由于成孔α亚基（hSloalpha）与不同β亚基的结合导致通道性质改变。由于hSloalpha单独产生功能性BKCa通道，我们使用了双顺反子表达方法来确保α和β亚基都表达，β亚基过量。使用这种方法，我们研究了四个β亚基（β 1至β 4）对克隆的BKCa通道的影响。将四个P亚基单独克隆到具有插入内部核糖体进入位点下游的hSloot cDNA的载体中。将构建体与表达绿色荧光蛋白的构建体一起瞬时转染到HEK 293细胞中，作为转染的标记。荧光细胞表达BKCa通道，其电流从内向外或由外向外的补丁记录。我们使用该表达系统测量的电流与Brenner等人在非洲爪蟾单核细胞中表达的电流相似。
Large-conductance Ca2+-activated K+ (BKCa) channels are sensitive to both voltage and internal [Ca2+] and are found in many tissues. Their physiological roles range from causing relaxation of smooth muscle to regulating the frequency of action potential firing. There is considerable variation between different tissues in their Ca2+ - and voltage-dependence. Much of this variation results from the association of the pore-forming alpha subunit (hSloalpha) with different beta subunits leading to altered channel properties. Since hSloalpha alone produces functional BKCa channels, we have used a bicistronic expression method to ensure that both alpha and beta subunits are expressed, with the beta subunit being in excess. Using this method we have investigated the effect of four beta subunits (beta1 to beta4) on cloned BKCa channels. The four P subunits were individually cloned into a vector that had hSloot cDNA inserted downstream of an internal ribosome entry site. The constructs were transiently transfected into HEK293 cells together with a construct that expresses green fluorescent protein, as a marker for transfection. Fluorescent cells expressed BKCa channels whose currents were recorded from inside-out or outside-out patches. The currents we measured using this expression system were similar to those expressed in Xenopus onocytes by Brenner et al.