Lippiat J D, Standen N B, Harrow I D, Phillips S C, Davies N W
Ion Channel Group, Dept. Cell Physiology & Pharmacology, University of Leicester, Leicester LE1 9HN, UK.
J Membr Biol. 2003 Mar 15;192(2):141-8. doi: 10.1007/s00232-002-1070-0.
Large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels are sensitive to both voltage and internal [Ca(2+)] 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 Ca(2+)- 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 BK(Ca) 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 BK(Ca) channels. The four beta subunits were individually cloned into a vector that had hSloalpha 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 BK(Ca) 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 oocytes by Brenner et al. (Brenner, R., Jegla, T.J., Wickenden, A., Liu, Y., Aldrich, R.W. 2000. Cloning and functional expression of novel large-conductance calcium-activated potassium channel beta subunits, hKCNMB3 and hKCNMB4. J. Biol. Chem.275:6453-6461.)
大电导钙激活钾（BK(Ca)）通道对电压和细胞内[Ca(2+)]均敏感，存在于多种组织中。其生理作用范围从引起平滑肌舒张到调节动作电位发放频率。不同组织之间其钙依赖性和电压依赖性存在相当大的差异。这种差异很大程度上源于形成孔道的α亚基（hSloα）与不同的β亚基结合，导致通道特性改变。由于单独的hSloα就能产生功能性的BK(Ca)通道，我们采用了双顺反子表达方法来确保α亚基和β亚基都能表达，且β亚基过量表达。利用这种方法，我们研究了四种β亚基（β1至β4）对克隆的BK(Ca)通道的影响。将这四种β亚基分别克隆到一个载体中，该载体在内部核糖体进入位点下游插入了hSloα cDNA。这些构建体与一个表达绿色荧光蛋白的构建体一起瞬时转染到HEK293细胞中，绿色荧光蛋白作为转染的标记物。荧光细胞表达BK(Ca)通道，从内向外或外向内的膜片上记录其电流。我们使用该表达系统测量的电流与Brenner等人在非洲爪蟾卵母细胞中表达的电流相似（Brenner, R., Jegla, T.J., Wickenden, A., Liu, Y., Aldrich, R.W. 2000. 新型大电导钙激活钾通道β亚基hKCNMB3和hKCNMB4的克隆与功能表达。《生物化学杂志》275:6453 - 6461.）
